amino acid. ASV avian sarcoma virus ..... to divide them into âcopy-and-pasteâ or. âcut-and-pasteâ ... However, strict borders cannot be drawn, as some elements ...
Helsinki 2006
Characterization of the Molecular Components and Function of the BARE-1, Hin-Mu and Mu Transposition Machineries
19/2005 Anssi Rantakari Characterisation of the Type Three Secretion System in Erwinia carotovora 20/2005 Sari Airaksinen Role of Excipients in Moisture Sorption and Physical Stability of Solid Pharmaceutical Formulations 21/2005 Tiina Hilden Affinity and Avidity of the LFA-1 Integrin is Regulated by Phosphorylation 22/2005 Ari Pekka Mähönen Cytokinins Regulate Vascular Morphogenesis in the Arabidopsis thaliana Root 23/2005 Matias Palva Interactions Among Neuronal Oscillations in the Developing and Adult Brain 24/2005 Juha T. Huiskonen Structure and Assembly of Membrane-Containing dsDNA Bacteriophages 25/2005 Michael Stefanidakis Cell-Surface Association between Progelatinases and ß2 Integrins: Role of the Complexes in Leukocyte Migration 26/2005 Heli Kansanaho Implementation of the Principles of Patient Counselling into Practice in Finnish Community Pharmacies 1/2006 Julia Perttilä Expression, Enzymatic Activities and Subcellular Localization of Hepatitis E Virus and Semliki Forest Virus Replicase Proteins 2/2006 Tero Wennberg Computer-Assisted Separation and Primary Screening of Bioactive Compounds 3/2006 Katri Mäkeläinen Lost in Translation: Translation Mechanisms in Production of Cocksfoot Mottle Virus Proteins 4/2006 Kari Kreander A Study on Bacteria-Targeted Screening and in vitro Safety Assessment of Natural Products 5/2006 Gudrun Wahlström From Actin Monomers to Bundles: The Role of Twinfilin and a-Actinin in Drosophila melanogaster Development 6/2006 Jussi Joensuu Production of F4 Fimbrial Adhesin in Plants: A Model for Oral Porcine Vaccine against Enterotoxigenic Escherichia coli 7/2006 Heikki Vilen Mu in vitro Transposition Technology in Functional Genetics and Genomics: Applications on Mouse and Bacteriophages 8/2006 Jukka Pakkanen Upregulation and Functionality of Neuronal Nicotinic Acetylcholine Receptors 9/2006 Antti Leinonen Novel Mass Spectrometric Analysis Methods for Anabolic Androgenic Steroids in Sports Drug Testing 10/2006 Paulus Seitavuopio The Roughness and Imaging Characterisation of Different Pharmaceutical Surfaces 11/2006 Leena Laitinen Caco-2 Cell Cultures in the Assessment of Intestinal Absorption: Effects of Some Co-Administered Drugs and Natural Compounds in Biological Matrices 12/2006 Pirjo Wacklin Biodiversity and Phylogeny of Planktic Cyanobacteria in Temperate Freshwaters 13/2006 Antti Alaranta Medication Use in Elite Athletes
ANNA-HELENA SAARIAHO
Recent Publications in this Series:
ISSN 1795-7079 ISBN 952-10-3182-4
Characterization of the Molecular Components and Function of the BARE-1, Hin-Mu and Mu Transposition Machineries
ANNA-HELENA SAARIAHO
Institute of Biotechnology, and Division of Genetics Department of Biological and Environmental Sciences Faculty of Biosciences, and Viikki Graduate School in Biosciences University of Helsinki
Dissertationes bioscientiarum molecularium Universitatis Helsingiensis in Viikki 14/2006
14/2006
CHARACTERIZATION OF THE MOLECULAR COMPONENTS AND FUNCTION OF THE BARE-1, HIN-MU AND MU TRANSPOSITION MACHINERIES
ANNA-HELENA SAARIAHO
Institute of Biotechnology and Division of Genetics, Department of Biological and Environmental Sciences, Faculty of Biosciences and Viikki Graduate School in Biosciences University of Helsinki
ACADEMIC DISSERTATION To be presented, with the permission of the Faculty of Biosciences of the University of Helsinki, for public criticism in the auditorium 1041 of the Biocenter, Viikinkaari 5, Helsinki, on the June 9th, 2006, at 12 o’clock noon.
Supervisor Docent Harri Savilahti Institute of Biotechnology University of Helsinki
Reviewers Docent Tero Ahola Institute of Biotechnology University of Helsinki Professor Kristiina Mäkinen Department of Applied Biology Faculty of Agriculture and Forestry University of Helsinki
Opponent Professor Maia Kivisaar Department of Genetics Institute of Molecular and Cell Biology Tartu University and Estonian Biocentre Estonia
ISBN 952-10-3182-4 (paperback) ISBN 952-10-3183-2 (PDF, http://ethesis.helsinki.fi/) ISSN 1795-7079 (paperback) ISSN 1795-8229 (PDF, http://ethesis.helsinki.fi/) Cover figure: a schematic illustration of Mu core machinery. Edita Prima Oy Helsinki 2006
To the memory of my father and my son
TABLE OF CONTENTS ABBREVIATIONS ORIGINAL PUBLICATIONS A. SUMMARY ........................................................................................................................ 1 B. INTRODUCTION............................................................................................................... 2 1. TRANSPOSABLE ELEMENTS UBIQUITOUS RESIDENTS OF GENOMES.......................................................... 2 2. CLASSIFICATION OF ELEMENTS ....................................................................... 2 2.1 RETROELEMENTS (CLASS I) ....................................................................... 3 2.2 DNA-ELEMENTS (CLASS II) ......................................................................... 5 3. UNITY IN TRANSPOSITION ................................................................................. 5 3.1 SIMILARITY OF CHEMICAL REACTIONS ................................................ 5 3.2 DIVERSITY OF MECHANISMS ..................................................................... 6 3.2.1 Non-replicative transposition ................................................................... 6 3.2.2 Replicative transposition ........................................................................... 8 3.3 SIMILARITY OF CATALYZING ENZYMES: TRANSPOSASES AND INTEGRASES ......................................................................................... 9 3.4 TRANSPOSITION MACHINERIES ............................................................ 10 3.4.1 Functional and structural differences of transposition machineries . 11 4. PLANT RETROELEMENTS .................................................................................. 12 4.1 GENERAL STRUCTURE OF PLANT LTR- RETROTRANSPOSONS ... 13 4.2 RETROTRANSPOSON LIFE CYCLE ......................................................... 13 4.2.1 From transcription to integration ......................................................... 14 4.3 IDENTIFICATION AND ACTIVITY STUDIES OF PLANT RETROTRANSPOSONS ................................................................................ 16 4.4 BARE-1, A BARLEY RETROTRANSPOSON FAMILY ............................. 16 4.4.1 Structure of BARE-1 ............................................................................... 17 4.4.2 Distribution and activity of BARE-1 family ......................................... 18 5. DNA TRANSPOSONS: TRANSPOSABLE BACTERIOPHAGES ................... 18 5.1 PHAGE MU: A VIRUS AND A TRANSPOSON ........................................... 18 5.1.1 Replicative transposition of mu and function Of mu transposition machinery ............................................................. 20 5.1.1.1 DNA COMPONENTS OF THE MACHINERY ............................. 20 5.1.1.2 PROTEIN COMPONENTS OF THE MACHINERY .................... 21 5.1.1.3 ASSEMBLY AND FUNCTION OF THE MACHINERY ............... 23 5.1.1.4 DISASSEMBLY OF THE MACHINERY: TRANSITION FROM TRANSPOSOSOME TO REPLISOME ......................................... 24 5.1.1.5 STRUCTURE FUNCTION RELATIONSHIPS OF THE MACHINERY ....................................................................... 24 5.1.2 “Non-replicative” transposition of Mu ................................................. 24 5.2 MU AS A TRANSPOSITION MODEL SYSTEM: IN VITRO ASSAYS ..... 25 5.3 OTHER TRANSPOSABLE BACTERIOPHAGES ..................................... 26 C. AIMS OF THE PRESENT STUDY ................................................................................ 28
D. E.
MATERIALS AND METHODS ..................................................................................... 29 RESULTS AND DISCUSSION ....................................................................................... 30 1. IDENTIFICATION AND CHARACTERIZATION OF BARE-1 AND HIN-MU TRANSPOSITION MACHINERY COMPONENTS (I, II) ................ 30 1.1 IDENTIFICATION OF BARE-1 VLP MACHINERY COMPONENTS (I) .......................................................................................... 30 1.1.1 BARE-1 GAG and IN are expressed and processed into mature sizes in vivo (I) ........................................................................... 31 1.1.2 BARE-1 GAG, IN and cDNA are present with RT-activity in middle fractions of the sucrose gradient (I) .......................................... 31 1.1.3 VLP-like structures are formed (I) ...................................................... 32 1.2 IDENTIFICATION AND CHARACTERIZATION OF HIN-MU CORE MACHINERY COMPONENTS (II) ................................................. 33 1.2.1 Identification of ends: Hin-Mu is a full-length Mu-like prophage (II) ............................................................................. 33 1.2.2 Identification of transposase: MuAHin is structurally similar to MuA (II) .................................................................................. 34 1.2.3 Identification of binding sites: Hin-Mu ends are conserved and contain putative transposase binding sites (II) .................................... 34 1.2.4 Interactions between DNA and protein components of Hin-Mu and Mu core machineries (II) ................................................. 35 1.2.5 General features of Mu and Hin-Mu binding sites (II) ....................... 36 2. FUNCTION OF HIN-MU AND MU TRANSPOSITION CORE MACHINERIES (II, III) ............................................................................. 36 2.1 FUNCTION OF HIN-MU MACHINERY (II) .............................................. 37 2.1.1 Catalytically competent Hin-Mu transpososomes are assembled (II) .................................................................................... 37 2.2 FUNCTION OF MU MACHINERY (II, III) ................................................. 38 2.2.1 MuA catalyzes hairpin processing reaction preferentially with longer hairpin loops (III) ........................................................................ 38 2.2.2 MuA hairpin processing shares similarities with cleavage reaction (III) ............................................................................. 39 2.2.3 Hairpin processing takes place within Mu transpososome (III) ......... 40 2.3 FLEXIBILITY OF MU MACHINERY (II, III) ............................................ 40 3. FUNCTION OF BARE-1, HIN-MU AND MU TRANSPOSITION MACHINERIES IN VIVO (I, II, III) ..................................................................... 41 3.1 IS BARE-1 TRANSPOSITIONALLY ACTIVE IN VIVO? (I) ..................... 41 3.2 IS HIN-MU TRANSPOSITIONALLY ACTIVE IN VIVO? (II) ................. 42 3.3 DOES MUA CATALYZE HAIRPINNING OF MU DNA IN VIVO? (III) .. 42 4. MINIMAL COMPONENT IN VITRO TRANSPOSITION ASSAY AS A TOOL (II, III) ...................................................................................................43 F. CONCLUSIONS AND FUTURE PROSPECTS ........................................................... 44 G. ACKNOWLEDGEMENTS ............................................................................................. 46 H. REFERENCES ................................................................................................................ 48
Abbreviations A aa ASV ATP BARE bp C CDC cDNA DEP DNA DR dsDNA env/ENV gag/GAG Hin-Mu HIV HTH HU IAS IgG IHF in/IN IS L-end LER LINE LTR kb kDa MITEs MLV MuA MuB
adenine amino acid avian sarcoma virus adenosine triphosphate barley retroelement base pair(s) cytosine cleaved donor complex complementary deoxyribonucleic acid double-ended integration product deoxyribonucleic acid direct repeat double-stranded DNA envelope gene/ protein gene encoding structural capsid protein/ structural retroviral capsid protein Haemophilus influenzae Rd Mu-like prophage human immunodeficiency virus helix-turn-helix E. coli DNA binding protein, accessory protein in Mu transposition internal activating sequence (transposition enhancer element in Mu genome) immunoglobulin G E.coli integration host factor protein integrase gene/protein insertion sequence left end three site (Left end-Enhancer-Right end) synaptic intermediate in Mu transposition long interspersed repeated element long terminal repeat kilobase(s) kilodalton(s) miniature inverted-repeat transposable elements murine leukemia virus bacteriophage Mu transposase protein A bacteriophage Mu transposition protein B
MRF mRNA nt ORF PAGE PBS pol/POL REMAP PCR PIC PPT
Mu replication factor messenger ribonucleic acid nucleotide(s) open reading frame polyacrylamide gel electrophoresis retroelement minus strand priming binding site polymerase gene / protein retrotransposon microsatellite amplified polymorphism polymerase chain reaction preintegration complex retroelement plus strand primer binding site, a polypurine tract pr/PR protease gene/ protein R-end right end RAG1/RAG2 recombination activating gene proteins 1 and 2 rh gene encoding RNaseH RNA ribonucleic acid RNaseH ribonuclease H RSV Rous sarcoma virus rt/RT reverse transcriptase gene/protein SGS strong gyrase site in Mu genome SEP single-ended integration product SINE short interspersed repeated element SIV simian immunodeficiency virus SSC stable synaptic complex ssDNA strong stop DNA SSRs simple sequence repeats STC strand transfer complex TCC target capture complex TE transposable element TIR terminal inverted repeat TEM transmission electron microsopy Tn transposon tRNA transfer ribonucleic acid UTL untranslated leader sequence VLP virus like particle V(D)J variable (diversity) joining
ORIGINAL PUBLICATIONS The thesis is based on the following publications, which are referred to in the text by their Roman numerals. I
Jääskeläinen, M., Mykkänen, A.-H., Arna, T., Vicient, C. M., Suoniemi, A., Kalendar, R., Savilahti, H. and Schulman, A. H. (1999) Retrotransposon BARE1: expression of encoded proteins and formation of virus-like particles in barley cells. Plant J., 20: 413-422.
II
Saariaho, A.–H., Lamberg, A., Elo, S. and Savilahti, H. (2005) Functional comparison of the transposition core machineries of phage Mu and Haemophilus influenzae Mu like prophage Hin-Mu reveals interchangeable components. Virology, 331: 6-19
III
Saariaho, A- H., and Savilahti, H. (2006) Characteristics of MuA transposasecatalyzed processing of model transposon end DNA hairpin substrates. Nucleic Acids Research, in press.
Summary
A. SUMMARY A wide variety of transposable elements use a fundamentally similar mechanism called transpositional DNA recombination (transposition) for the movement within and between the genomes of their host organisms. Although transposable elements inhabit the genomes of a diversity of organisms, the DNA breakage and joining reactions that underlie their transposition are chemically similar in virtually all known transposition systems. The similarity of the reactions is also reflected in the structure and function of the catalyzing enzymes, transposases and integrases. The transposition reactions take place within the context of a transposition machinery, which can be particularly complex, as in the case of the VLP (virus like particle) machinery of retroelements, which in vivo contains RNA or cDNA and a number of element encoded structural and catalytic proteins. Yet, the minimal core machinery required for transposition comprises a multimer of transposase or integrase proteins and their binding sites at the element DNA ends only. Although the chemistry of DNA transposition is fairly well characterized, the components and function of the transposition machinery have been investigated in detail for only a small group of elements This work focuses on the identification, characterization, and functional studies of the molecular components of the transposition machineries of BARE-1, Hin-Mu and Mu. For BARE-1 and HinMu transpositional activity has not been
shown previously, whereas bacteriophage Mu is a general model of transposition. For BARE-1, which is a retroelement of barley (Hordeum vulgare), the protein and DNA components of the functional VLP machinery were identified from cell extracts. In the case of Hin-Mu, which is a Mu-like prophage in Haemophilus influenzae Rd genome, the components of the core machinery (transposase and its binding sites) were characterized and their functionality was studied by using an in vitro methodology developed for Mu. The function of Mu core machinery was studied for its ability to use various DNA substrates: Hin-Mu end specific DNA substrates and Mu end specific hairpin substrates. The hairpin processing reaction by MuA was characterized in detail. New information was gained of all three machineries. The components or their activity required for functional BARE-1 VLP machinery and retrotransposon life cycle were present in vivo and VLP-like structures could be detected. The HinMu core machinery components were identified and shown to be functional. The components of the Mu and Hin-Mu core machineries were partially interchangeable, reflecting both evolutionary conservation and flexibility within the core machineries. The Mu core machinery displayed surprising flexibility in substrate usage, as it was able to utilize Hin-Mu end specific DNA substrates and to process Mu end DNA hairpin substrates.
1
Introduction
B. INTRODUCTION 1. TRANSPOSABLE ELEMENTS - UBIQUITOUS RESIDENTS OF GENOMES Transposable elements (TEs) were initially dicovered in maize by Barbara McClintock in the 1940’s (McClintock 1956, 1987). Today, the number of identified elements as well as the knowledge and understanding of these “jumping genes” have reached a completely different level. Also, these elements are no longer thought as “junk” or “selfish” DNA. Instead, it is now generally accepted that the contribution of TEs to the generation of variability has an important role in genome evolution. TEs are discrete DNA segments that are able to move or copy themselves from one locus to another within or between their host genome(s) without a requirement for DNA homology. TEs move by a mechanism called transpositional recombination or simply transposition (for reviews see Mizuuchi 1992, 1997, Mizuuchi and Baker 2002). Certain viruses too, such as bacteriophage Mu and retrovirus HIV-1, utilize transpositional recombination during their life cycle. TEs are abundant residents in virtually all the genomes studied, but the number of families, the copy number, and the proportion of TEs in different genomes vary substantially (for reviews see Hua-Van et al. 2005, Kidwell and Lisch 2002, Kumar and Bennetzen 1999). For instance, the genomic portion of TEs is approximately 3% in Saccharomyces cerevisiae, 45% in humans, and apparently more than 70% in some plant genomes such as maize and barley. Although many, if not most, of the elements are no longer active and inhabit the genome as silent residents, the mobility of the active elements often causes 2
deleterious effects in the genome, such as various types of genome rearrangements, instability, and mutations. Transposition may be destructive to both the host and the element, unless tightly regulated. TEs not only play an important role in the evolution of their host genomes, but also co-evolve with their hosts, a feature that is essential for their long-term survival. This co-evolution has led to the generation of sophisticated regulation mechanisms beneficial for both the host and the element (for reviews see Kidwell and Lisch 2000, 2002, Labrador and Corces 1997). TEs may also benefit their hosts over evolutionary time by creating a source of genetic variation. For instance, in prokaryotes, TEs promote the spreading of drug resistance genes, virulence factors etc. by lateral DNA transfer (reviewed in Bushman 2002). In plants (Grandbastien 1998, Kumar and Bennetzen 1999, Wessler 1996) and in yeast (Lesage and Todeschini 2005), transposition is often triggered by cellular stress, when TEs can provide genome plasticity essential for survival and for adaptation to unusual situations. In some cases, TEs may have evolved into functional host genes. For instance, V(D)J recombination, a process that generates diversity in the vertebrate immune system by assembling immunoglobulin and T-cell receptor genes by a DNA rearrangement reaction, shows striking similarities with transposition (Zhou et al. 2004) and has been suggested to derive from an ancestral transposition system (Agrawal et al. 1998, Hiom et al. 1998). 2. CLASSIFICATION OF ELEMENTS During the past ten years, the data from
Introduction
the genome sequencing projects has enabled the identification of a multiplicity of new previously undetectable elements. Somewhat paradoxically, as the number, variety and the detailed knowledge of the elements have increased, the classification of the elements has become more “blurry” (Capy 2005). TEs can be divided in categories according to their host, the mechanism by which they move, the enzymes catalyzing the chemical reactions, or the structure of the element. However, one major distinguishing feature among the TEs is, whether their transposition includes an RNA intermediate stage (Class I, collectively called retroelements) or whether it relies exclusively on DNA intermediates (Class II, called DNA transposons). These two classes, the retroelements and DNA transposons, can further be divided into several subclasses (reviewed in Craig et al. 2002) of which some will be described here. The DNA elements are found in both prokaryotes and eukaryotes, whereas the RNA elements (retroviruses and retrotransposons) appear to be restricted to eukaryotic organisms. Transposition of DNA and retroelements is mediated by an element-encoded recombinase protein, a transposase or an integrase, respectively (Haren et al. 1999). Both classes of elements include autonomous elements that code for their own transposition and non-autonomous elements that lack this ability and usually depend on autonomous elements from the same or a different family to provide a transposase or integrase in trans. 2.1 RETROELEMENTS (CLASS I) Structurally retroelements are divided into those that carry long-terminal repeats (LTR) at their genome ends, including
retroviruses and LTR retrotransposons, and to those that do not i.e. non-LTR retrotransposons. Retroviruses are RNA viruses that share similar genome organization and carry closely related genes (for reviews see Coffin et al 1997, Craigie 2002). Retroviruses usually have three open reading frames (ORFs; Fig. 1) gag (encoding structural capsid proteins), pol (encoding enzymes: protease, PR; integrase, IN; reverse transcriptase, RT; and RNaseH), and env (envelope glycoprotein) polyproteins. When a retrovirus enters a cell as a retroviral particle, its RNA genome is reverse transcribed into a double stranded (ds) DNA copy that terminates with LTRs, which are subsequently recognized and bound by the viral integrase. As a result, preintegration complexes (PICs), containing retroviral DNA, IN, and other protein factors, are formed and transferred to the nucleus (e.g. in case of HIV-1), where the viral DNA is integrated into the host genome. Alternatively, PICs of some viruses (e.g. MLV, murine leukemia virus) enter the nucleus during the mitosis, when the nuclear envelope breaks down. The integrated viral DNA copy (provirus) is stably maintained and replicated along cellular DNA. The provirus DNA terminates invariantly with the retroviral terminal consensus 5’TG…CA3’ at both ends (for review see Hindmarsh and Leis 1999). LTR retrotransposons resemble a proviral form of retroviruses in their structure (Fig.1), coding capacity, and life cycle (reviewed in Boeke and Stoye 1997). They have virtually identical LTRs at their DNA ends which terminate with 5’TG…CA-3 and usually enclose a single gag-pol ORF or two ORFs, gag and pol. The LTR retrotransposons are subdivided into Ty1/copia and Ty3/gypsy groups 3
Introduction
on the basis of their gene order and their sequence similarity (Xiong and Eickbush 1990). The Ty3/gypsy elements have a gene order identical to retroviruses, whereas in the Ty1/copia elements the in gene is located between the pr and rt (see Fig. 1). Although some exeptions exist (see below), the LTR retrotransposons are generally distinguished from retroviruses by the lack of an env gene that is required for formation of extracellular infectious virus particles and for spreading from cell to cell (Xiong and Eickbush 1990). In general, the
life cycle of LTR-retrotransposons follows that of retroviruses, except that they are not infectious. Although nucleoprotein capsids, called virus-like-particles (VLPs), which contain the “viral” RNA or cDNA (Garfinkel et al. 1985, Shiba and Saigo 1983) are generated, they are left marooned inside the host cell and are not infectious. However, some Ty3/gypsy group elements, and recently also few Ty1/copia group elements have been shown to contain an env-like gene encoding a protein with an unknown function (for reviews see Levin
Figure 1. Major types of TEs and overall organization of Class I and Class II elements. Class I: Retroviruses (e.g. HIV-1), LTR retrotransposons (including Ty3/gypsy and Ty1/copia groups), and non-LTR retrotransposons (e.g. LINE and SINE). Most LTR-retrotransposons have two open reading frames, ORFs (depicted by white rectangles), the first encoding GAG and the second POL polyprotein. Retroviruses have a third ORF that encodes the structural envelope (ENV) protein required for cell-to-cell transmission. Some LTR retrotransposons also have a third ORF (dashed line rectangle) encoding an ENV-like protein (see text for details). The genes and their encoded products are: gag, structural virion core proteins; env, structural envelope protein; pr, protease; rt, reverse transcriptase; rh, RNaseH; in, integrase. Long terminal repeats (LTRs) at each end are depicted by black arrows. Class II: DNA transposons at simplest (e.g. IS elements) encode a transposase protein only and contain terminal inverted repeats (TIRs, gray boxes) at each end that function as transposase binding sites. The flanking host DNA is not shown for clarity. Drawn according to Bennezen 2000 and Schmidt 1999.
4
Introduction
2002, Peterson-Burch et al. 2000). In most cases these elements have not been shown to be infectious, except the gypsy element of D. melanogaster (Kim et al. 1994, Song et al. 1994). Non-LTR retrotransposons (also known as LINE-type retrotransposons, retroposons, or polyA elements) have a structure similar to mRNA (Fig. 1; reviewed in Craig et al. 2002). They lack LTRs, but are often terminated by an A-rich region at their 3’ end. They simply reverse transcribe a cDNA copy of their RNA transcript directly onto the chromosomal target site. They often contain two ORFs encoding GAG and POL. This class includes also several nonautonomous elements that lack coding functions for an IN or RT. The best-known members of the non-LTR family are the autonomous LINEs (long interspersed repeated elements) and nonautonomous SINEs (short interspersed repeated elements).
transposition e.g. genes encoding enzymes responsible for antibiotic resistance. Composite transposons are composed of two IS-elements with an internal sequence (e.g.Tn5, Tn10). Many eukaryotic DNA elements (e.g P-elements, hAT superfamily and Tc1/mariner family members) are more complex and contain introns. Nonautonomous MITEs (miniature invertedrepeat transposable elements) have only conserved TIRs but no coding potential. In general, DNA transposition can be replicative or non-replicative by nature, and one way to classify the elements is to divide them into “copy-and-paste” or “cut-and-paste” elements, respectively, according to the pathway utilized. However, strict borders cannot be drawn, as some elements can utilize both of these transposition modes (e.g. IS903; Tavakoli and Derbyshire 2001, Weinert et al. 1984). 3. UNITY IN TRANSPOSITION
2.2 DNA-ELEMENTS (CLASS II) DNA elements range from simple insertion sequences (ISs) to complex viral genomes such as bacteriophage Mu (for reviews see Craig et al. 2002, Saedler and Gierl 1996, Sherrat 1995). Characteristically, these elements have specific sequences at their DNA ends, called terminal inverted repeats (TIRs; Fig. 1). Autonomous DNA elements encode a transposase protein that specifically recognizes the TIRs (or transposase binding sites in case of bacteriophage Mu and Tn7) at element ends and catalyzes the chemical reactions of transposition. In the case of the simplest autonomous prokaryotic transposons, IS-elements, the transposase is the only element-encoded protein. However, many DNA transposons encode also additional sequences required for transposition or genes nonessential for
Development of defined in vitro systems for various elements has enabled detailed studies of the transposition mechanism and revealed the striking similarity of the chemical reactions of transposition. The biochemistry of reactions has been examined in great detail e.g. for bacterial transposition systems of Tn5, Tn7, Tn10, and Mu as well as for HIV-1 and V(D)J recombination systems (reviewed in Craig et al. 2002). 3.1 SIMILARITY OF CHEMICAL REACTIONS Despite of the diversity of the TEs, virtually all elements studied utilize similar chemistry for the DNA breakage and joining reactions underlying transposition (for review see Curcio and Derbyshire 2003, Graig 1995, Haren et al. 1999). 5
Introduction
In general, two common steps, a donor cleavage and a strand transfer, are involved in the reaction series (Fig. 2; for reviews see Mizuuchi 1992, Mizuuchi 1997, Mizuuchi and Baker 2002). In the first step, a pair of site-specific endonucleolytical cleavages expose the reactive 3’OHs at the element’s ends (or in some cases at flanking DNA ends), and in the second step, a pair of strand esterification reactions covalently join the newly exposed element’s 3’ end into the new target DNA. These two reactions are chemically very similar to each other: in the cleavage step a H2O molecule serves as the attacking nucleophile that hydrolyses the phosphodiester bond at the transposon end, whereas in the strand transfer the exposed 3’OH acts as the nucleophile that attacks into a phosphodiester bond at the target DNA, in a similar manner. Some elements use a reaction mechanism that includes two intermediate steps between
Figure 2. Transposition reactions. 1) Donor cleavage and 2) strand transfer. Shown is the replicative transposition reaction in which the flanking DNAs (light gray) remain attached to the 5’ end of the transposon. In non-replicative transposition the flanking DNAs are removed. Only short stretches of flanking DNA are shown for clarity.
6
the donor cleavage and strand transfer; a hairpin formation and resolution (opening), which chemically are virtually identical to donor cleavage and strand transfer (see next chapter). All these steps are catalyzed by the element-encoded transposase or integrase protein(s). 3.2 DIVERSITY OF MECHANISMS Depending on the mechanistic details of the transposition reaction series, the outcome of transposition can be either non-replicative or replicative by nature and lead to a formation of either a simple insertion or a cointegrate (for reviews see Curcio and Derbyshire 2003, Craig 1995, Haren et al. 1999). Mechanistically, the main distinguishing feature between the two pathways is whether a doublestrand cleavage (non-replicative; Fig. 3, A-D) releasing the complete element, or a single-strand nick (replicative; Fig. 3, E-G) exposing only the reactive 3’ end/s, occurs at the ends of the transposon before integration (Turlan and Chandler 2000). In addition, some elements have intermediate steps or structures between the cleavage and strand transfer reactions. The strand transfer reaction is virtually identical in all the systems studied. The subsequent steps following these transposition reactions, which involve several host cell repair and/ or replication factors, are not described here in detail. 3.2.1 Non-replicative transposition Non-replicative (cut-and-paste) transposons have evolved several strategies to release themselves from the flanking host DNA prior to strand transfer. In case of Tn7, a double strand break is made by using two distinct protein species, TnsB and TnsA cleaving the 3’ and 5’ ends, respectively (Fig. 3, A; Sarnovsky et al. 1996). Alternatively, a transposon can be
Introduction
excised from the host DNA by the action of a single protein species, via a DNA hairpin intermediate. In Tn10 and Tn5 transposition systems (Fig. 3, B) hairpins are formed by the transposase at transposon DNA end after the initial hydrolytic cleavage, when the exposed 3’OH attacks the phosphate backbone of the 5’ end of the non-transferred strand and joins the 3’OH to a scissile phosphate on the non-
transferred strand (Bhasin et al. 1999, Kennedy et al. 1998). The transposase then opens the newly formed hairpin by a hydrolytic cleavage and regenerates a 3’OH residue that is subsequently used for strand transfer. The V(D)J recombination mediated by RAG1 and RAG2 recombinases is mechanistically similar to the transposition of Hermes element (a member of hAT
Figure 3. Unity in transposition mechanisms. All transposable elements (black lines) share the two critical chemical reactions: a donor cleavage (indicated by small black vertical arrows) and a strand transfer to the target DNA (dark gray lines). In non-replicative transposition (A-D) the elements undergo a double-strand cleavage, either without (A, D) or by way (B, C) of a hairpin intermediate that liberates the element from flanking host DNA (dashed lines) and eventually results in simple insertion. In replicative transposition (E) only a single strand nick is introduced at the element 3’ ends (E-G) and the 5’ end(s) remain attached to the flanking DNA. Transposition of Mu and Tn3 generates a branched structure that is replicated to yield a co-integarte, or sometimes alternatively repaired to yield a simple insertion. In retroelement integration (F) the integrated DNA is first replicated by transcription and reverse transcription after which the 3’ ends are generally cleaved (end processing) and joined to the new target. IS911 (G) uses a variation of replicative transposition: a single 3’ end is nicked and a circular intermediate is generated by a mechanism similar to hairpin formation. This intermediate is resolved by replication to yield first an excised circular transposon, and then by the following second cleavage, a linear transposon. The strand transfer reaction is identical in all cases (A-F) covalently joining the element 3’ ends to the new target DNA, cleaved in a staggered manner. As a result, the elements are flanked by short gaps that reflect the staggered positions of target cleavage and joining. Finally, the host DNA repair functions repair these gaps and generate the end product, in most cases a simple insertion with short target site duplications (small white rectangles). This figure was inspired by Craig 1995, Haren et al. 1999, Curcio and Derbyshire 2003. For details and for references, see text.
7
Introduction
family; Zhou et al. 2004); and in vitro RAG1 and RAG2, indeed, perform DNA transposition reaction (Agrawal et al. 1998, Hiom et al. 1998). In these two systems, hairpins are generated at flanking DNA by a mechanism similar to Tn5 and Tn10 (Fig. 3, C; McBlane et al. 1995, Zhou et al. 2004). The major difference is that in these systems the 5’ ends of the element are cleaved first (instead of 3’ ends), and the reactive 3’OH nucleophiles are generated at the flanking DNA (paraller to signal sequences in V(D)J recombination), not at the transposon DNA (coding sequences in V(D)J recombination). Direct transesterification reaction by the 3’OH on the opposing strand results in a hairpin at the flanking DNA with concomitant release of the linear transposon. Polymorphism detected in the junctions (P-nucleotides) is generated by imprecise opening of the hairpins, and by subsequent repair. In V(D)J recombination, the hairpins are formed and opened by RAG1 and RAG2 recombinases (Besmer et al. 1998, Shockett and Schatz 1999). In Hermes system the hairpins are formed by its transposase but the opening reaction is yet uncharacterized. Most probably, on the basis of the characteristic footprints left behind after excision, other members of the hAT transposon family too utilize a hairpinning mechanism similar to Hermes. However, some non-replicative elements exist, such as Mos1 (a Tc1/mariner group element; Fig 3, D), that cleave their 5’ ends first, but for which the second strand cleavage occurs by a yet uncharacterized mechanism that does not involve hairpins (Dawson and Finnegan 2003). 3.2.2 Replicative transposition Replicative DNA transposition (copyand-paste, Fig. 3, E) is used e.g. by the Tn3 family of prokaryotic transposons 8
(reviewed in Grindley 2002), IS6 family of insertion elements (Chandler and Mahillon 2002) and transposing bacteriophages, such as phage Mu, during their lytic lifecycle (Chaconas and Harshey 2002). Their transposases cleave only at the 3’ ends of the transposon DNA and transfer these ends to the new target. As the 5’ ends of the transposon DNA are not processed at this stage, they remain attached to the flanking DNA, resulting in a branched DNA structure commonly known as the Shapiro intermediate (Shapiro 1979), which contains a copy of the transposon joined both to the target and to the flanking host DNA. Replication of the Shapiro intermediate by the host’s replication machinery completes the steps of replicative transposition and leads to a cointegrate structure that eventually results in a new copy of a transposon in the target DNA. Alternatively, the Shapiro intermediate can be nicked by nucleases and repaired to yield a simple insertion. In the case of Tn3, an element-encoded sitespecific recombination system (resolvase) further processes the cointegrate to generate a simple insertion into a target DNA and to regenerate the donor (see Grindley 2002). The integration of retroelements (for reviews see, Boeke and Stoye 1997, Brown 1997, Craigie 2002) is always replicative by nature, as these elements (Fig. 3, F) are separated from the flanking host DNA by the synthesis of a full-length mRNA transcript. Reverse transcription of the RNA intermediate yields a doublestranded cDNA copy, generally a few basepairs longer than the final integrated copy. The extra bases at the 3’ ends are removed during end-processing (reaction identical to donor cleavage) by integrase, prior to integration (strand transfer) into the host genome. The short 5’ end
Introduction
extensions of viral DNA are presumably then removed by host repair enzymes. IS911 (and possibly members of IS3, IS30, IS256 and IS21 families; for review see Rousseau et al. 2002) uses a variation of a replicative mechanism (Fig.3, G). Initially its transposase (OrfAB) makes a single-strand nick at one 3’ transposon end which is then transferred to the same strand of the opposite end. This circularizes a single transposon strand, leaving the complementary strand attached to the donor backbone. The host factors then resolve the second transposon strand by replication. As a result a circular transposon copy, in which the transposon ends lie alongside, is generated. Upon target capture transposase cleaves the transposon ends and finally joins the 3’ ends to the new target (for details see Duval-Valentin et al. 2004). 3.3 SIMILARITY OF CATALYZING ENZYMES: TRANSPOSASES AND INTEGRASES The establishment of defined in vitro transposition systems for elements such as Tn5, Tn7, Tn10, Mu, HIV-1 and Ty1 (reviewed in Craig et al. 2002) has not only allowed the characterization of the biochemical steps of transposition but also functional studies of transposases and integrases. Subsequent structural studies of these enzymes have revealed remarkable similarity in their structure, especially within the catalytic domain and in the active site organization (reviewed in Mizuuchi and Baker 2002). Transposases and integrases are multifunctional, multidomain proteins that share several structural and functional similarities (for reviews see Haren et al 1999, Polard and Chandler 1995). The most important functions of these enzymes are: to recognize the specific sequences at the element end, pair the ends to form a
synaptic complex, capture the target DNA and cleave it in a staggered manner, and to catalyze the critical chemical reactions. Structurally, the most important units of the transposases and integrases include a catalytic core and a DNA-binding domain responsible for catalysis and transposon DNA end recognition, respectively. Other domains may provide functions for protein-protein interactions with accessory proteins and specific proteinDNA interactions with accessory DNA sites or for unspecific interactions with target DNA. Transposases and integrases often function as multimers and their monomeric forms are catalytically inactive. In catalysis they use a one step transesterification mechanism and require divalent metal ions, but do not require any external energy source or utilize covalent protein-DNA intermediates (Mizuuchi 1992, Mizuuchi 1997). The X-ray crystal structures of the catalytic core domains of MuA and Tn5 transposases (Davies et al. 2000, Rice and Mizuuchi 1995) as well as HIV-1, avian sarcoma virus (ASV), Rous sarcoma virus (RSV), and simian immunodeficiency virus (SIV) integrases (Bujacz et al. 1995, 1996, Chen et al. 2000a, 2000b, Dyda et al.1994, Goldgur et al. 1998, Wang et al. 2001, Yang et al. 2000) have revealed a remarkable similarity within their catalytic core domains (see also reviews Grindley and Leschiziner 1995, Rice et al. 1996, Rice and Baker 2001). However, the domains outside the catalytic core do not share structural similarity. The Cterminal domain seems to be especially diverse, or in some cases it is absent, whereas the N-terminal domains, usually involved in DNA binding, show more structural similarity and often contain helix-turn-helix (HTH) motifs (Rice and Baker 2001). The structural studies of 9
Introduction
these enzymes also revealed that they are members of a larger superfamily of polynucleotidyl transferases that include e.g. RNaseH and a Holliday junction resolving enzyme RuvC (Davies et al. 2000, Dyda et al 1994, Rice and Mizuuchi 1995). Recently crystal structures of two eukaryotic transposases, Hermes and Mos1, have been solved (Hickman et al. 2005, Richardson et al. 2006). They share some structural similarities with prokaryotic transposases and integrases in their catalytic core (e.g. RNaseH like fold), but also show apparent differences from the prokaryotic transposases (especially the Hermes transposase). All these and many other transposases and integrases form a family of DDE transposases/integrases, because their active site contains three phylogenetically conserved acidic residues (Asp, Asp, Glu) called the DDE motif, which has been proposed to coordinate the divalent metal ions essential for catalysis (Doak et al 1994, Fayet et a. 1990, Kulkosky et al. 1992, for review see Haren et al 1999). In addition to the DDE motif, at least the IS4 family transposases (including e.g. Tn5 and Tn10) appear to have additional conserved residues, a motif called “YREK signature” (Rezsohazy et al. 1993), in their active site. The YREK motif seems to be especially important for the DNA hairpin mechanism used by these proteins (Allingham et al. 2001, Davies et al 2000, Reznikoff 2003). Also, the RAG1 recombinase involved in the initiation of V(D)J recombination appears to be a distantly related member of the DDE-transposase family (Fugmann et al. 2000, Kim et al. 1999, Landree et al. 1999). 3.4 TRANSPOSITION MACHINERIES Transposition and retroviral integration 10
proceed within higher order nucleoprotein complexes (often called transpososomes), which are the molecular machineries of transposition (Chaconas et al. 1996, Gueguen et al. 2005). While these complexes may also contain other proteins, the components of the minimal catalytic core (the core machinery) are the element ends and a few transposase/ integrase protomers only. In particular, in the case of retro-elements the transposition machineries are often elaborate. For instance, the nucleoprotein particles of retroviruses and the VLPs of LTR-elements contain all the components required for retroelement life cycle and can be considered as giant retroviral machinery (VLP machinery). Retroviral machinery can also be defined as a large nucleoprotein complex, PIC, which contains retroelement cDNA, integrase proteins and other yet unidentified protein components and that is capable of correct integration both in vivo and in vitro (Bowerman et al. 1989). The smallest entity, capable of catalysis in vitro, is the minimal core machinery which includes fragments of LTR ends and a multimer of integrase proteins only. In general, transposition is initiated when the element-encoded transposase or integrase recognizes and binds the specific DNA sequences at the element ends, and pairs them into a highly organized synaptic nucleoprotein complex via specific proteinprotein and protein-DNA interactions. Only after formation of this complex these enzymes become catalytically activated and catalyze the chemical reactions of transposition which take place within the context of this specific protein-DNA complex (Mizuuchi 1992, Mizuuchi and Baker 2002). During the assembly and catalytic steps, these complexes go through several conformational changes and structural transitions. In the Mu system
Introduction
the assembly of these complexes functions as a key regulatory step (Mizuuchi et al. 1992, Wang et al. 1996) and after the assembly the transposition proceeds through a complex series of ordered steps showing consecutive increase in the complex stability (Chaconas et al. 1996). The requirement for proper assembly of such a complex prior to catalytic activation assures that the appropriate DNA substrates (element ends and sometimes target DNA) are present and promote the coordination of the reaction steps (Gueguen et al. 2005). So far, little is known about the detailed structure and structure-function relationships of transposition machineries. The recently solved three-dimensional structure of the Tn5 synaptic complex has given information about how the transposase active site engages its DNA substrates and about the mechanism of hairpinning (Davies et al. 2000). Also, a recent reconstructed 3D image of Mu cleaved donor complex has shed light on structure-function relationships in the Mu transpososome (Yuan et al. 2005). Both structures have provided structural reasons for catalysis of cleavage and strand transfer in trans (i.e. a transposase bound at one end catalyses reactions at the other end, and vice versa) (Aldaz et al. 1996, Namgoong and Harshey 1998, Naumann and Reznicoff 2000, Savilahti and Mizuuchi 1996, Williams et al. 1999), which may be a general characteristic of the transpososomes. 3.4.1 Functional and structural differences of transposition machineries Although the DDE motif of the enzymes and the shared chemistry seem to be common themes in most transposition systems, the detailed ways in which
the elements assemble the individual nucleoprotein complexes for catalytic steps and the components of these machineries can vary and result in important functional differences. In addition to catalysis, other functions, such as target immunity, target site selection, and regulation of transposition, are also mediated by or through the transposition machineries. Most elements assemble their core machineries of a single transposase protein in its multimeric form. Dimeric (Tn10; Reznicoff 2002, Tn5; Haniford 2002), tetrameric (Mu; Chaconas and Harshey 2002, HIV-1; Li et al. 2006) and hexameric (Hermes; Hickman et al. 2005) machineries have been described. The transposition machinery of Tn7 is exceptional. Tn7 encodes five separate proteins, each with a specific function, which are assembled into a heteromeric complex TnsABCDE (Waddel and Craig 1988). Of these proteins only two are required for catalytic functions, TnsA and TnsB for cleavage of transposon 3’ and 5’ ends, respectively. TnsB is also responsible of recognition and binding of the element ends as well as of strand transfer (Sarnovsky et al. 1996). Both TnsA and TnsB contain a DDE motif, but only TnsB belongs to the DDE transposase/integrase family, whereas TnsA structurally resembles type II restriction endonucleases (Hickman et al. 2000). Interestingly but logically, inactivation of TnsA converts the normally non-replicative Tn7 transposition machinery into a Mulike replicative system (May and Craig 1996). Also, transposition reaction under artificial conditions generates circularized forms of Tn7 DNA (Biery et al. 2000), similar to those detected for IS911 (Polard et al. 1992, Polard and Chandler 1995). The main mechanistic differences between the systems arise from the nature of the initial cleavage (see Fig. 3.): a single 11
Introduction
strand nick versus double strand cut, either via or without a (transposon end or flanking end) hairpin. Additional differences are, whether the initial cleavage (at 3’ or 5’ end of the transposon) occurs in particular order as in Tn10 transposition (the 3’ strand cleavage precedes 5’ strand cleavage; Bolland and Kleckner 1996) or in both ends simultaneously. Also, the target can be brought in before (Tn7; Bainton et al. 1993) or after (Tn10; Sakai and Kleckner 1997) the initial cleavage. Transposition machineries also mediate target site selection (reviewed in Craig 1997). Either the transposase itself interacts with the target DNA (as in case of Tn10) or this function is mediated through an accessory protein (e.g. MuB in the Mu system see B.5.1.1). In Tn7 system TnsD+TnsE proteins function in target site selection, the former directing transposition into a specific site in the E.coli chromosome called attTn7 and the latter into various sites. In general target site selection can be relatively random (e.g. Mu, HIV-1), but usually a preferred consensus can be found (as for Mu; Haapa-Paananen et al. 2002, Mizuuchi and Mizuuchi 1993). Some elements have strict target sequence requirements (e.g. Tc1, Ty3; van Luenen and Plasterk 1994, reviwed by Sandmeyer et al. 2002) whereas for others an aberrant DNA conformation such as bent DNA (HIV-1; Milot et al. 1994), DNA mismatches (Mu; Yanagihara and Mizuuchi 2002, RAG1/RAG2; Tsai et al. 2003) or triple helix DNA (Tn7; Rao et al. 2000) can function as a hot spot for targeting in vitro. In some systems, target immunity functions (i.e. the ability to avoid integration into itself) are dissected into separate accessory proteins (Mu, MuB; Tn7, TnsC, for reviews see Chaconas and Harshey 2002, Craig 2002), which mediate 12
these functions by interacting with the transposase protein, whereas in others, larger transposase proteins deal with multiple functions, including immunity (e.g. Tn3; Grindley 2002). Regulation of the machinery has been most thoroughly studied in the case of Mu and some aspects of it will be discussed in B.5.1.1. In general, regulation can occur at several levels and time points along the assembly pathway or during catalysis and it may be mediated by element encoded proteins or by host factors alternatively. 4. PLANT RETROELEMENTS In plants, retrotransposons represent the most abundant and widespread class of TEs and consists of both the LTR retrotransposons and non-LTR retrotransposons, the former including Ty1/copia and Ty3/gypsy groups (Xiong and Eickbush 1990, see also Fig. 1) and the latter autonomous LINEs and nonautonomous SINEs (for review see Schmidt 1999). Retroviruses have not been identified in plants yet. Traditionally the LTR-retrotransposons have been distinguished from retroviruses by the lack of the env gene. However, recently several gypsy (Vicient et al. 2001b, Wright and Voytas 2002) and copia-like (Kapitonov and Jurka 1999, Laten et al. 1998, PetersonBurch 2000) plant retroelements have been identified that contain an env-like gene encoding a putative ENV protein. The function of this protein is yet unknown, as these elements have not been shown to be infectious. Although retrotransposons constitute a major portion of plant genomes (Flavell et al. 1992, 1994, Voytas et al. 1992), they are much less studied than their relatives in Drosophila, yeast, or mammals. Because of the replicative nature of retrotransposition,
Introduction
these elements may rapidly increase their copy number and can thereby increase plant genome size significantly. However, today most plant retrotransposons appear to be inactive or defective copies. Structurally and functionally plant retrotransposons are highly similar to the retrotransposons and retroviruses of other eukaryotic organisms. However, there are important differences in the genomic organization of retrotransposons in plants compared to some other eukaryotes including their often high copy numbers, extensively heterogeneous populations, and chromosomal dispersion patterns (for review see Bennetzen 1996, Kumar and Bennetzen 1999). 4.1. GENERAL STRUCTURE OF PLANT LTRRETROTRANSPOSONS Plant retrotransposons closely resemble retroviruses in their structure and function (reviewed in Boeke and Stoye 1997, Eickbush and Malik 2002, Kumar and Bennetzen 1999). In general, plant LTR retrotransposons carry two LTRs that can vary from 100 bp to over 5 kb in length and are in direct orientation relative to each other. In an active element the LTRs are identical in sequence and usually terminate with retroviral consensus termini 5’TG… CA3’. The LTRs are required for the initiation and termination of transcription, for priming the reverse transcription, and for binding of IN (when in cDNA form) during the integration. The LTRs can be divided into three functional domains, consecutively U3 (3’ unique to the 3’ end of the mRNA), R (repeated terminus of the transcript), and U5 (5’ unique to the 5’ end of the mRNA). The 5’ LTR functions as a transcription promoter and contains a minus strand priming binding site PBS (see Fig. 4) immediately internal
to the 5’ LTR. The 3’ LTR functions as a transcription terminator and contains a PPT, which resembles the retroviral plus strand priming site, adjacent to the 3’ LTR. Transcription initiates at the 5’ end of the R in the 5’ LTR and terminates in the 3’ end of the R in the 3’ LTR. The LTRs usually flank a 5-7 kb internal protein-encoding domain, which in most cases contains a single gagpol ORF or in some cases two separate ORFs, gag and pol (e.g. Ty1 element of Saccharomyces cerevisiae) (Boeke et al 1985). The gag encodes (GAG) proteins that make up the major structural component of a cytoplasmic VLP in which reverse transcription occurs. The pol encodes enzymes in the following order (in Ty1/copia elements): PR, IN and RTRNaseH, that are involved in generating a dsDNA copy of the retrotransposon mRNA and inserting it into the host genome. 4.2 RETROTRANSPOSON LIFE CYCLE Very little is known about the life cycle of plant retroelements, mostly due to a low number of active elements characterized. However, yeast Ty-elements of are typically active and much information about retrotransposon life cycle has been therefore gained from studies of e.g. Ty1 and Ty3 elements. In general, the LTR retrotransposons share several functional similarities with retroviruses in gene expression and in their life cycle (for review see Boeke and Stoye 1997). The encoded enzymatic machineries of nonplant LTR retroelements and retroviruses are highly similar (e.g. Ty1 and HIV-1). The integration of HIV-1 and Ty1 has been studied in detail with various in vitro assays (Li et al. 2006, Moore and Garfinkel 2000, reviewed in Craigie 2002, Voytas and Boeke 2002). 13
Introduction
4.2.1 From transcription to integration In general, the retrotransposon life cycle (Fig. 4) begins when RNA polymerase II initiates transcription from the retrotransposon 5’ LTR and terminates it at 3’ LTR (for review see Boeke and Stoye 1997, Voytas and Boeke 2002). In case of many LTR retrotransposons, including those studied in plants (Hirochika 1993, Hirochika et al. 1996a, Pouteau et al. 1991), the initiation of transcription is believed to be a key regulating step limiting retrotransposition (Boeke and Stoye 1997). Following transcription, the resulting mRNA is transported from the nucleus to cytoplasm and translated into proteins that carry out replication and integration. The gene products of retroviruses and retrotransposons are expressed as polyproteins, which then undergo endoproteolytic cleavage (maturation) into functional units by the self-encoded PR (Wellink and van Kammen 1988). The stoiciometry of protein expression is critical: expression of excess structural GAG relative to catalytic POL is required for VLP formation. This is generally achieved by translational frameshifting (reviewed in Jacks 1990) or by transcriptional splicing of the POL sequences (Brierley and Flavell 1990, Yoshioka et al. 1990). Most retroelements use -1 ribosomal frameshifting, but e.g. Ty1 uses a +1 frameshifting mechanism to synthesize GAG-POL fusion protein (Voytas and Boeke 2002). The VLPs are the functional units of retrotransposition that carry out the reverse transcription and integration, and thus are obligate intermediates of the retrotransposon life cycle (Eichinger and Boeke 1988, Garfinkel et al. 1985). As with retroviruses, the VLP assembly phase is the least well understood and 14
has been mainly studied with Ty1 and Ty3 elements (for reviews see Roth 2000, Sandmeyer et al. 2002, Voytas and Boeke 2002). In general, PR specifically cleaves the GAG and GAG-POL polyproteins into mature GAG, PR, RT, and IN proteins, which nucleate around the retrotransposon mRNA to form the VLP. Also packaged within the particle is a cellular tRNA that primes first-strand DNA synthesis during reverse transcription. In both retroviral particles and Ty1 VLPs, the mRNA is dimeric, consisting of two identical plusstrand RNAs joined by noncovalent bonds (Feng et al. 2000). Ty1 VLPs have been visualized by electron microscopy where they appear as oval, electron-dense structures showing polydispersity and ranging from 15-60 nm in diameter (Burns et al 1992, Garfinkel et al 1985, Mellor et al. 1985, for review see Roth 2000). In cells that express GAG proteins with Cterminal truncation the VLPs formed are smaller and less polydispersed (Al-Khayat et al. 1999, Burns et al. 1992). In general, the Ty1 VLPs appear to be icosahedral with a porous and spiky shell. The VLPs subsequently undergo a maturation process that consists of architectural reorganization and reverse transcription. Retroviral and retroelement RT has two distinct activities: 1) a DNA polymerase (RT) that uses either RNA or DNA as a template and 2) a nuclease (RNaseH) that specifically degrades RNA strand of RNA/DNA duplexes. Reverse transcription takes place within VLPs and converts the retroelement RNA into ds cDNA. DNA synthesis (minus-strand) initiates near the 5’ end of the element at PBS, using host-encoded tRNA as a primer. Minus strand synthesis extends to the 5’ end of the mRNA and generates a short minus strand strong stop DNA (ssDNA). The RNaseH degrades the RNA
Introduction
of the RNA/DNA hybrid. Because the 3’ terminus of the ssDNA is complementary to the R region located at the 5’ end of the mRNA, the ssDNA is subsequently transferred (1st jump) to the 3’ end of another mRNA molecule, where the minus-strand synthesis proceeds to the 5’ end of the mRNA. Again, RNaseH removes the RNA from the RNA/DNA duplex, leaving only a short PPT, an oligoribonucleotide, which primes the plus-strand DNA synthesis towards the template end. After this the plus strand ssDNA is transferred (2nd jump) to the 5’ end of the cDNA. The completetion of the plus-strand synthesis results in a linear cDNA. In most retroviruses the PBS and PPT are separated from the 5’ and 3’ LTRs
by 2 bp. The end of the minus strand ssDNA primed from the tRNA begins with the two nucleotides located between the PBS and the 5’ LTR. After reverse transcription, these two nucleotides are found at the end of the element. Similarly, priming of the plus strand at the PPT results in addition of two nucleotides at its 5’ end that are copied upon minus-strand completion. Thus the extrachromosomal linear cDNA, unlike the integrated sequence, usually possesses two extra nucleotides at each end (for review and illustration see Feuerbach et al. 1997, Sandmeyer et al. 2002, Voytas and Boeke 2002). As VLPs are formed predominantly in the cytoplasm, after reverse transcription the linear cDNA and the IN bound
Figure 4. Retrotransposon life cycle. The integrated retroelement is transcribed into mRNA (black line) and exported to the cytoplasm (1), where it is translated into GAG and POL polyproteins (2) that are processed into functional units by an element-encoded protease (PR). These units and cellular tRNA, which acts as a primer for reverse transcription, are assembled into VLPs (3) together with the transcript (mRNA depicted by black curved line) that is then converted to cDNA (depicted by grey curved line) by reverse transcriptase (RT) within the VLPs (4). This cDNA is finally transferred into the nucleus in the context of a preintegration complex that also contains integrase (IN) that finally integrates the cDNA as a new copy into the host genome (5). Figure is modified from Grandbastien 1998.
15
Introduction
to its ends at LTRs are thought to be transported (most probably in the context of a nucleoprotein complex, similar to retroviral PICs) to the nucleus, where the IN catalyzed integration to the host genome takes place. In the case of retroviruses, prior to integration the two terminal bases from each 3’ end of these blunt-ended molecules are removed by end processing and a linear recessed 3’ end intermediate is generated. End processing has been shown to take place also during the integration of yeast Ty3 and tobacco Tnt1 elements (Feuerbach et al. 1997, Sandmeyer et al. 2002), but it is not a feature of Ty1 or Ty5 integration (Moore et al. 1995, see also Voytas and Boeke 2002). The catalytic steps of integration are identical to the cleavage and strand transfer reactions of retroviruses (for review see Brown 1997, Craigie 2001, 2002). 4.3 IDENTIFICATION AND ACTIVITY STUDIES OF PLANT RETROTRANSPOSONS In general, Ty1/copia group elements have been searched e.g. with PCR by using primers that are designed according to the most conserved regions in the rt or in gene sequence (Flavell et al. 1992, Hirochika et al. 1992, Voytas et al. 1992). Ty3/ gypsy elements have been screened with PCR by using primers designed for the rt-in junction, in order to distinguish the gypsy elements from the copia according to the difference in the order of the rt and in genes (Suoniemi et al. 1998a). Some plant LTR retrotransposons have been discovered because of their ability to transpose or inactivate gene function (reviewed in Grandbastien 1998). During recent years, analyses of the data from the genome sequencing projects have revealed new uncharacterized elements whose transpositional activities can be 16
evaluated according to their sequence conservation, similarity of their LTRs, existence of target site duplications, and by their insertional polymorphism (Grandbastien 1998). Despite the large amount of descriptive data about a wide variety of plant retroelements, little is known about the natural behavior of these elements, e.g. transpositional activity, life cycle, or regulation of the activity. Most retrotransposon sequences in plants appear to be defective or inactive under normal growth conditions (for review see Grandbastien 1998, Wessler et al. 1995, Wessler 1996). Only a few active retrotransposons have been characterized in plants (for reviews see Grandbastien, 1998, Kumar and Bennetzen, 1999). Direct evidence for transposition has been obtained for Tnt1 (Grandbastien 1989), Tto1 (Hirochika 1993), and Tos17 elements only (Hirochika et al. 1996). Some elements are activated under stress conditions, such as tissue culture (Hirochika 1993, Hirochika et al 1996), infection by bacteria (Pouteau et al 1994) or viruses (Hirochika et al. 1995). During the past ten years, transcriptional or translational activity or reverse transcription have been demonstrated for an increasing number of elements. So far, no in vitro integration assays have been established for plant retrotransposons mostly because their normal state of transpositional activity appears to be virtually undetectable. 4.4 BARE-1, A BARLEY RETROTRANSPOSON FAMILY BARE-1 was the first complete retrotransposon described for barley (Hordeum vulgare) (Manninen and Schulman 1993, for review see Vicient et al. 1999a). In general, barley retrotransposon population is comprised of a highly heterogeneous set of retrotransposons,
Introduction
mostly Ty1/copia elements, including a collection of sequences which are closely related to BARE-1 (Gribbon et al. 1999, Kalendar et al. 2004, Schulman and Kalendar 2005, Shcherban’ and Vershinin 1997, Vicient et al. 2005). In fact, a large fraction of the barley Ty1/ copia elements are BARE-1 elements. The BARE-1 retrotransposon family is present in ~1-2x104 full-length copies dispersed throughout the genome, and therefore represents about 2.9% of the barley genome (Vicient et al. 1999b). In addition, an even larger population of BARE-1 solo LTRs is present (Suoniemi et al. 1996b, Vicient et al. 1999b). Intramolecular homologous recombination between BARE-1 LTRs has been suggested to explain the large excess (7-42 fold) of solo LTRs, and to function as a regulatory mechanism that reduces the number of functional retrotransposons in the host genome (Vicient et al. 1999b). 4.4.1 Structure of BARE-1 The first full-length BARE-1 element (Manninen and Schulman 1993), named BARE-1a is 12088 bp long, but containes a 3135-bp insertion in its 3’
LTR. Therefore, the canonical BARE-1 element (Fig. 5) is predicted to be around 8.9 kb in length, including relatively long (approximately 1.8-1.9 kb) and highly conserved LTRs (Manninen and Schulman 1993, Suoniemi et al 1996a, Vicient et al 1999b). Structurally the BARE-1 element contains all the components of a functional retrotransposon (Manninen and Schulman 1993). The BARE-1 internal region encodes a predicted polyprotein bearing the key residues, structural motifs and conserved regions associated within retroviral and retrotransposon polypeptides (Suoniemi et al. 1997). The predicted polyprotein (1301 residues) contains well-conserved GAG-PR-IN-RT-RH segments. Especially the BARE-1 IN is highly conserved and its modeled tertiary structure shows structural similarities with HIV-1 and ASV INs (Suoniemi 1998b). The BARE-1 LTRs contain 6 bp imperfect inverted repeats at their ends with the canonical 5’TG…CA3’ terminal sequences. The genomic direct repeat flanking BARE-1insertion site (target site duplication) is 5 bp (Suoniemi 1997). Two TATA boxes have been identified
Figure 5. Organization of a full-length 8.9-kb BARE-1 element with 1.8-kb LTRs (long terminal repeats). The organization of BARE-1 is 5´-LTR-UTL-gag-pr-in-rt-rh-UTR-LTR-3’, where UTL is the 5´untranslated leader, gag encodes the structural GAG protein, in integrase, rt-rh both the reverse transcriptase and RNaseH, and UTR is the 3´untranslated region. The PBS indicates a minus strand priming site and PPT a plus strand priming site. The LTRs are divided in three contiguous regions organized U3-R-U5 (3’ unique-repeated- 5’ unique). The terminal 5’TG and CA3’ of the LTRs are shown.
17
Introduction
inside the BARE-1 LTRs and shown to be functional (Suoniemi et al. 1996a). The PBS of BARE-1 is complementary to tRNA iMet and the PPT of BARE-1 is highly conserved (Suoniemi et al. 1997). The 5’ untranslated leader sequence (UTL) in BARE-1 is unusually long (2 kb), but still conserved among the BARE-1 population. It has been suggested that the BARE-1 UTL might function in the regulation of retrotransposition activity (Suoniemi et al. 1996a, Vicient et al. 1999a). 4.4.2 Distribution and activity of the BARE-1 family The BARE-1 retroelement family is abundant throughout the Hordeum (Vicient 1999a,b) and also widely distributed within the Triticeae tribe (Gribbon et al. 1999, Kalendar et al. 1999, Vicient et al. 2001a). BARE-1 is also closely related to RIRE1 elements found in the phylogenetically distant rice (Noma et al. 1997). BARE-1 has been shown to be transcriptionally active in various tissues and tissue cultures of barley (Suoniemi et al 1996a) and in other Triticae species (Pearce et al. 1997). The BARE-1 insertions within barley and in other Triticae species are highly polymorphic, indicating transposition in the recent evolutionary past (Gribbon et al. 1999, Kalendar et al. 1999, Waugh et al. 1997). Its recent activity is also supported by the observation that all BARE-1 insertions examined appear to be flanked with a perfectly conserved 5bp target site duplication (Shirasu et al. 2000). The BARE-1 family has also been suggested to be stress induced in the wild, by e.g. drought (Kalendar et al. 2000).
18
5. DNA TRANSPOSONS: TRANSPOSABLE BACTERIOPHAGES 5.1 PHAGE MU: A VIRUS AND A TRANSPOSON Bacteriophage Mu was first described in the 1960’s as a temperate phage of Escherichia coli and other Gram negative bacteria with an extraordinary capacity to induce mutations (Taylor 1963), thus its name Mu (i.e. mutator). The life cycle of Mu can proceed through two distinct pathways: lysogenic that leads to a stable lysogen of a prophage or lytic that proceeds by generation of progeny phage particles (reviewed in Symonds et al. 1987). Mu is an exceptional virus as it uses DNA transposition efficiently during the distinct stages of its life cycle (Fig. 6). It is also exceptional as a transposon, as the outcome of its transposition can be either non-replicative, as during initial integration into the host genome (Akroyd and Symonds 1983, Chaconas et al. 1983, Harshey 1984, Liebart et al. 1982) or replicative, as during the lytic propagation and phage genome amplification (Chaconas et al. 1981). The 36,717-bp genome of the bacteriophage Mu (Morgan et al. 2001) is one of the largest, most efficient, and most complex transposons known (for reviews see Chaconas and Harshey 2002, Mit’kina 2003, Mizuuchi 1992). Despite its complexity, phage Mu has served as a model system for transposition studies primarily due to its high efficiency of in vivo transposition (Symonds et al. 1987) and an early development and establishment of a defined and efficient in vitro transposition assay (Craigie et al. 1985, Mizuuchi 1983). A number of subsequent studies with purified components have presented a detailed
Introduction
Figure 6. Life cycle of phage Mu and related Mu-like bacterial viruses. Upon infection, phage adsorbs to its host and injects its linear dsDNA genome into the cell. The phage encoded Nprotein, injected along the phage DNA, circularizes the phage genome with heterogeneous host DNA present at its ends (Harshey and Bukhari 1983, Gloor and Chaconas 1986). Note that the N-protein is not shown here, and the phage is drawn in linear form for simplicity. The phage genome is then integrated at random sites of the host chromosome by non-replicative transposition (Akroyd and Symonds 1983, Chaconas et al. 1983, Harshey 1984, Liebart et al. 1982), catalyzed by virus-encoded transposase. The infection and the following integration can lead (with low frequency) to lysogeny or (with high frequency) to lytic cycle (Howe and Bade 1975). In lysogeny, the virus genome remains integrated in the chromosome as a prophage and its lytic functions are repressed during successive cell divisions. In the lytic cycle, the transposase (along with other factors) catalyzes several rounds of replicative transposition in which multiple new copies of the originally integrated phage genome become inserted into new locations along the host chromosome (Chaconas et al. 1981). The phage is also able to enter the lytic cycle through induction of a prophage (Howe and Bade 1975). Following phage DNA replication via transposition, and phagespecific protein expression, the host chromosome is cleaved and phage DNA packaged into virus particles. The packaged single molecule of dsDNA in each particle is linear and contains the ~37 kb phage genome flanked by short variable regions of host DNA (approximately 50-150 bp at left and 0.5-3 kb at right genome end in case of Mu) that results from a “headfull” DNA packaging system (Bukhari et al. 1976). Some 100 phage particles per cell are liberated during lysis.
19
Introduction
description of an in vitro transposition pathway that recapitulates the in vivo phage replicative pathway (reviewed in Chaconas and Harshey 2002). 5.1.1 Replicative transposition of Mu and function of Mu transposition machinery Phage Mu encodes one of the most complex, but thoroughly characterized transposition machineries that carries out the transposition (for reviews see Baker 1995, Chaconas and Harshey 2002, Chaconas et al. 1996, Mizuuchi 1992, Yuan et al. 2005). The assembly of this machinery is an elaborate process, which requires a number of phage specific DNA factors, several phage- and hostencoded proteins, and a complex circuit of cooperative protein-protein and proteinDNA interactions on a supercoiled DNA substrate. Also, bending of the DNA and intertwining of the domains from separate monomers of the transposase are required for the construction of functional active sites. In general, Mu transposition proceeds within a specific protein-DNA complex, called a Mu transpososome (Craigie and Mizuuchi 1987, Surette et al. 1987), which forms via a multistep assembly pathway and synapses the transposon ends (for reviews see Chaconas et al 1996). In its core, this functional unit contains a tetramer of MuA transposase that catalyzes the transposition steps leading to the Shapiro intermediate and subsequently to a formation of a cointegrate. Subsequent to MuA-catalyzed steps, the completion of replicative Mu transposition involves an unknown number of host encoded proteins that include protein remodeling and DNA replication factors (for review see Nakai et al. 2001) and as a result, a 5-bp target
20
site duplication is generated (Allet 1979, Kahmann and Kamp 1979). 5.1.1.1 DNA COMPONENTS OF THE MACHINERY The Mu genome ends contain three MuA binding sites each, designated L1, L2, L3 and R1, R2, R3 in the order of their distance from the left (L) and right (R) end, respectively (Craigie et al. 1984). The arrangement and orientation of these binding sites in the R- and L-end are different (see Fig 7). The sites share a 22bp consensus sequence, with no obvious internal symmetry and their binding affinity to MuA as well as their relative importance in transposition varies (Allison and Chaconas 1992, Craigie et al 1984, Groenen and van de Putte 1986, Lavoie et al 1991). In addition to transposase binding sites, another important DNA factor is the transposition enhancer (E), also called an internal activating sequence (IAS), located ~1 kb from the left end of the Mu genome (see Fig. 7; Leung et al. 1989, Mizuuchi and Mizuuchi 1989, Surette et al. 1989). The enhancer contains three operator sites (O1-O3) and also an E.coli integration host factor (IHF) binding site between the O1 and O2. The enhancer increases the transposition efficiency both in vivo and in vitro (Leung et al. 1989, Surette et al. 1989) and plays an essential role in the transpososome assembly (Mizuuchi et al. 1992, Surette and Chaconas 1992). Negative supercoiling also plays an important role in Mu transposition (Craigie and Mizuuchi 1986, Craigie et al. 1985) and in vivo, supercoiling is thought to be modulated via a strong gyrase site (SGS) located at the center of the Mu genome (Pato et al. 1990, Pato 1994, Pato et al. 1995, Pato and Banerjee 1996, 1999). In addition to these specific sites, the
Introduction
terminal endmost nucleotides of the Mu genome 5’CA3’ (especially the terminal A) are essential in transposition, both in vivo and in vitro, and mutations in these nucleotides have been shown to affect the formation and stability of the transposition complexes, donor cleavage and strand transfer in vitro (Burlingame et al.1986, Coros and Chaconas 2001, GoldhaberGordon et al. 2003, Lee and Harshey 2001, 2003, Surette et al. 1991, Yanagihara and Mizuuchi 2003). 5.1.1.2 PROTEIN COMPONENTS OF THE MACHINERY The most important protein for transposition is the phage-encoded 75-kDa (663-aa) MuA transposase, organized in three major domains to which various
functions have been mapped (see Fig. 8; Nakayama et al. 1987). The N-terminal domain (I) mediates the specific binding to both the enhancer, via subdomain Iα (Clubb at al 1994, 1996, Leung et al 1989, Mizuuchi and Mizuuchi 1989) and to MuA binding sites at Mu ends, via subdomains Iβγ (Clubb et al. 1997, Kim and Harshey 1995, Leung et al 1989, Nakayama et al. 1987, Namgoong et al. 1998b, Zou et al. 1991). Subdomains Iγ and Iβ bind to separate half sites (basepairs 1-11 and basepairs 12-22, respectively) of the 22 bp binding site. The subdomain IIα of the central domain (II) is involved in catalysis, and contains the phylogenetically conserved acidic amino acids (Asp269-Asp336-Glu392) of the DDE motif (Baker and Luo 1994, Kim et al 1995, Krementsova et al. 1998, Rice
Figure 7. The organization of the DNA regions of the Mu genome essential in transposition. Mu genes (1-55) are numbered according to Morgan et al. (2001). Shown are the genes A and B encoding transposase proteins MuA and MuB, respectively. The substructures of the Mu genome left (L) end, enhancer (E) region, and right (R) end are enlarged. MuA binding sites (L1-L3, R1-R3, white arrows) and their location at each end are shown with numbering in the 5’-3’ orientation. In the upper enlargement are shown the operator region and the binding site for E.coli integration host factor (IHF; white rectangle). The operator region consists of three distinct operator segments (O1-O3, light gray rectangles) that serve as binding sites for c repressor. Internal activation sequence (IAS) acts as a transpositional enhancer and interacts with MuA transposase. Binding sites are drawn according to Craigie et al. 1984, operator region according to Allison and Chaconas 1992 and Baker 1995. The figure is not drawn to scale and the host DNA flanking the Mu genome is not shown for clarity.
21
Introduction
and Mizuuchi 1995). Subdomains IIβ and IIIα appear to participate in nonspecific DNA binding, and to be associated with transpososome assembly (Baker et al. 1993, Krementsova et al. 1998, Mariconda et al. 2000, Naigamwalla et al. 1998, Nakayama et al 1987, Rice and Mizuuchi 1995, Wu and Chaconas 1995). Subdomain IIIα also possesses nuclease activity, possibly interacts with the Mu-host junction and the DDE motif, and may be involved in the structural catalytic transitions that occur in the transpososome between the cleavage and strand transfer steps (Kuo et al. 1991, Naigamwalla et al 1998, Namgoong et al. 1998a, Wu and Chaconas 1995, Yang et al. 1995). Subdomain IIIβ interacts with MuB (Baker et al 1991, Leung and Harshey. 1991, Wu and Chaconas 1994) and ClpX proteins (Levchenko et al 1995). Efficient transposition requires another phage-encoded protein, MuB (312aa), which is an ATP-dependent sequence independent DNA binding protein involved in transpososome activation (Baker et al 1991, Surette and Chaconas 1991, Surette et al. 1991), in selection and delivery of proper targets (Maxwell et al.
1987, Naiganwalla and Chaconas 1997, Yamauchi and Baker 1998), and in target immunity (Adzuma and Mizuuchi 1988, 1989). MuB also appears to suppress a variety of defects that compromise transpososome assembly or catalysis, by a yet unknown mechanism (Coros and Chaconas 2001, Lee and Harshey 2001, 2003, Mizuuchi et al. 1995, Namgoong et al. 1998, Surette and Chaconas 1991, Surette et al. 1991, Wu and Chaconas 1992). In addition, two host-encoded DNAbending and -binding proteins, HU and IHF function during the early steps of transposition and play important roles in transpososome assembly (Craigie and Mizuuchi 1987, Craigie et al. 1985, Surette et al. 1987, Watson and Chaconas 1996). HU binds as a dimer, in a sequence independent manner, between the L1 and L2 binding sites and is incorporated into Mu transpososomes (Lavoie and Chaconas 1990). HU is the only host protein required for Mu transposition in vitro under standard conditions, but at lower levels of Mu DNA supercoiling IHF is required (Craigie et al. 1985). IHF stimulates strand
Figure 8. Domain organization of MuA transposase. Shown are the domains I, II, and III that were originally identified by partial proteolysis (Nakayama et al. 1987). Subdomain division (Greek letters, N-terminal residues indicated by numbers) is based on various structural and functional studies (see text for details and references). The DDE motif (Asp269-Asp336-Glu392) in the catalytic domain II is shown. The figure is drawn according to Krementsova et al. 1998 and Schumacher et al. 1997).
22
Introduction
transfer step in vitro by binding to the IAS and by inducing a sharp bend required for enhancer function (Higgins et al. 1989, Surette and Chaconas 1992, Surette et al. 1989). 5.1.1.3 ASSEMBLY AND FUNCTION OF THE MACHINERY At an early stage of Mu transposition MuA binds as a catalytically inert monomer to the binding sites at the Mu genome ends (Craigie et al. 1984, Kuo et al. 1991), thereby initiating an elaborate Mu transpososome assembly pathway involving all six MuA binding sites and the enhancer. As a result a complex circuit of interactions between MuA subunits, between the L end, the enhancer, and R end activates the formation of the first complex detected in vitro, the transient three-site synaptic complex called LER (Watson and Chaconas 1996). The assembly of the LER requires DNA supercoiling and precisely positioned DNA bends induced by the host proteins HU and IHF (Wang and Harshey 1994). These multiple contacts and strict requirements ensure the proper architecture of the complex before catalytic steps. After the formation of this unstable, short-lived LER, transposition of Mu proceeds through a series of increasingly stable proteinDNA complexes (transpososomes), in which MuA protomers have tetramerized and become catalytically activated (Baker and Mizuuchi 1992, Craigie and Mizuuchi 1987, Mizuuchi et al. 1992, Lavoie et a. 1991, Surette et al. 1987). The LER is rapidly converted into stable synaptic complex (SSC, also known as type 0 complex), in which the Mu ends are stably synapsed and coordinated within the active site in the transpososome, and in which the enhancer is no longer needed (Mizuuchi et al. 1992). In the SSC, the Muhost junction is protected, and MuA binds
tightly to only three of the binding sites: L1, R1, and R2 (Kuo et 1991, Lavoie et al. 1991, Mizuuchi et al. 1991, 1992). The formation of the SSC is the rate limiting step of the following cleavage reaction and acts as a critical control point that ensures the proper coordination of the ends before the inert MuA monomers become activated and the catalysis of chemical reactions takes place (Mizuuchi et al. 1992, Wang et al. 1996). If the terminal basepairs of Mu are mutated, the LER is not converted into the SSC and LERs accumulate (Naigamwalla et al. 1998). Upon addition of Mg2+ or Mn2+, the donor cleavage rapidly occurs and as a result, the SSC is converted into more stable cleaved donor complex (CDC, also known as a type 1 complex; Craigie and Mizuuchi 1987, Surette et al. 1987), in which the exposed 3’OHs at the transposon ends are held in proper orientation for the strand transfer to take place (Craigie and Mizuuchi 1987, Mizuuchi et al 1992). In general, both cleavage and strand transfer reactions are rapid compared to the assembly step, and the reactions are carried out by two MuA subunits (at R1 and L1) acting in trans i.e. the MuA monomer bound at one end catalyses reactions at the other end, and vice versa (Aldaz et al. 1996, Namgoong and Harshey 1998, Savilahti et al. 1995, Williams et al. 1999, Yuan et al. 2005). In the presence of Ca2+, the donor cleavage does not take place and the SSCs accumulate (Baker and Luo 1994, Mizuuchi et al. 1992). When MuB recruits a proper target to the transpososome, target capture complexes (TCC) are formed. Mu transpososomes are remarkably flexible in target capture as the target DNA can be captured at LER, SSC, or CDC stages (Naigamwalla and Chaconas 1997). In many other transposition systems target 23
Introduction
DNA can be brought in at a particular stage only. During the strand transfer, the newly exposed 3’OHs act as nucleophiles that attack the phosphodiester bond of the target DNA, allowing simultaneous onestep cleavage and joining (Mizuuchi and Adzuma 1991). The resulting strand transfer complex (STC or type 2 complex), is the most stable of the Mu transpososomes (Lavoie et al. 1991, Mizuuchi et al. 1992, Surette et al. 1987). 5.1.1.4 DISASSEMBLY OF THE MACHINERY: TRANSITION FROM TRANSPOSOSOME TO REPLISOME In the STC MuA remains very tightly bound to the DNA ends (Surette et al. 1987). Other machinery components, HU and MuB, also remain bound to the branched Shapiro intermediate that will serve as a template for Mu DNA replication (Lavoie and Chaconas 1990). The tightly bound STC prevents access and function of the DNA replication machinery. It is destabilized by ClpX that weakens the transpososome interaction with DNA by interacting with MuA IIIβ subdomain (Burton et al. 2001, 2003, Kruklitis et al 1996, Kruklitis and Nakai 1994, Nakai and Kruklitis 1995, Levchenko et al 1997, Nakai et al 2001). As a result, the STC is converted into a more fragile complex, called STC2 (or type 3 complex). The STC2 in turn promotes formation of another nucleoprotein complex (prereplisome) by yet unidentified host factors, called Mu replication factors (MRFs) which displace the transpososome in an ATP-dependent reaction. These steps are subsequently followed by the recruitment of more host proteins involved in formation of the preprimosome and eventually the replisome, capable for replication and formation of the cointegrate (for review 24
see Nakai et al. 2001). As a result, single strand gaps are repaired by the host factors and a 5-bp target site duplication is generated (Allet 1979, Kahmannn and Kamp 1979). 5.1.1.5 STRUCTURE FUNCTION RELATIONSHIPS OF THE MACHINERY A recent 3D reconstruction of the Mu transpososome from electron micrographs shows how DNA and MuA monomers are positioned in relation to each other, and reveals structural explanations for several biochemical activities described above (Rice 2005, Yuan et al. 2005). For instance, Mu transpososome appears to be held together mostly by DNAprotein interactions. The small number of protein-protein interactions explains why the presence of DNA is required for MuA tetramerization. This 3D model also provides structural evidence of contribution of only two of the four MuA monomers (not four as originally suggested; Baker et. al. 1993, 1994) in catalytic steps and of catalysis in trans (see above), also further explaining why monomeric MuA is inactive (Aldaz et al. 1996, Namgoong and Harshey 1998, Savilahti and Mizuuchi 1996, Williams et al. 1999). 5.1.2 “Non-replicative” transposition of Mu When Mu enters its host cell and integrates into the host chromosome, Mu genome is not replicated (Fig. 3; Harshey 1984). Therefore, the initial integration is referred as “non-replicative”. However, very little is known about the mechanism of this “non-replicative” integration leading to a simple insertion (Akroyd and Symonds 1983, Chaconas et al. 1983, Harshey 1984, Liebart et al. 1982). Some of the protein and DNA factors essential during replicative transposition, such as
Introduction
MuB (Roldan and Baker 2001), ClpX (Mhammedi-Alaoui et al. 1994) and SGS (Sokolsky and Baker 2003), have been shown to be less important during the initial integration. Although transposition of Mu has been thoroughly studied in vitro, conservative integration of infecting Mu DNA by cut-and-paste mechanism has not been observed in vitro (Chaconas and Harshey 2002). Thus it is unclear whether the distinction between replicative and non-replicative transposition in the case of Mu reflects a true difference in the mechanism (single-strand versus doublestrand cut) or whether it is the different processing (by repair) of the Shapiro intermediate of the replicative pathway that yields to a simple insertion (Craigie and Mizuuchi 1985). Very recent results by Au et al. (2006) support the latter option, as these studies incdicated that the flanking DNA remains linked to Mu DNA at the time of integration. They suggest that the initial Mu integration following infection proceeds through a cointegrate pathway, followed by repair, and shares similarities with retroviral integration. 5.2 MU AS A TRANSPOSITION MODEL SYSTEM: IN VITRO ASSAYS Phage Mu was the first TE for which an in vitro transposition system was developed (Mizuuchi 1983). While the original assay relied on E.coli cell extracts and plasmid substrates with Mu genome ends, the system described by Craigie et al. (1985) utilized purified protein and DNA components and resulted in more detailed dissection of this process. The early development of Mu in vitro assay enabled the detailed study of the transposition steps, components, and mechanism, and turned Mu into a general model of transposition. The defined Mu
in vitro transposition assay by Craigie et al. (1985) involves a model superhelical plasmid substrate that contains the critical Mu DNA sequences (the binding sites and IAS) in a proper orientation, MuA, MuB, at least one of the DNA bending proteins (HU), and a target plasmid DNA. Nowadays, much more simplified versions of the assay have been developed. The use of dimethlysulfoxide and glycerol in the reaction has relaxed the requirements for transpososome assembly in vitro i.e. the requirements for the DNA supercoiling, HU, and IAS (Baker and Mizuuchi 1992, Craigie and Mizuuchi 1986, Craigie et al. 1985, Mizuuchi and Mizuuchi 1989). In addition, the use of R-end substrates only, containing the R1 and R2 binding sites, instead of L- and R-ends has further increased the efficiency of the reaction (Craigie and Mizuuchi 1987, Namgoong et al. 1994). At its simplest, Mu transposition reaction can be performed with purified MuA and a short R-end DNA segments containing R1 and R2 binding sites as the only macromolecular components (Savilahti et al. 1995). This minimal in vitro reaction faithfully reproduces transpososome assembly, donor cleavage and strand transfer steps (Fig. 9; Savilahti et al. 1995) and has been used effectively in detailed mechanistic studies of transpososome function and organization (reviewed in Chaconas and Harshey 2002). With the minimal in vitro assay, specific questions about the Mu core machinery components (MuA or DNA substrates) can be asked. For instance, the effects of reaction conditions can be monitored, requirements for particular reaction steps can be determined, and reaction products analyzed along the reaction pathway. Also, usage of DNA substrates with specific
25
Introduction
flanking sequences or structures can be studied and effects of mutations either in DNA substrates or in MuA investigated. 5.3 OTHER TRANSPOSABLE BACTERIOPHAGES Only few Mu-like phages have been characterized as viruses (DuBow 1987), including the closest relative of Mu, the coliphage D108 (Hull et al. 1978) and the Pseudomonas aeroginosa phage D3112 (Wang et al. 2004). However, data from several bacterial genome sequencing projects have led to the discovery and
identification of new Mu-like phages as DNA sequences within bacterial genomes where they represent integrated prophages or their remnants. Approximately 20 Mu-like prophage sequences have been identified (reviewed in Braid et al. 2004) and the number appears to be constantly increasing. Some of the complete Mu-like prophages have been sequenced, studied for their genomic organization, and named by using a very diverse “nomenclature” (Table 1). Before this study none of these prophages had been characterized for their transpositional properties.
Figure 9. Minimal in vitro transposition assay reaction schematics. Mu R-end donor DNA fragments (50 bp) that contain R1 and R2 binding sites are assembled with MuA transposases into a transposition complex (stable synaptic complex, SSC). Under reaction conditions with Mg2+, the complex then executes successive donor DNA cleavage and strand transfer reactions, to yield a cleaved donor complex (CDC) and a strand transfer complex (STC), respectively. By using precleaved donor DNA fragments, the cleavage step can be bypassed. Strand transfer into circular target DNA generates two major products. A double-ended integration product (DEP) is generated when both donor DNA fragments are properly transferred to the target DNA, and concomitantly the target DNA becomes linearized. A single-ended integration product (SEP) is generated in cases when only one of the donor DNA fragments is transferred into the target DNA and the supercoiled circular target DNA becomes relaxed. If the donor DNA fragments are radiolabeled, the label is incorporated in the DNA product and the reaction products can be analyzed by agarose gel electrophoresis and visualized by autoradiography.
26
Introduction
Table 1. Mu, D108 and recently identified complete Mu-like prophages PHAGE/ PROPHAGE HOST
length (bp)
References:
Mu
Escherichia coli
36 717
Morgan et al. 2002
D108
Escherichia coli
ND
Hull et al. 1978
FluMu/ Hin-Mu
Haemophilus influenzae Rd
34 676
Morgan et al 2001, this study (II)
Pnm1
Neisseria meningitidis Z2491
39 314
Morgan et al 2002, Klee et al 2000
MuMenB
Neiseria meningitidis MC58
34 539
Masignani et al. 2001
D3112
Pseudomonas aeruginosa
37 611
Wang et al. 2004
B3
Pseudomonas aeruginosa
38 439
Braid et al 2004
BcepMu **
Burkholderia cenocepacia
36 748
Summer et al. 2004
MuEb
Escherichia blattae
33 339
Andres et al. 2004
MuSo1
Shewanella oneidensis
34 551
Heidelberg et al. 2002
MuSo2
Shewanella oneidensis
35 666
Heidelberg et al. 2002
SfV *
Shigella flexneri
37 074
Allison et al. 2002
* Genomic organization similar to lambda phage, but tail assembly and structural genes homologous to Mu-like phages, a functional bacteriophage ** Represents more distantly related family of Mu-like phages, a functional bacteriophage (see Summer et al. 2004) ND, Not determined
27
Aims of the Present Study
C. AIMS OF THE PRESENT STUDY In spite of the detailed knowledge of the chemical reactions of DNA transposition, little is known about the machineries that perform these critical reactions and carry out other tasks involved in transposition. For only a handful of elements, the components, structure, and function of their transposition machineries have been characterized in detail. In the case of plant retrotransposons, these machineries are expected to be similar to those of better-studied nonplant retroelements, but this remains to be characterized as no machineries (VLP, PIC, or core machinery) in plants have been identified yet. The BARE-1 element was a good candidate for fulfilling the requirements of an active element with a productive life cycle due to its high copy number, structural conservation, and transcriptional activity. We decided to take a step forward in identification of a plant VLP transposition machinery, and aimed to identify the individual protein (GAG, IN, RT) and DNA (cDNA) components of BARE-1 VLP machinery, and finally to reveal the presence of VLPs. One of the best characterized transposition machineries belongs to bacteriophage Mu. However, as only few Mu like phage has been identified
28
as viruses, it has not been possible to conduct comparative studies with other Mu-like phages. Recently, identification of Mu-like prophages as byproducts in bacterial genome sequencing projects has increased the number of Mu relatives. Some of these prophages have been studied for their genomic organization, but none of them has been characterized for their transpositional properties. We aimed to identify the components of the Haemophilus influenzae prophage HinMu core machinery, the transposase and its binding sites. We also aimed to evaluate their conservation compared to Mu, and to study their functionality in vitro. Phage Mu is an exceptional transposon as it uses both non-replicative and replicative transposition during its life cycle. Biochemical and structural comparisons together with evolutionary considerations suggest that the Mu transposition machinery might share functional similarities with machineries of the systems that employ a hairpin intermediate during the catalytic steps of transposition. We aimed to characterize whether Mu machinery can accommodate and process DNA end hairpins, similar to those found in Tn5 and Tn10 systems.
Materials and Methods
D. MATERIALS AND METHODS The bacterial strains, plasmids and oligonucleotides used in this study are described in the original publications. The experimental methods used in this study are described in original publications and summarized in Table 2. References to published methods can be found in the articles. Table 2. Methods used in this study Method
Described and used in
Agarose gel analysis of DNA-protein transposition complexes
II
III
Agarose gel analysis of transposition reaction products
II
III
II
III III
Annealing of oligonucleotides Antibody production
I
Autoradiography
I
II
Bacterial expression
I
II
CsCl gradient centrifugation
III
DNA sequencing Electron microscopy
I
Electrophoresis techniques
I
Electrotransformation Immunoblotting
III
II
III
II
III
I
Isolation of genomic DNA Molecular cloning tehcniques
II
II I
II
III
Mu in vitro minimal transposition assay
II
III
Oligonucleotide gel purification
II
III
II
III
PCR primers and reactions
I
Plant cell culture extraction
I
Plant cell culture, extraction and fractionation
I
Preparation of plant material
I
Probes
II
Protein purification
I
REMAP technique
I
Reverse transcriptase -assay
I
SDS-polyacrylamide gel electrophoresis
I
II
Sequence anayses and comparisons
II
Solid phase DNase I footprinting
II
Southern blotting and hybridization
II
Sucrose gradient ultracentrifugation Urea polyacryl amide gel electrophoresis
I II
III
29
Results and Discussion
E. RESULTS AND DISCUSSION 1. IDENTIFICATION AND CHARACTERIZATION OF BARE-1 AND HIN-MU TRANSPOSITION MACHINERY COMPONENTS (I, II) 1.1 IDENTIFICATION OF BARE-1 VLP MACHINERY COMPONENTS (I) Retroelement encoded protein components required for formation of functional VLP machinery are: GAG, IN, RT-RNaseH and PR. These are also the predicted components of the BARE-1 ORF encoded polyprotein (Manninen and Schulman 1993). The replication cycle of retroelements begins with the transcription of RNA that is translated into a polyprotein(s) and which also serves as a template for subsequent reverse transcription. After translation, the polyprotein is cleaved by PR and VLPs are assembled. The RNA is reverse transcribed into cDNA in the context of VLPs before integration. 1.1.1 BARE-1 GAG and IN are expressed and processed into mature sizes in vivo (I) At the time we began this study, only few plant retrotransposons had been shown to be transcriptionally active (Hirochika 1993, Pouteau et al. 1991, Royo et al. 1996, Suoniemi et al. 1996b). Today the number of transcriptionally active elements has increased. However, transcription of most characterized plant retrotransposons appear to be non-constitutive, BARE-1 and Orge being rare exceptions (Neumann et al. 2003, Suoniemi et al. 1996b, for review see Grandbastien 1998). Because in lifecycle an active retroelement the transcription is followed by translation, we searched for the translated BARE-1 GAG and IN proteins in vivo. 30
The expression of BARE-1 GAG and IN proteins in vivo was investigated by using antisera raised to subcloned and expressed GAG and IN components of the predicted BARE-1a polyprotein. No crossreactivity was detected between anti-IN IgG and expressed GAG, and vice versa (I, Fig. 1A). With these antisera, GAG and IN translation products were recognized in various barley tissue extracts and in cell cultures (I, Fig. 1BC). Detection of mature sized GAG (32.5 kDa) and IN (34.3 kDa) polypeptides and the absence of full-length polyprotein on the immunoblots suggested that the full-length polyprotein was already cleaved by PR. In general, GAG showed stronger immune response than IN, even though anti-IN and anti-GAG antibodies had shown similar response sensitivities to E. coli expressed IN and GAG, thus indicating that in vivo GAG was expressed in higher quantities than IN. This is not surprising, since GAG is needed in greater amounts for construction of the VLPs than the other components of the polyprotein, as demonstrated for several non-plant retrotransposon systems studied (Haoudi et al. 1997, Voytas and Boeke 1993). In the case of BARE-1, as both GAG and IN are components of the same polyprotein, the putative frameshift in the pr region of BARE-1a might permit greater expression of GAG relative to IN, similarly as in many retroviral systems (Swanstrom and Wills 1997). Our study demonstrated for the first time that translation products of a plant retrotransposon can be sufficiently abundant to be detected immunologically in vivo. Later BARE-1 translation products have been detected immunologically in several Graminae species (Vicient et al. 2001a). The high translational activity of
Results and Discussion
BARE-1 in vivo suggests that in the case of BARE-1 post-translational mechanisms may play an important role in regulation of BARE-1 retrotransposition. A postintegrational control by intrachromosomal homologous recombination between BARE-1 LTRs has been suggested to be in operation and to reduce the number of internal BARE-1 sequences (Vicient et al. 1999B). 1.1.2 BARE-1 GAG, IN and cDNA are present with RT-activity in middle fractions of the sucrose gradient (I) In the life cycle of retroelements the step following translation is the formation of VLPs. The VLPs of non-plant retrotransposons (Eichinger and Boeke 1988, Hajek and Friesen 1998) and retroviruses (Frankel and Young 1998) have generally been shown to contain IN, RT, and an RNA intermediate that is subsequently reverse transcribed to cDNA, within a capsid constructed of GAG. As it is likely that plant retrotransposon VLPs are similar in that respect, we studied the association of BARE-1 GAG and IN together with RT-activity and BARE-1 cDNA. The cell culture extracts that showed the strongest GAG expression, were concentrated and fractionated by ultracentrifugation on sucrose gradients. The migration of BARE-1 GAG and IN proteins and reverse transcriptase activity in the gradient was studied. We examined the fractions for the presence of light scattering material, for total protein content, for immunoreactive GAG and IN, and for RT-activity (I, Fig. 2). Since mature VLPs should contain cDNA as a result of reverse transcription, the presence of BARE-1 cDNA in the fractions was studied by PCR. Primers expected to be fairly specific for BARE-1 gag region, due to
its low degree of conservation, were used (I, Fig. 3A). As a control, the presence of contaminating genomic DNA was assayed by using a PCR-based marker technique REMAP, which amplifies regions between microsatellites or simple sequence repeats (SSRs) and the LTR ends in the genome (Kalendar et al. 1999). The co-migration of the detected VLP components and the presence of contaminating genomic DNA in fractions are summarized in Figure 10. In general, the sucrose gradients presented a complex picture with GAG, IN, cDNA, and RT activity in several peaks, which do not fully coincide. However, the peaks of RT activity, the presence of GAG and IN responses, and cDNA in these fractions were reproducible over the course of many gradients. Majority of the total protein remained in the top fractions (I, Fig. 2B). Strong GAG immunoresponse detected in the top fractions in the absence of BARE-1 IN, RT-activity, or BARE-1 cDNA (Fig. 2CDE) suggested that a large fraction of GAG was not assembled into VLPs, but was present as free monomers or small multimers. Several middle fractions contained all VLP components (II, Fig. 2 and 3; Fig. 10) which suggested that they are assembled into VLPs. In addition to mature VLPs, the presence of VLP assembly intermediates or PICs lacking some of the VLP components may have given positive results for individual components in the gradient fractions (see Fig. 10). The presence of BARE-1 cDNA together with RT-activity in several fractions without contaminating genomic DNA, indicated that BARE-1 RNA had been reverse transcribed into cDNA. Although the RT activity detected in the fractions could not directly be linked to BARE-1, the migration of GAG and IN in the same fractions suggested that the activity (or at 31
Results and Discussion
least some of it) could derive from BARE-1 VLPs. The strong RT activity in the pellet, together with GAG, IN and BARE-1 cDNA, suggested that BARE-1 VLPs were formed, aggregated, and precipitated into a pellet during ultrcentrifugation, which is a common phenomenon during virus or VLP preparation. 1.1.3 VLP-like structures are formed (I) No VLPs have been identified in plant systems yet. However, yeast Ty1 VLPs have been studied in detail e.g. with electron microscopy (for review see Roth 2000). As co-migration of the putative BARE-1 VLP components in the gradient (Figure 10) suggested that VLPs might be present and detectable by transmission electron microscopy (TEM), the fractions
6 and 10 were subjected to TEM and visualized by negative staining. VLP-like structures were detected in both fractions. In fraction 6 (I, Fig. 4A) single type of particles, highly similar in size (~40 nm) and morphology to those of gypsy MDG4 from Drosophila (Syomin et al. 1993), were detected. In fraction 10 (I, Fig. 4B, C), two types of structures were present: smaller (10.0±0.3 nm) and larger (~35 nm), the appearance of which was highly similar to negatively stained yeast Ty1 VLPs (Burns et al. 1992, Palmer et al. 1997, Roth 2000). In the Ty-1 system, VLPs show a pronounced variation in size and e.g. VLPs composed of only one GAG polyprotein are smaller and show narrower size range (11-16 nm) than those that contain IN and RT (15-39 nm) (Burns et al 1992, Roth 2000).
Figure 10. Co-migration of BARE-1 VLP components in the sucrose gradient. The presence of BARE-1 GAG and IN, RT-activity, and BARE-1 cDNA, as well as contaminating genomic DNA (gDNA) in gradient fractions (1-20) and in pellet (P) are indicated by “-” not detected, (+) barely detectable, and +, ++, +++ indicating the level of positive detection. The open arrows on the left indicate presence of light scattering bands. The fractions highlighted in gray were studied by transmission electron microscopy for the presence of VLPs.
32
Results and Discussion
While the structures visualized in this study could not be positively identified as BARE-1 VLPs, their co-migration with the BARE-1 VLP components (GAG, IN, and cDNA) and RT activity suggested that at least some are indeed BARE-1 VLPs. In addition, when considering the number of BARE-1 elements present in barley, it is highly likely that some of the structures would be BARE-1 VLPs. However, it is possible that some of the VLP-like structures as well as RT activity may derive from other elements identified in barley genome, such as e.g. romani (Suoniemi et al 1998a) or BAGY-2 (Shirasu et al. 2000, Vicient et al. 2001b) or from another yet unidentified active element. Recently, in addition to BARE-1, the transcription and translation in vivo as well as presence of cDNA has been demonstrated for Tto1 element (Takeda et al. 2001). However, for Tto1 no VLP-like structures have been reported. Taken together, this study indicated that BARE-1 polyprotein was translated in vivo, and subsequently cleaved to functional units of predicted size, which can associate with cDNA to form VLPs. Most probably the complex migration pattern of VLP components in sucrose gradients and the size variation in VLP-like structures detected by TEM indicated the presence of a variety of assembly intermediates, or variations in the composition or structure of VLPs, which may further reflect e.g. mutations in GAG (Brookman et al. 1995, MartinRendon et al. 1996, Merkulov et al. 1996). Ty1 VLPs too, show polydispersity and certain mutations in Ty1 GAG have been shown to alter the size and morphology of VLPs drastically. As VLPs undergo structural changes during the maturation process (Kirchner and Sandmeyer 1993), their appearance in TEM as well as their
migration in sucrose gradient may differ considerably. In general, the VLP assembly of Ty1 appears to be very flexible and a wide range of structural variations are tolerated in VLPs (reviewed in Roth 2000). It is likely that plant retrotransposon VLPs would be similar in that respect. 1.2 IDENTIFICATION AND CHARACTERIZATION OF HIN-MU CORE MACHINERY COMPONENTS (II) In the beginning of this study (II), the completion of the sequencing project of Haemophilus influenza Rd genome had revealed an integrated Mu-like prophage sequence (Fleischman et al 1995), that we named Hin-Mu. The approximate genomic location of Hin-Mu was known, but the exact length and the precise ends of this prophage were unidentified. The significant homology shared with the genome of bacteriophage Mu (Morgan et al. 2001) and a presence of Mu gene A homolog encoding a putative transposase protein in Hin-Mu suggested that Hin-Mu core machinery components, the putative transposase and its binding sites at the ends of Hin-Mu genome, could be identified and their functionality studied in vitro. 1.2.1 Identification of ends: Hin-Mu is a full-length Mu-like prophage (II) By DNA sequence comparison between the H. influenzae Rd (containing Hin-Mu prophage) and another H. influenzae strain without a prophage at the corresponding locus we determined that Hin-Mu prophage was 34,676 bp long. In the H. influenzae Rd genome it was flanked on both sides by a 5-bp target site duplication that is a hallmark of Mu transposition (Allet 1979, Kahman and Kamp 1979). Its genome ends carried the terminal nucleotides 5’CA3’ that are conserved among wide variety 33
Results and Discussion
of elements and have been shown to be important for efficient Mu transposition (see B.5.1.1.1). Therefore, Hin-Mu was a full-length Mu-like prophage, which had been integrated into the H. influenzae Rd genome by transposition. 1.2.2 Identification of transposase: MuAHin is structurally similar to MuA (II) The chemical reactions of transposition are catalyzed by the element encoded transposase protein. Phage Mu encoded MuA transposase is a 633-aa product (II, Fig.1A) of gene A. Hin-Mu prophage contained a similar A gene encoding a homologous putative 687-aa transposase Therefore the protein (MuAHin). conservation and possible functionality of putative MuAHin transposase were evaluated by aligning and comparing the amino acid sequences of MuA and MuAHin with regard to structural and functional characteristics based on the information available for MuA (Clubb et al. 1994, 1997, Rice and Mizuuchi 1995, Schumacher et al. 1997). The comparison of these two proteins revealed a co-linear domain organization, significant amino acid similarity (>31%) in each domain and subdomain suggesting structural similarity between MuAHin and MuA (II, Fig. 1 and II, Table 1). The three phylogenetically conserved acidic amino acids (Asp 279, Asp 344, Glu 400) of the DDE motif were identified in MuAHin domain II and they aligned well with those of MuA (Asp 269, Asp 336, Glu 392). The high degree of conservation within the central catalytic domain (63%), especially in and around the DDE motif, suggested that MuAHin might have retained at least some degree of catalytic activity. The N-terminal DNA binding domain I was also conserved (41%), but contained 34
some insertions and deletions that might affect its DNA binding properties. As transposases and their binding sites in DNA are expected to evolve as a pair, it is likely that the observed conservation in this domain reflects the conservation of the transposase binding sites. Domain III, responsible for nonspecific DNA binding as well as interactions with MuB and ClpX proteins, was the least similar (32%). This was not surprising, since contacts between this domain and its binding partners are expected to be less specific or less conserved, and thus more amino acid changes can be tolerated within this domain. 1.2.3 Identification of binding sites: Hin-Mu ends are conserved and contain putative transposase binding sites (II) Instead of simple TIRs found at the ends of most DNA transposons, bacteriophage Mu and its closest relative D108 contain three transposase binding sites at each end of their genome (R1-R3 and L1-L2, at L-and R-end, respectively; Craigie et al. 1984, Symonds et al. 1987). As Hin-Mu was a full-length Mu-like prophage, we searched for binding sites with a similar organization at its ends. Putative transposase binding sites at Hin-Mu ends were identified and their potential functionality was evaluated by aligning and comparing the Hin-Mu R- and L-ends to the Mu and D108 end sequences (II, Fig. 2). Of the six Hin-Mu binding sites, two at each end (R1-R2 and L1-L2) were easily identified and were very similar to those of Mu and D108 both in sequence and in location. For the third binding sites two alternative locations (R3/R3* and L3/L3*) were determined and further analyses performed.
Results and Discussion
Sequence comparisons of Mu, D108, and putative Hin-Mu transposase binding sites to a 22-bp consensus sequence derived from six Mu sites (R1-R3 and L1-L3; II, Fig. 3) and scoring of each binding site according to their similarity to the consensus sequence, revealed a high degree of conservation within the binding sites of these phages. Of the three phages the Hin-Mu binding sites were the most variable (the lowest scores). The sequences of putative R3* and L3* binding sites matched better to the consensus than those of R3 and L3 sites (II, Fig. 3), although the positions of the latter sites matched better to positions of Mu R3 and L3 sites (II, Fig. 2), respectively. 1.2.4 Interactions between DNA and protein components of Hin-Mu and Mu core machineries (II) Interaction of transposase molecules with its specific binding sites at the transposon ends represents an important early step in DNA transposition. This initial transposase binding can be studied by using various footprinting techniques under reaction conditions that allow monomeric transposase binding but do not promote transpososome assembly. As we were able to identify conserved putative binding sites at Hin-Mu ends, the interactions of purified MuAHin with these binding sites were investigated by using a solid phase DNase I footprinting technique. Hin-Mu ends were footprinted with MuAHin, and Mu ends with MuA (as a positive control). In addition, as Mu and Hin-Mu machinery components (binding sites and transposase proteins) shared significant similarity, we decided to study the interchangeability of the components, by footprinting Hin-Mu ends with MuA, and vice versa. Despite our extensive trials, MuAHin was not able to produce footprints on any
of the substrates used. On the contrary, MuA generated clear footprints on both Mu ends, not only confirming the existing data (Craigie et al. 1984, Zou et al., 1991) but also revealing some new information of its binding sites (see 1.2.5, II, Fig. 4AB). Furthermore, MuA generated clear footprints on Hin-Mu R-end substrates that correlated well with our binding site predictions (II, Fig. 4A). Relative to the Mu R-end, the affinity of MuA for the Hin-Mu R-end appeared to be lower and a somewhat smaller area was protected, indicating fewer protein-DNA contacts. Nevertheless, MuA showed a characteristic protection pattern on Mu and Hin-Mu substrates, with one DNase I sensitive site in each binding site. These sensitive sites were in identical positions in each binding site, indicating qualitatively similar contacts with all binding sites (II, Fig, 6). By using the positions of the DNaseI sensitive sites (II, Fig. 6) and the positions of DNase I protection areas at the HinMu R end (II, Fig. 4A) together with our previous sequence analyses (II Fig. 2,3) we predicted that of the two alternative R3 binding sites (R3 or R3*) the R3* represented the major binding site. However, no footprints were obtained for Hin-Mu L-end with MuA (nor with MuAHin) and the predicted binding sites could not be confirmed experimentally. We found that data from earlier mutation studies (Groenen and van de Putte, 1986) that delineated essential and non-essential nucleotides within MuA binding sites (II, Fig. 3) was in correlation with our results. Within the Hin-Mu R-end binding sites, all essential nucleotides were conserved and MuA generated clear footprints with these binding sites, while in each Hin-Mu L-end binding site at least one essential nucleotide was mutated, probably explaining the lack of detectable footprints in this end. 35
Results and Discussion
1.2.5 General features of Mu and HinMu binding sites (II) MuA specifically binds to its binding sites through its N-terminal domain (Nakayama et al. 1987). This proteolytically defined domain contains three independently folded subdomains, Iα, Iβ, and Iγ. The 22bp binding sites can also be divided in two half sites, the first half (nt 1-11) interacting with subdomain the Iγ and the second half (12-22) with Iβ. Our sequence analyses (II, Fig. 2 and 3) and footprinting experiments (II, Fig. 4) revealed some common features about the binding sites of these phages. Alignment of the Mu, D108 and HinMu binding sites revealed that in general the first half (II, Fig. 4, nt 1-11) showed less conservation than the second half site (nt 12-22), suggesting functionally less important contacts between MuA Iβ subdomain and the first half-site. The general conservation of all binding sites was in good agreement with the data of Groenen and van de Putte (1986) who determined the conservation of certain nucleotides within the binding sites (see I, Fig. 3) as either essential or nonessential for transpositional functionality. Our binding site analysis also revealed that in the case of Hin-Mu, the nucleotide replacements in the Hin-Mu binding sites compared to consensus sequence were not random. Instead, in each nucleotide position (1-22) a particular nucleotide change appeared to dominate (Table 3) probably reflecting coevolution of the Hin-Mu binding sites with
MuAHin transposase. Comparison of the MuA footprints on Mu and Hin-Mu R-ends (II, Fig. 5A) revealed some new details. Within MuA footprints two previously undetected DNase I-sensitive sites were detected. The alignment of all DNase I-sensitive sites detected in this study with those detected in earlier studies revealed a unity in positions of these sites (II, Fig. 6). In general, DNase I-sensitive sites in footprints indicate distortions in DNA structure (Leblanc and Moss 1994). While the nature of this anomaly in Mu has not been established, our results indicate that, despite sequence differences, MuA distorts both Mu and Hin-Mu binding sites in a similar manner. It is known that MuA can effectively bend its DNA binding site up to 90 degrees (Zou et al. 1991, Kuo et al. 1991). Thus, it may be that MuA-mediated bending generates the DNase I-sensitive sites, which in turn, are detected by DNase I footprinting. 2. FUNCTION OF HIN-MU AND MU TRANSPOSITION CORE MACHINERIES (II, III) The function of bacteriophage Mu transposition machinery can be studied by a minimal-component in vitro transposition assay, in which purified transposase and short Mu end DNA substrates containing the critical transposase binding sites are the only macromolecular components of the reaction (Savilahti et al. 1995). In this study, this assay was employed for both
Table 3. Types of nucleotide replacements at Hin-Mu binding sites compared to consensus sequence nt position consensus R1 R2 R3 L1 L2 L3
36
1 C/T
2 G
A
A
A
A T T
3 T A A A A
4 T G A G
5 T C C
6 C
8 9 10 11 C/T G/T T/A A/G
12 A
13 A
G G
G
A C T
A
7 A
T A
A
T
G
14 15 16 A/G C/T G/A C C A C A C A A C
17 C
A A A
18 G
19 A
20 A
21 A
22 G/A
Results and Discussion
Mu (III) and Hin-Mu (II), with reaction conditions identical to those generally used for Mu. In the case of Hin-Mu the functionality of the identified and isolated core machinery components (binding sites and transposase) was investigated, and compared to those of Mu (II). Also, interchangeability of the Hin-Mu and Mu machinery components was studied by using this assay. In addition, in the case of Mu, DNA hairpin processing by MuA was studied (III) by assembling transpososomes with synthetic DNA model substrates that contain hairpin ends and critical transposase binding sites. As it was difficult to make a priori predictions of potentially suitable hairpin substrates for the Mu machinery, several types of hairpin substrates (III, Fig. 1) were used in this assay, to monitor the successful assembly of stable protein-DNA complexes and to detect identifiable hairpin processing reaction products In both studies (II, III), the purified transposase proteins and the short ~50-bp radiolabeled transposon R-end substrates (see materials and methods II, III) were assembled into DNA-protein complexes, either in the presence (II) or in the absence (III) of divalent metal ions. The assembled stable protein-DNA complexes (Savilahti et al. 1995) were analyzed by native agarose gel electrophoresis and visualized by autoradiography. Transposition reaction products (strand transfer products) were generated in the presence of divalent metal ions and a plasmid target. Following the disassembly of protein-DNA complexes, the transposition reaction products were analyzed by agarose gel electrophoresis and autoradiography. Two species of reaction products involving the target plasmid were expected: the double-ended reaction product (DEP) and the single-
ended reaction product (SEP), resulting from the utilization of one or two donor substrate molecules, respectively (II Fig. 5A and III Fig 2A, Goldhaber-Gordon et al. 2002a,b, Krementsova et al. 1998, Lee and Harshey 2001). 2.1 FUNCTION OF THE HIN-MU MACHINERY (II) 2.1.1 Catalytically competent Hin-Mu transpososomes are assembled (II) The relationship between the transposase binding site affinities and transpososome assembly is not known. Neither are the number, nature, or order of the specific DNA-protein and protein-protein interactions responsible for the successful assembly known. Therefore, although MuAHin did not generate detectable footprints with any of the substrates studied, we examined whether MuAHin was nevertheless able to promote the assembly of catalytically competent complexes by using minimal in vitro transposition assay. The analyses (II, Fig. 5C) revealed that MuAHin was able to assemble stable complexes with Hin-Mu end specific fragments, but not with any of the Mu end fragments. Somewhat surprisingly, MuA efficiently assembled stable complexes with all the (Mu and Hin-Mu) fragments studied. Subsequent analysis of strand transfer reaction products (II, Fig. 5D) demonstrated that the catalytic activities of MuA and MuAHin were in clear correlation with the assembly results, suggesting that transpososome assembly or stability was the limiting step of the reaction. While MuAHin generated reaction products with Hin-Mu end specific fragments only, MuA successfully generated reaction products (SEPs and DEPs) with all the fragments studied. With precut Hin-Mu substrates
37
Results and Discussion
MuAHin generated both SEPs and DEPs in substantial quantities, whereas with uncut and frayed Hin-Mu substrates (see II, Fig. 5D) only a limited number of SEPs were detected. To summarize, Hin-Mu is the first Mulike prophage for which transpositional activity has been demonstrated. The assembly and the activity assays together showed that catalytically competent HinMu transpososomes were assembled, even though MuAHin was unable to produce footprints on any of the substrates, underscoring the importance of reaction conditions, and demonstrating that the lack of footprints in binding studies does not necessarily indicate a non-functional transposase. The results demonstrated that MuAHin had retained the strand transfer activity of its catalytic core, but almost undetectable MuAHin activity with both uncut and frayed substrates suggested that the donor cleavage activity of MuAHin may be compromised. Finally, cloning and sequencing of several transposontarget DNA junctions of MuAHin and MuA transposition reaction products (II, Fig. 5D) verified that the 3’ transposon end was accurately joined to the target DNA in each case and that the 5-bp target site duplication generated during transposition was present in all the DEPs studied. 2.2 FUNCTION OF THE MU MACHINERY (II, III) The transposition reaction mechanisms of the non-replicative transposons Tn10 and Tn5 involve the formation and opening of a transposon end DNA hairpin intermediate (see B 3.2.1; Bhasin et al. 1999, Kennedy et al. 1998), which evidently is reflected in the proficiency of their respective transposition machineries to accommodate and process hairpin
38
substrates. A high degree of unity in DNA transposition reactions in general (Craig 1995), the fact that Mu can transpose non-replicatively (Harshey 1984), and the previously discovered flexibility in MuAcatalyzed reactions (Goldhaber-Gordon et al. 2002a,b, this study II) prompted us to investigate whether the Mu machinery also could accommodate and process transposon DNA end hairpins. 2.2.1 MuA catalyzes hairpin processing reaction preferentially with longer hairpin loops (III) In order to study hairpin processing by MuA, minimal in vitro transposition reactions were performed with various hairpin and standard substrates in precut and uncut configurations (III, Fig. 1B). As the length of the transposon flanking DNA within the donor DNA segment, as well as its end configuration, have been shown to be critical variables in determining the assembly, stability, and activity characteristics of Mu transpososomes (Savilahti et al. 1995), we also investigated the effect of hairpin loop length and loop sequence on hairpin processing by using substrates with loops of variable lengths or sequences in transposition reactions (III, Fig. 1, Fig. 5). Our analyses of protein-DNA complexes and reaction products clearly demonstrated that MuA was able to assemble DNA-protein complexes, process hairpins, use the opened ends for subsequent strand transfer, and generate the expected reaction products, SEPs and DEPs (III, Fig. 2 and 5). Hairpins with longer loops were more productive or more stable in complex assembly than those containing only a few unpaired nucleotides, possibly indicating that in hairpins with longer loops the critical contacts required
Results and Discussion
for the transpososome assembly and stability are more optimally positioned. In addition, longer loops may allow more flexibility in the DNA at the vicinity of the endmost transposon nucleotides shown to be important in complex assembly (Davies et. al 2000, Savilahti et al. 1995), thereby facilitating the proper conformation of the active site. Hairpins with longer loops were also processed more efficiently by MuA, as they generated more strand transfer (especially DEPs) and hairpin opening products (III, Fig. 5 and 6). The preferential processing of these substrates may in turn reflect more suitable protein-DNA contacts between the MuA and the critical endmost transposon nucleotides. Shorter loops were also applicable substrates for catalysis, but they appeared to uncouple the coordinated action of MuA within the transpososome, as they yielded mainly SEPs. Possibly, the accommodation of such suboptimal substrates induces conformational and functional changes within Mu transpososome so that productive reactions in both transposon ends become more difficult or even impossible to execute. In general, the ability of each substrate to generate reaction products correlated well with the substrate’s ability to yield stable complexes. This observation together with the fact that extended incubation time did not increase the amount of reaction products suggests that assembly or stability of the complexes was probably the limiting factor in hairpin processing. The hairpins were openend at the exact 3’ end of the transposon, as evidenced by detection of 50-nt hairpin opening products with denaturing ureaPAGE gel electrophoresis analysis (III, Fig. 6A). As the extension of incubation time did not increase the yield of these cleaved
intermediates we concluded that these opened hairpins are intermediates, which are subsequently used for strand transfer. Subsequent sequencing of transposontarget junctions of reaction products from several independent molecules verified that hairpins were joined to the target DNA exactly at the 3´OH end of the transposon DNA as a result of Mu transposition chemistry with the hallmark 5-bp target site duplication (III, Table 1). 2.2.2 MuA hairpin processing shares similarities with cleavage reaction (III) Certain characteristics of the hairpin processing reaction were determined in this study. First, we showed that hairpin processing took place only in the presence of catalytically active MuA, since no reaction products were generated without MuA or with an active-site mutant MuAE392Q (III, data not shown) deficient in both cleavage and strand transfer (Baker and Luo 1994). These results also suggest that the hairpin processing reactions utilize the same active site that is used for the donor DNA cleavage and strand transfer steps. Second, we determined the divalent metal ion requirements for the hairpin processing reaction. In Mu system in the presence of Mg2+ or Mn2+ both cleavage and strand transfer reactions take place, whereas Ca2+ supports the strand transfer only. Hairpin processing took place in the presence of Mg2+ or Mn2+, but not with Ca2+ revealing that the divalent metal ion requirements for hairpin processing were similar as for the cleavage reaction (Savilahti et al. 1995), and further suggesting that hairpin opening reaction by MuA mimics the donor cleavage reaction. In addition, Mn2+ appeared to stimulate the coordination between the reactions at each transposon end, since in
39
Results and Discussion
the presence of Mn2+ the reaction products were predominantly DEPs, whereas Mg2+ yielded mainly SEPs. Third, we found that hairpin processing reaction did not require MuB, but its presence appeared to increase the amount of reaction products generated for currently unknown reason (III, Fig. 2B), possibly by enhancing the transpososome assembly, increasing their stability, or by stimulating the catalytic step/s. Similar effects with MuB has been observed in several in vitro studies previously (reviewed in Chaconas and Harshey 2002, Coros and Chaconas 2001, Lee and Harshey 2001, 2003). Finally, the effects of DNA sequence in the hairpin loop and at transposon end were studied. The lack of assembly and strand transfer products (III, 5AB) with mutated Mu hairpin donors and non-Mu hairpin donors indicated that MuA processed only hairpins that contained the Mu end sequence. However, the sequence in the hairpin loop had no effect on complex assembly or hairpin processing (III, Fig. 5AB). 2.2.3 Hairpin processing takes place within Mu transpososome (III) We believed that hairpin processing takes place within transpososomes for the following reasons: 1) MuA monomers are inert prior to tetramerization into an active transpososome (Baker and Mizuuchi 1992), 2) the active sites within the Mu transpososome are formed by combining structural elements from two different MuA protomers (Yuan et al. 2005), 3) the catalytically productive conformation in the Mu transpososome entails a crisscrossed architecture (Yuan et al. 2005), within which 4) the catalysis of both cleavage and strand transfer occurs in trans (Aldaz et al. 1996, Namgoong and Harshey 1998, Savilahti and Mizuuchi
40
1996, Williams et al. 1999). Also, for structural reasons, and because the strand transfer step is included, it is reasonable to assume that catalysis of the hairpin processing reactions detected in this study also must occur in trans. However, this was confirmed by an experiment with mixed transpososomes (III, see fig. 4), in which labeled hairpin donor and unlabelled precut donor fragments were assembled within transpososomes. The relative increase in the amount of detectable DEPs upon addition of unlabeled precut donor DNA (Figure 4B) indicated that hairpin processing took place within the Mu transpososome. This was further supported by our complex assembly results, which indicated that formation of stable hairpin complexes correlated with the amount of strand transfer products generated. 2.3 FLEXIBILITY OF MU MACHINERY (II, III) Within Mu transpososome the transposase binding sites are bound specifically by the N-terminal domain I of MuA (Nakayama et al. 1987), whereas the cleavage site (transposon 3’ end) must be engaged within MuA’s active site in domain II (Rice and Mizuuchi 1995). This study revealed two types of flexibility within MuA and Mu core machinery: 1) flexibility in substrate binding (recognition sites) and 2) flexibility within the active site. The MuA’s ability to generate footprints on Hin-Mu R end, to assemble functional transpososomes, and subsequently to catalyze transposition reactions with Hin-Mu substrates, revealed an unexpected flexibility of MuA with respect to its binding sites (II). MuA accommodated a surprising level of sequence variation within its binding
Results and Discussion
sites. Similar flexibility in substrate usage and binding site recognition has been reported by Goldhaber-Gordon et al. (2002a), although in their study the Mu transpososomes used contained a pair of one wild type Mu end and one altered “pseudo-Mu end”. They suggested that MuA binding sites play an important role in positioning the cleavage site (3’ end) properly in respect to the active site (Goldhaber-Gordon 2002b). Indeed, it would be rational to think that MuA would allow more flexibility within the binding sites in order to ensure proper and more critical positioning of the cleavage site within the active site. It remains to be elucidated how MuA in fact recognizes the proper binding sites and their position relative to cleavage site, if such flexibility within binding site sequence is allowed and still the distance to the cleavage site is critical. This study also revealed that MuA can accommodate a wide range of hairpin substrates and encage them in the active site for productive catalysis (III) reflecting flexibility within its active site. Based on information from the crystal structure of the Tn5 transpososome, in which the active site appears relatively crowded (Davies et al. 2000, Reznikoff 2003), the observed degree of flexibility within the Mu transpososome active site is somewhat surprising. In the Tn5 transpososome, the hairpin forms via dramatic bending of the DNA backbone with concomitant flipping of a thymidine base and interactions from the conserved YREK motif (see 3.3, Rezsohazy et al. 1993) appear to be directly involved in the process (Davies et al. 2000, Naumann and Reznikoff 2002). Such a motif has not been identified in Mos1 transposase that performs dsDNA break without a hairpin intermediate
(Richardson et al. 2006). We were also unable to identify such a motif in MuA so that it is difficult to postulate how the Mu transpososome accommodates hairpins. In the case of longer loops, MuA’s active site might actually function in a similar fashion and conformation as it does for donor cleavage, and no additional conformational changes in the protein and DNA structures need to be postulated, a scenario that is also consistent with the metal ion analysis. However, to encage shorter DNA loops in a catalytically competent conformation, structural flexibility in MuA within the transpososome may be important. 3. FUNCTION OF BARE-1, HINMU AND MU TRANSPOSITION MACHINERIES IN VIVO (I, II, III) 3.1 IS BARE-1 TRANSPOSITIONALLY ACTIVE IN VIVO? (I) To summarize the results of our and other studies: BARE-1 is transcriptionally active in barley (Suoniemi et al 1996a) and in other Triticae species (Pearce et al. 1997). This study demonstrated that BARE-1 GAG and IN are translated and cleaved into mature sized polypeptides in vivo. We also showed that BARE-1 GAG and IN, RT activity and BARE-1 cDNA comigrated to a certain extent in the sucrose gradient together with VLP-like structures. Furthermore, taking into account the abundance of BARE-1 elements in barley, the highly polymorphic nature of the BARE-1 insertions, and the observation that all BARE-1 insertion sites studied have invariably been flanked by perfect 5bp target site duplications it is likely that BARE-1 is transpositionally active in vivo. At least some of the BARE-1 copies may be active and they may provide functional
41
Results and Discussion
VLP components also for defective copies. Further, when considering the high translational activity of BARE-1 in vivo, a population of variably defective BARE-1 elements could complement each others by providing components in trans and thus result in a population of chimeric functional VLPs. 3.2 IS HIN-MU TRANSPOSITIONALLY ACTIVE IN VIVO? (II) Hin-Mu is highly similar to phage Mu with regard to DNA sequence and genomic organization (Morgan et al. 2001). Such a high degree of conservation along with the fact that the Hin-Mu transpositional machinery has retained its catalytic activity strongly suggests an evolutionarily recent integration. However, our recent results indicate that Hin-Mu may not be functional in vivo, as virus plaques could not be generated by plating several H. influenzae strains with supernatants of the H. influenzae Rd strain (even after treatment with chloroform; Saariaho AH and Savilahti H., unpublished results). In addition, the lack of MuAHin footprints and the low catalytic activity of Hin-Mu transpososomes also suggest that HinMu may not be transpositionally active in vivo. 3.3 DOES MUA CATALYZE HAIRPINNING OF MU DNA IN VIVO? (I, II, III) Although MuA was able to process preformed model DNA hairpins in vitro, Mu has not been shown to use hairpinning mechanism in vivo or in vitro. Theoretically, hairpinning could release the Mu genome from the flanking DNA
42
during initial integration and thus provide an alternate explanation for the nonreplicative integration through the repair of a cointegrate (Craigie and Mizuuchi 1985). Although recent results by Au et al. (2006) support this “repair pathway” and we only have evidence of hairpin processing in Mu system the formal possibility remains that MuA might use hairpinning as an alternative catalytic mechanism in certain situations in vivo or under special reaction conditions in vitro. Interestingly, in artificial conditions in vitro, MuA has been shown to produce DNA hairpins at target DNA (Au et al. 2005, P. Rice, H. Savilahti and K. Mizuuchi, unpublished results). Another possibility is that MuA can only process hairpins but not form them, representing an evolutionary remnant of the hairpinning mechanism, or alternatively evolutionary recently gained yet incomplete novel activity of MuA. Nevertheless, even if hairpins would never be introduced to Mu in the wild and hairpin processing would occur only in artificial settings, hairpin processing activity represents a natural biological property of MuA, which may be evolutionary important. As donor cleavage and hairpin opening appear to be virtually identical reactions in their characteristics, the distinction between cleavage and hairpin opening reactions is arbitrary, and in fact hairpin opening (at least with longer loops) could be regarded as a form of donor cleavage. The MuA’s ability to process short hairpin loops (although relatively poorly) suggests that evolutionarily the non-hairpinning and the hairpinning mechanisms may be more related than previously anticipated.
Results and Discussion
4. MINIMAL COMPONENT IN VITRO TRANSPOSITION ASSAY AS A TOOL (II, III) In this study we showed that the minimal component in vitro transposition assay can successfully be used for activity studies of not only Mu (III) but also other Mu-like phages, such as Hin-Mu (II). The advantage of this type of assay is that no phage
propagation is required, which enables the characterization of the transpositional activity of both functional and defective prophages. In fact, also partial activities of machinery components (e.g. transposase) can be studied using this assay. This is in striking contrast to previous in vivo transposition assays that are able to detect full activities only.
43
Conclusions and Future Prospects
F. CONCLUSIONS AND FUTURE PROSPECTS In this study, the DNA and protein components of the VLP machinery of BARE-1 (I) and the core machinery of Hin-Mu (II) were identified using different approaches. The BARE-1 VLP machinery components were detected in vivo, their co-migration in the sucrose gradient was studied, and VLP-like structures visualized. The Hin-Mu core machinery components were identified and their functionality and activity was investigated in vitro (II) by using a minimal component assay developed for Mu. With this particular assay, also the ability of Mu transposition core machinery to use Hin-Mu specific substrates (II) and Mu DNA end hairpin substrates (III) was studied. This study (I) revealed that the BARE-1 VLP machinery components necessary for carrying out the life cycle of an active retrotransposon were present. The migration of the VLP components in the sucrose gradient, the detected VLPlike structures in fractions containing the machinery components, and the number of full length BARE-1 copies within the barley genome together indicate that BARE-1 VLPs are assembled. Our results, together with the abundance of BARE-1 in barley and its transcriptional activity suggest that in future the transpositional activity of BARE-1 in vivo or the transpositional competence of BARE-1 VLPs in vitro could be examined and detected. This study also revealed (II) that HinMu core machinery components were present, functional, and assembled into catalytically competent transpososomes. Hin-Mu is the first Mu-like prophage for which transpositional activity has been demonstrated. We also showed that the Mu minimal component in vitro transposition assay is not only an efficient tool when 44
studying the function of the Mu machinery, but it is also applicable to other Mu-like prophages (II). In principle, with this assay it should be possible to investigate the transpositional activities of any Mulike phages identified (Table 1) if putative A gene and phage genome ends could be identified. In addition, transpositional activities of partial or defective prophages, and partial activities of the transposases (e.g. cleavage mutants) could also be investigated. In future, additional features of HinMu machinery and MuAHin activity can be studied, e.g. the target site selectivity. Further, a comparative study of binding sites and transposase sequences of a larger group of Mu-like prophages could reveal conservation between the binding sites and transposases, which in turn would provide more information about the important transposase-binding site contacts. Also, as the Mu minimal in vitro assay revealed the functionality of Hin-Mu components and transposition activity of the core machinery, a similar approach could be used for a larger group of prophages carrying conserved A gene and binding sites. The interchangeability of their machinery components combined with comparative analyses of their transposase and binding site sequences might reveal new important aspects about the evolutionary relationships of these phages and their transposition machineries. Our studies with Mu (II, III) revealed unexpected flexibility of the Mu core machinery with respect to substrate usage. MuA was able to bind Hin-Mu R-end specific binding sites, assemble stable complexes and perform the reaction chemisty with Hin-Mu substrates indicating flexibility in substrate binding. MuA was
Conclusions and Future Prospects
also able to assemble transpososomes with Mu end hairpin substrates, process a variety of different hairpin substrates, and catalyze the following strand transfer reactions, indicating surprising flexibility within its active site. These results
revealed new uncharacterized aspects of MuA’s function that may be evolutionary and mechanistically important, and which arise new questions about the degree of flexibility of the Mu machinery and MuA for further studies.
45
Acknowledgements
G.
ACKNOWLEDGEMENTS
This work was carried out in the DNA Recombination Laboratory at the Institute of Biotechnology, in the Research program in Cellular Biotechnology and at the Department of Biosciences, Division of genetics, University of Helsinki. The work was financially supported by the Academy of Finland, the Viikki Graduate School in Biosciences and the University of Helsinki grant. The director of the Institute of Biotechnology, Prof. Mart Saarma is acknowledged for the high quality and good spirit in BI, and the present and former Heads of the division of Genetics Prof. Tapio Palva and Pekka Heino for helping with the PhD bureaucracy. I am grateful to my supervisor Docent Harri Savilahti for the guidance to the transposon world, for being present whenever scientific questions arose, and for sharing “the battles” with each article prior to publication. I also highly appreciate his understanding in that people tend to have a life outside the lab too. I warmly thank Prof. Kristiina Mäkinen and Docent Tero Ahola for their careful reviews and valuable suggestions on this manuscript. Professors Dennis Bamford and Teemu Teeri are also warmly thanked for being in my follow-up group, which has also served as a “support group” during the difficult times. All the co-authors are acknowledged: Alan Schulman and people in his lab, especially Marko Jääskeläinen for showing me the world of BARE-1, and for teaching plant biology techniques, Arja Lamberg for the groundwork of the Hin-Mu project and Seija Elo for Hin-Mu Southerns. The Viikki Graduate School in Biosciences is acknowledged not only for the salary and courses I have had the 46
privilege to enjoy, but also for taking care of the progression of my thesis. Especially for the Head of the school Prof. Marja Makarow and ex-coordinator Dr. Nina Saris, I wish to express my gratitude for their optimism and encouraging discussions with me. Also, coordinators Ritva Niemelä, Eeva Sievi and Anita Tienhaara are acknowledged for generously helping and taking care of all practical things. Timo Päivärinta is thanked for the layout of this thesis. All the present (Maria, Anja, Hilkka, Tiina, Lotta, Eini, Heikki, Sari, Pirjo) members of our group are thanked for nice moments, fruitful discussions, and practical help. Auli, Pirjo, Sari and Danielle are especially acknowledged for the excellent technical support as well as for maintaining the order in the lab. The former members Saija and Eini are thanked for fruitful scientific and nonscientific discussions, unofficial parties, co-operation in gardening and dog business, and for their friendship, Suvi for cynisim and for everyday jokes, Arja for optimism and funny moments, Juha-Matti and Anna for fruitful discussions and for the voice of reason. I also wish to express my appreciation here to Prof. Mirja Salkinoja-Salonen for taking me in her project as a “green” 19year old biology student and for guiding me to the field of research. Also, former members of MSS-project, especially Joanna, Raine and Katri are thanked for keeping contact with me through the years in the form of parties, coffee hours, telemark holidays, and sauna sessions. I warmly thank ALL my friends (too many to name here). First of all, I wish to express my deepest gratitude for those people, who where there for us when
Acknowledgements
our loved baby Eetu died and our world collapsed. You helped us to continue our fragile life. Also, thank you for friends in KÄPY ry, for your unconditional support. My friends in the field of research are thanked for sharing similar thoughts, feelings, and things related to science, including the moments of success and frustration. For all my non-scientific friends I am grateful, for not being involved so much in science and for bringing contrast to my life in the form of other activities. Thank you for your friendship and for making my life fun and rich also during the stress and downhills during these years! Especially warm hugs belong to Kaisa, Jone and Mari. I am deeply grateful to my parents for supporting me in life in all goals, not only in professional ones. It was my late father Seppo’s interest in nature and the disease we shared that guided me to the field of biology. I thank my parents for the never-ending optimism and “sisu”. My mother Sirkka-Liisa is especially warmly thanked for ALL help during the writing of this thesis. My sister Susanna, Richard and India are acknowledged for the sunny holidays in France and for the hospitality during our visits. Finally, my greatest thanks belong to my family. They have helped me through the hard days and cleared me the most important things in life. My husband
Kalle, who has shared 15 years of coevolution with me, has been my friend, therapist, IT-supervisor, cook, baby-sitter, and irreplaceable support during these years. I thank my children for all my heart for being the way they are. Touko, our 4year old “Pelle Peloton”, has made several inventions to help our daily life: e.g. “a migraine vacuum”, which he developed for my migraine days, “a rechargeable sleepener” (“nukutin”) for Emma for the restless nights, “material machine” for himself which keeps him in materials for all building jobs, and not to forget the energyelectrical-cleaning-of water systems etc. running in our living room from day to day. Emma Aurora, our “valon tuoja”, has brought a lot of light and joy into our life with the most wonderful smile, big hugs with wet wet kisses and gentle words “äiti, isi, Tonto”. Our secret “stress releasers”, Tua and Viikka basenjis have provided everyday source of pressure release by being always present, understanding, listening, comforting, soft, warm, and loving…and sometimes, ah, so stubborn! I’ve needed all these things, people, and animals to finish my thesis. And now… it is finished! Helsinki, May 2006
47
References
H. REFERENCES Adzuma K, Mizuuchi K (1988). Target immunity of Mu transposition reflects a differential distribution of Mu B protein. Cell 53:257-66.
and inactivation of the gene region encoding B12-dependent glycerol dehydratase by a new mu-like prophage. J Mol Microbiol Biotechnol 8:150-68.
Adzuma K, Mizuuchi K (1989). Interaction of proteins located at a distance along DNA: mechanism of target immunity in the Mu DNA strand-transfer reaction. Cell 57:41-7.
Au TK, Agrawal P, Harshey RM (2006). Chromosomal integration mechanism of infecting mu virion DNA. J Bacteriol 188:1829-34.
Agrawal A, Eastman, QM, Schatz DG (1998). Transposition mediated by RAG1 and RAG2 and its implications for the evolution of the immune system. Nature 394:744-51.
Au TK, Pathania S, Harshey RM (2004). True reversal of Mu integration. EMBO J 23:340820.
Akroyd JE, Symonds N (1983). Evidence for a conservative pathway of transposition of bacteriophage Mu. Nature 303:84-6. Al-Khayat HA, Bhella D, Kenney JM., Roth JF, Kingsman AJ, Martin-Rendon E, Saibil HR (1999). Yeast Ty retrotransposons assemble into virus-like particles whose T-numbers depend on the C-terminal length of the capsid protein. J Mol Biol 292:65-73. Aldaz H, Schuster E, Baker, TA (1996). The interwoven architecture of the Mu transposase couples DNA synapsis to catalysis. Cell 85:257-69. Allet B (1979). Mu insertion duplicates a 5 base pair sequence at the host inserted site. Cell 16:123-9. Allingham JS, Wardle SJ, Haniford DB (2001). Determinants for hairpin formation in Tn10 transposition. EMBO J 20:2931-42. Allison RG, Chaconas G (1992). Role of the A protein-binding sites in the in vitro transposition of Mu DNA. A complex circuit of interactions involving the Mu ends and the transpositional enhancer. J Biol Chem 267:19963-70. Allison GE, Angeles D, Tran-Dinh N, Verma NK (2002). Complete genomic sequence of SfV, a serotype-converting temperate bacteriophage of Shigella flexneri. J Bacteriol 184:1974-87. Andres S, Wiezer A, Bendfeldt H, Waschkowitz T, Toeche-Mittler C, Daniel R (2004). Insights into the genome of the enteric bacterium Escherichia blattae: cobalamin (B12) biosynthesis, B12-dependent reactions,
48
Bainton RG, Kubo KM, Feng J, Craig NL (1993). Tn7 transposition: target DNA recognition is mediated by multiple Tn7encoded proteins in a purified in vitro system. Cell, 72: 931-43. Baker TA (1995). Bacteriophage Mu: a transposing phage that integrates like retroviruses. Seminars in Virology 6:53-63. Baker TA, Luo L (1994). Identification of residues in the Mu transposase essential for catalysis. Proc Natl Acad Sci USA 91:665458. Baker TA, Mizuuchi K (1992). DNApromoted assembly of the active tetramer of the Mu transposase. Genes Dev 6:2221-32. Baker TA, Kremenstova E, Luo L (1994). Complete transposition requires four active monomers in the Mu transposase tetramer. Genes Dev 8:2416-28. Baker TA, Mizuuchi M, Mizuuchi K (1991). MuB protein allosterically activates strand transfer by the transposase of phage Mu. Cell 65:1003-13. Baker TA, Mizuuchi M, Savilahti H, Mizuuchi K (1993). Division of labor among monomers within the Mu transposase tetramer. Cell 74:723-33. Bennetzen JL (1996). The contributions of retroelements to plant genome organization, function and evolution. Trends Microbiol 4:347-53. Bennetzen JL (2000). Transposable element contributions to plants gene and genome evolution. Plant Mol Biol 42:251-69.
References
Besmer E, Mansilla-Soto J, Cassard S, Sawchuk DJ, Brown G, Sadofsky M, et al. (1998). Hairpin coding end opening is mediated by RAG1 and RAG2 proteins. Mol Cell 2:817-28 Bhasin A, Goryshin IY, Reznikoff WS (1999). Hairpin formation in Tn5 transposition. J Biol Chem 52:37021-29. Biery MC, Lopata M, Craig NL (2000). A minimal system for Tn7 transposition: the transposon-encoded proteins TnsA and TnsB can execute DNA breakage and joining reactions that generate circularized Tn7 species. J Mol Biol 297:25-37. Bowerman B, Brown PO, Bishop JM, Varmus HE (1989). A nucleoprotein complex mediates the integration of retroviral DNA. Genes Dev 3:469-78. Boeke JD, Garfinkel DJ, Styles CA, Fink GR (1985). Ty elements transpose through an RNA intermediate. Cell 40:491-500. Boeke JD, Stoye JP (1997). Retrotransposons, endogeneous retroviruses, and the evolution of retroelements. In Coffin JF, Hughes SH, Varmus HE (eds.), Retroviruses, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 343-436.
necessary for formation of the Ty1 virus-like particle structure. Virology 212:69-76. Bujacz G, Jaskolski M, Alexandratos J, Wlodawer A, Merkel G, Katz RA, Skalka AM (1995). High-resolution structure of the catalytic domain of avian sarcoma virus integrase. J Mol Biol 253:333-46. Bujacz G, Jaskolski M, Alexandratos J, Wlodawer A, Merkel G, Katz RA, Skalka AM (1996). The catalytic domain of avian sarcoma virus integrase: conformation of the active-site residues in the presence of divalent cations. Structure 4:89-96. Bukhari AI, Froshauer S, Botchan M (1976). Ends of bacteriophage Mu DNA. Nature 264:580-3. Burlingame RP, Obukowicz MG, Lynn DL, Howe MM (1986). Isolation of point mutations in bacteriophage Mu attachment regions cloned in a lambda: mini-Mu phage. Proc Natl Acad Sci USA. 83:6012-6. Burns NR, Saibil HR, White NS, Pardon JF, Timmins PA, Richardson SM, et al. (1992). Symmetry, flexibility and permeability in the structure of yeast retrotransposon virus-like particles. EMBO J 11:1155-64.
Bolland S, Kleckner N (1996). The three chemical steps of Tn10/IS10 transposition involve repeated utilization of a single active site. Cell 84:223-33.
Burton BM, Baker TA. (2003). Mu transpososome architecture ensures that unfolding by ClpX or proteolysis by ClpXP remodels but does not destroy the complex. Chem Biol 10:463-72.
Braid MD, Silhavy JL, Kitts CL, Cano RJ, Howe MM (2004). Complete genomic sequence of bacteriophage B3, a Mu-like phage of Pseudomonas aeruginosa. J Bacteriol 86:6560-74.
Burton BM, Williams TL, Baker TA. (2001) ClpX-mediated remodeling of Mu transpososomes: selective unfolding of subunits destabilizes the entire complex. Mol Cell 8:449-54.
Brierley C, Flavell AJ (1990). The retrotransposon copia controls the relative levels of its gene products post-transcriptionally by differential expression from its two major mRNAs. Nucleic Acids Res 18:2947-51.
Bushman F (2002). Lateral DNA transfer, Mechanisms and consequences. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Brown PO (1997). Integration. In Coffin JF, Hughes SH, Varmus HE (eds.), Retroviruses, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp.161-203. Brookman JL, Stott AJ, Cheeseman PJ, Adamson CS, Holmes D, Cole J, Burns NR (1995). Analysis of TYA protein regions
Capy P (2005) Classification and nomenclature of retrotransposable elements. Cytogenet Genome Res 110:457-61. Chaconas G, Harshey RM (2002). Transposition of phage Mu DNA. In Craig NL, Craigie R, Gellert M, Lambowitz AM (eds.), Mobile DNA II. American Society for Microbiology, Washington DC, pp. 384-402.
49
References
Chaconas G, Kennedy DL, Evans D (1983). Predominant integration end products of infecting bacteriophage Mu DNA are simple insertions with no preference for integration of either Mu DNA strand. Virology 128:48-59. Chaconas G, Lavoie BD, Watson MA (1996). DNA transposition: Jumping gene machine, some assembly required. Current Biology 6:817-20. Chaconas G, Harshey RM, Sarvetnick N, Bukhari AI (1981). Predominant end-products of prophage Mu DNA transposition during the lytic cycle are replicon fusions. J Mol Biol 150:341-59. Chandler M, Mahillon J (2002). Insertion sequences revisited. In Craig N., Craigie R, Gellert M, Lambowitz AM (eds.), Mobile DNA II. American Society for Microbiology, Washington DC, pp. 305-363. Chen Z, Yan Y, Munshi S, Li Y, ZugayMurphy J, Xu B, et al. (2000b). X-ray structure of simian immunodeficiency virus integrase containing the core and C-terminal domain (residues 50-293) -an initial glance of the viral DNA binding platform. J Mol Biol 296:52133. Chen JC, Krucinski J, Miercke LJ, FinerMoore JS, Tang AH, Leavitt AD, Stroud RM (2000a). Crystal structure of the HIV1 integrase catalytic core and C-terminal domains: a model for viral DNA binding. Proc Natl Acad Sci USA 97: 8233-8. Clubb RT, Omichinsk JG, Savilahti H, Mizuuchi K, Gronenborn AM, Clore GM (1994). A novel class of winged helix-turnhelix protein: the DNA-binding domain of Mu transposase. Structure 2:1041-8. Clubb RT, Mizuuchi M, Huth JR, Omichinski JG, Savilahti H, Mizuuchi K, et al. (1996). The wing of the enhancer-binding domain of Mu phage transposase is flexible and is essential for efficient transposition. Proc Natl Acad Sci USA 93:1146-50. Clubb RT, Schumacher S, Mizuuchi K, Gronenborn AM, Clore GM (1997). Solution structure of the I gamma subdomain of the Mu end DNA-binding domain of phage Mu transposase. J Mol Biol 273:19-25.
50
Coffin JF, Hughes SH, Varmus HE (eds.) (1997). Retroviruses. Cold Spring Harbor Laboratory Press, New York, USA. Coros CJ, Chaconas G (2001). Effect of mutations in the Mu-host junction region on transpososome assembly. J Mol Biol 310:299309. Craig NL (1995). Unity in transposition reactions. Science 270:253-4. Craig NL (1997). Target site selection in transposition. Annu Rev Biochem 66:437-74. Craig NL (2002). Tn7. In Craig, NL, Craigie, R, Gellert, M, Lambowitz, AM (eds.), Mobile DNA II. American Society for Microbiology, Washington DC, pp. 423-456. Craig NL, Craigie R, Gellert M, Lambowitz AM (eds.) (2002). Mobile DNA II. American Society for Microbiology, Washington DC. Craigie R (2001). HIV integrase, a brief overview from chemistry to therapeutics. J Biol Chem 276:23213-16. Craigie R (2002). Retroviral DNA integration. In Craig, NL, Craigie R, Gellert M, Lambowitz AM (eds.), Mobile DNA II. American Society for Microbiology, Washington DC, pp. 61330. Craigie R, Mizuuchi K (1985). Mechanism of transposition of bacteriophage Mu: structure of a transposition intermediate. Cell 41:867-76. Craigie R, Mizuuchi K (1986). Role of DNA topology in Mu transposition: mechanism of sensing the relative orientation of two DNA segments. Cell 45:793-800. Craigie R, Mizuuchi K (1987). Transposition of Mu DNA: joining of Mu to target DNA can be uncoupled from cleavage at the ends of Mu. Cell 51:493-501. Craigie R, Arndt-Jovin DJ, Mizuuchi K (1985). A defined system for the DNA strand-transfer reaction at the initiation of bacteriophage Mu transposition: protein and DNA substrate requirements. Proc Natl Acad Sci USA 82:7570-4. Craigie R, Mizuuchi M, Mizuuchi K (1984). Site-specific recognition of the bacteriophage Mu ends by the Mu A protein. Cell 39:38794.
References
Curcio JM, Derbyshire KM (2003). The outs and ins of transposition: from Mu to kangaroo. Mol Cell Biol 4:1-13. Davies DR, Goryshin IY, Reznikoff WS, Rayment I (2000). Three-dimensional structure of the Tn5 synaptic complex transposition intermediate. Science 289:77-85. Dawson A Finnegan DJ (2003). Excision of the Drosophila mariner transposon Mos1. Comparison with bacterial transposition and V(D)J recombination. Mol Cell 11:225-35. Doak TG, Doerder FP, Jahn CL, Herrick G (1994). A proposed superfamily of transposase genes: transposon-like elements in ciliated protozoa and a common ”D35E” motif. Proc Natl Acad Sci USA 91:942-6. DuBow MS (1987). Transposable Mu-like phages. In Phage Mu, eds. Symonds N, Touissaint A, van de Putte P, Howe MM, Cold Spring Harbor laboratory, Cold Spring Harbor, NY, pp. 201-214. Duval-Valentin G, Marty-Cointin B, Chandler M (2004). Requirement of IS911 replication before integration defines a new bacterial transposition pathway. EMBO J 23:3897-906. Dyda F, Hickman AB, Jenkins TM, Engelman A, Craigie R, Davies DR (1994). Crystal structure of the catalytic domain of HIV-1 integrase: similarity to other polynucleotidyl transferases. Science 266:1981-6. Eichinger DJ, Boeke JD (1988). The DNA intermediate in yeast Ty1 element transposition copurifies with virus-like particles: cell-free Ty1 transposition. Cell 54:955-66. Eickbush TH, Malik HS (2002). Origins and evolution of retrotransposons. In Craig NL, Craigie R, Gellert M, Lambowitz AM (eds.), Mobile DNA II. American Society for Microbiology, Washington DC, pp. 1111-46. Fayet O, Ramond P, Polard P, Prere MF, Chandler M (1990). Functional similarities between retroviruses and the IS3 family of bacterial insertion sequences? Mol Microbiol 4:1771-7. Feng YX, Moore SP, Garfinkel DJ, Rein A. (2000). The genomic RNA in Ty1 virus-like particles is dimeric. J Virol 74:10819-21.
Feuerbach F, Drouaud J, Lucas H (1997). Retrovirus-like end processing of the tobacco Tnt1 retrotransposon linear intermediates of replication. J Virol 71:4005-15. Flavell AJ, Pearce SR, Kumar A (1994). Plant transposable elements and the genome. Curr Opin Genet Dev 4:838-44. Flavell AJ, Dunbar E, Anderson R, Pearce SR, Hartley R, Kumar A (1992). Ty1-copia group retrotransposons are ubiquitous and heterogeneous in higher plants. Nucleic Acids Res 20:3639-44. Fleischmann RD, Adams MD, White O, Clayton RA, Kirkness EF, Kerlavage AR, et al. (1995). Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269:496-512. Frankel AD, Young JA (1998). HIV-1: fifteen proteins and an RNA. Annu Rev Biochem 67:1-25. Fugmann SD, Villey IJ, Ptaszek LM, Schatz DG (2000). Identification of two catalytic residues in RAG1 that define a single active site within the RAG1/RAG2 protein complex. Mol Cell 5:97-107. Garfinkel DJ, Boeke JD, Fink GR (1985). Ty element transposition: reverse transcriptase and virus-like particles. Cell 42:507-17. Gloor G, Chaconas G (1986). The bacteriophage Mu N gene encodes the 64kDa virion protein which is injected with, and circularizes, infecting Mu DNA. J Biol Chem 261:16682-8. Goldgur Y, Dyda F, Hickman AB, Jenkins TM, Craigie R, Davies DR (1998). Three new structures of the core domain of HIV-1 integrase: an active site that binds magnesium. Proc Natl Acad Sci USA 95:9150-4. Goldhaber-Gordon I, Williams TL, Baker TA (2002a). DNA recognition sites activate MuA transposase to perform transposition of nonMu DNA. J Biol Chem 277:7694-702. Goldhaber-Gordon I, Early MH, Gray MK, Baker TA (2002b). Sequence and positional requirements for DNA sites in a mu transpososome. J Biol Chem 277:7703-12.
51
References
Goldhaber-Gordon I, Early MH, Baker TA (2003). The terminal nucleotide of the Mu genome controls catalysis of DNA strand transfer. Proc Natl Acad Sci USA. 100:750914. Grandbastien MA (1998). Activation of plant retrotransposons under stress conditions. Trends Plant Science 3:181-87. Grandbastien MA, Spielmann A, Caboche M (1989). Tnt1, a mobile retroviral-like transposable element of tobacco isolated by plant cell genetics. Nature 337:376-80. Gribbon BM, Pearce SR, Kalendar R, Schulman AH, Paulin L, Jack P, Kumar A, Flavell AJ (1999). Phylogeny and transpositional activity of Ty1-copia group retrotransposons in cereal genomes. Mol Gen Genet 261:883-91. Grindley N (2002). The movement of Tn3like elements: Transposition and cointegrate resolution. In Craig NL, Craigie R, Gellert M, Lambowitz AM (eds.), Mobile DNA II. American Society for Microbiology, Washington DC, pp. 272-304. Grindley ND, Leschziner AE (1995). DNA transposition: from a black box to a color monitor. Cell 83:1063-6. Groenen MAM, van de Putte P (1986). Analysis of the ends of bacteriophage Mu using site-directed mutagenesis. J Mol Biol 189:597-602. Gueguen E, Rousseau P, Duval-Valentin G, Chandler M (2005). The transpososome: control of transposition at the level of catalysis. Trends Microbiol 13:543-9. Haapa-Paananen S, Rita H, Savilahti H (2002). DNA transposition of bacteriophage Mu. A quantitative analysis of target site selection in vitro. J Biol Chem 277:2843-51. Hajek KL, Friesen PD (1998). Proteolytic processing and assembly of gag and gag-pol proteins of TED, a baculovirus-associated retrotransposon of the gypsy family. J Virol 72:8718-24. Haniford DB (2002).Transposon Tn10. In Craig NL, Craigie R, Gellert M, Lambowitz AM (eds.), Mobile DNA II. American Society for Microbiology, Washington DC, pp. 457483.
52
Haoudi A, Rachidi M, Kim MH, Champion S, Best-Belpomme M, Maisonhaute C (1997). Developmental expression analysis of the 1731 retrotransposon reveals an enhancement of Gag-Pol frameshifting in males of Drosophila melanogaster. Gene 196:83-93. Haren L, Ton-Hoang B, Chandler M (1999). Integrating DNA: Transposases and retroviral integrases. Annu Rev Microbiol 53:245-281. Harshey RM (1984). Transposition without duplication of infecting bacteriophage Mu DNA. Nature (London) 311:580-81. Harshey RM, Bukhari AI (1983). Infecting bacteriophage Mu DNA forms a circular DNAprotein complex. J Mol Biol 167:427-41. Heidelberg JF, Paulsen K, Nelson E, Gaidos W, Nelson T, Read J, et al (2002). Genome sequence of the dissimilatory metal ionreducing bacterium Shewanella oneidensis. Nat Biotechnol 20:1118-23. Hickman AB, Li Y, Mathew SV, May EW, Craig NL, Dyda F (2000). Unexpected structural diversity in DNA recombination: the restriction endonuclease connection. Mol Cell 5:1025-34. Hickman, AB, Perez ZN., Zhou L, Musingarimi P, Ghirlando R, Hinshaw JE, et al. (2005). Molecular architecture of a eukaryotic DNA transposase. Nat Struct Mol Biol 12:715-21. Higgins NP, Collier DA, Kilpatrick MW, Krause HM (1989). Supercoiling and integration host factor change the DNA conformation and alter the flow of convergent transcription in phage Mu. J Biol Chem 264:3035-42. Hindmarsh P, Leis J (1999). Retroviral DNA integration. Microbiol Mol Biol Rev 63:83643. Hiom K, Melek M, Gellert M (1998). DNA transposition by the RAG1 and RAG2 proteins: A possible source of oncogenic translocations. Cell 94:463-70. Hirochika H (1993). Activation of tobacco retrotransposons during tissue culture. EMBO J 12:2521-8. Hirochika H, Otsuki H (1995). Extrachromosomal circular forms of the tobacco retrotransposon Tto1. Gene 165:22932.
References
Hirochika H, Fukuchi A, Kikuchi F (1992). Retrotransposon families in rice. Mol Gen Genet 233:209-16.
Kidwell MG, Lisch DR (2000). Transposable elements and host genome evolution. Trends Ecol Evol 15:95-9.
Hirochika H, Sugimoto K, Otsuki Y, Tsugawa H, Kanda M. (1996). Retrotransposons of rice involved in mutations induced by tissue culture. Proc Natl Acad Sci USA. 93:7783-8.
Kidwell MG, Lisch DR (2002). Transposable elements as sources in genomic variation. In Craig NL, Craigie R, Gellert M, Lambowitz AM (eds.), Mobile DNA II, Washington: ASM Press, pp. 384-402.
Howe MM, Bade EG (1975). Molecular biology of bacteriophage Mu. Science 190:624-32. Hua-Van A, Le Rouzic C, Maisonhaute P, Capy P (2005). Abundance, distribution and dynamics of retrotransposable elements and transposons: similarities and differences. Cytogenet. Genome Res 110:426-40. Hull RA, Gill GS, Curtiss R III (1978). Genetic characterization of Mu-like bacteriophage D108. J Virol 27:513-18. Jacks T (1990) Translational suppression in gene expression in retroviruses and retrotransposons. Curr Top Microbiol Immunol 157:93-124. Kahman R, Kamp D (1979). Nucleotide sequences of the attachment sites of bacteriophage Mu. Nature 280:247-50. Kalendar R, Grob T, Regina M, Suoniemi A, Sculman AH (1999). IRAP and REMAP. Two new retrotransposon based DNA fingerprinting techniques. Theor Appl genet 98:704-11. Kalendar R, Tanskanen J, Immonen S, Nevo E, Schulman AH (2000). Genome evolution of wild barley (Hordeum spontaneum) by BARE-1 retrotransposon dynamics in response to sharp microclimatic divergence. Proc Natl Acad Sci USA 97:6603-7. Kalendar R, Vicient CM, Peleg O,AnamthawatJonsson K, Bolshoy A., Schulman AH (2004). Large retrotransposon derivatives: abundant, conserved but nonautonomous retroelements of barley and related genomes. Genetics 166: 1437-50. Kapitonov VV, Jurka J (1999). The long terminal repeat of an endogenous retrovirus induces alternative splicing and encodes an additional carboxy-terminal sequence in the human leptin receptor. J Mol Evol 48:248-51.
Kim K, Harshey RM (1995). Mutational analysis of the att DNA-binding domain of phage Mu transposase. Nucleic Acids Res 23:3937-43. Kim K, Namgoong SY, Jayaram M, Harshey RM (1995). Step-arrest mutants of phage Mu transposase. Implications in DNA-protein assembly, Mu end cleavage, and strand transfer. J Biol Chem 270:1472-9. Kim DR, Dai Y, Mundy CL, Yang W, Oettinger MA (1999). Mutations of acidic residues in RAG1 define the active site of the V(D)J recombinase. Genes Dev 13:3070-80. Kim A, Terzian C, Santamaria P, Pelisson A, Purd’homme N, Bucheton A. (1994). Retroviruses in invertebrates: the gypsy retrotransposon is apparently an infectious retrovirus of Drosophila melanogaster. Proc Natl Acad Sci US A 91:1285-9. Kirchner J, Sandmeyer S. (1993). Proteolytic processing of Ty3 proteins is required for transposition. J Virol 67:19-28. Klee SR, Nassif X, Kusecek B, Merker P, Beretti JL, Achtman M, Tinsley CR (2000). Molecular and biological analysis of eight genetic islands that distinguish Neisseria meningitidis from the closely related pathogen Neisseria gonorrhoeae. Infect Immun 68:208295. Krementsova E, Giffin MJ, Pincus D, Baker TA (1998). Mutational analysis of the Mu transposase. Contributions of two distinct regions of domain II to recombination. J Biol Chem 273:31358-65. Kruklitis R, Nakai H (1994). Participation of the bacteriophage Mu A protein and host factors in the initiation of Mu DNA synthesis in vitro. J Biol Chem 269:16469-77.
Kennedy AK, Guhathakurta A, Kleckner N, Haniford DB (1998). Tn10 transposition via a DNA hairpin intermediate. Cell 95:125-34.
53
References
Kruklitis R, Welty DJ, Nakai H (1996) ClpX protein of Escherichia coli activates bacteriophage Mu transposase in the strand transfer complex for initiation of Mu DNA synthesis. EMBO J 15:935-44. Kulkosky J, Jones KS, Katz RA, Mack JP, Skalka AM. (1992). Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/ retrotransposon integrases and bacterial insertion sequence transposases. Mol Cell Biol 12:2331-8. Kumar A, Bennetzen JL (1999). Plant retrotransposons. Annu Rev Genet 33:479532. Kuo C-F, Zou A, Jayaram M, Getzoff E, Harshey R (1991). DNA-protein complexes during attachment-site synapsis in Mu DNA transposition. EMBO J 10:1585-91. Labrador M, Corces VG (1997). Transposable element-host interactions: Regulation of insertion and excision. Annu Rev Genet 31:381-404. Landree MA, Wibbenmeyer JA, Roth DB (1999). Mutational analysis of RAG1 and RAG2 identifies three catalytic amino acids in RAG1 critical for both cleavage steps of V(D)J recombination. Genes Dev 13:3059-69. Laten HM, Majumdar A, Gaucher EA (1998) SIRE-1, a copia/Ty1-like retroelement from soybean, encodes a retroviral envelope-like protein. Proc Natl Acad Sci USA 95:6897902. Lavoie BD, Chaconas G (1990). Immunoelectron microscopic analysis of the A, B, and HU protein content of bacteriophage Mu transpososomes. J Biol Chem 265:1623-7. Lavoie BD, Chan BS, Allison RG, Chaconas G. (1991). Structural aspects of a higher order nucleoprotein complex: induction of an altered DNA structure at the Mu-host junction of the Mu type 1 transpososome. EMBO J 10:30519. Leblanc B, Moss T (1994). DNase I footprinting. In DNA protein interactions; Principles and protocols. Kneale GG (ed.) New Jersey: Humana Press, pp. 1-10.
54
Lee I, Harshey RM (2001). Importance of the conserved CA dinucleotide at Mu termini. J Mol Biol 314:433-44. Lee I, Harshey RM (2003). The conserved CA/TG motif at Mu termini: T specifies stable transpososome assembly. J Mol Biol 330:26175. Lesage P, Todeschini AL (2005). Happy together: the life and times of Ty retrotransposons and their hosts. Cytogenet Genome Res 110:70-90. Leung PC, Harshey RM (1991). Two mutations of phage Mu transposase that affect strand transfer or interactions with B protein lie in distinct polypeptide domains. J Mol Biol 219:189-99. Leung PC, Teplow DB, Harshey RM (1989). Interaction of distinct domains in Mu transposase with Mu DNA ends and an internal transpositional enhancer. Nature 338:656-8. Levchenko I, Luo L, Baker TA (1995). Disassembly of the Mu transposase tetramer by the ClpX chaperone. Genes Dev 9:2399408. Levchenko I, Yamauchi M, Baker TA (1997). ClpX and MuB interact with overlapping regions of Mu transposase: implications for control of the transposition pathway. Genes Dev 11:1561-72. Levin HL (2002). Newly identified retrotransposons of the Ty3/gypsy class in fungi, plants and vertebrates. In Craig NL, Craigie R, Gellert M, Lambowitz AM (eds.), Mobile DNA II. American Society for Microbiology, Washington DC, pp. 684-704. Li M, Mizuuchi M, Burkem TR, Craigiem R (2006). Retroviral DNA integration: reaction pathway and critical intermediates. EMBO J 25:1295-304. Liebart JC, Ghelardini P, Paolozzi L (1982). Conservative integration of bacteriophage Mu DNA into pBR322 plasmid. Proc Natl Acad Sci USA 79:4362-6. Manninen I, Schulman AH (1993). BARE-1, a copia-like retroelement in barley (Hordeum vulgare L.). Plant Mol Biol 22:829-46.
References
Mariconda S, Namgoong SY, Yoon KH, Jiang H, Harshey RM (2000). Domain III function of Mu transposase analysed by directed placement of subunits within the transpososome. J Biosci 25:347-60. Martin-Rendon E Marfany G, Wilson S, Ferguson DJ, Kingsman, SM, Kingsman AJ (1996). Structural determinants within the subunit protein of Ty1 virus-like particles. Mol Microbiol 22:667-79. Masignani V, Giuliani MM, Tettelin H, Comanducci M, Rappuoli R, Scarlato V (2001). Mu-like Prophage in serogroup B Neisseria meningitidis coding for surfaceexposed antigens. Infect Immun 69:2580-8. May EW, Craig NL (1996). Switching from cut-and-paste to replicative Tn7 transposition. Science 272:401-4. Maxwell A, Craigie R, Mizuuchi K (1987). B protein of bacteriophage Mu is an ATPase that preferentially stimulates intermolecular DNA strand transfer. Proc Natl Acad Sci USA. 84:699-703. McBlane JF, van Gent DC, Ramsden DA, Romeo C, Cuomo CA, Gellert M, Oettinger MA (1995). Cleavage at a V(D)J recombination signal requires only RAG1 and RAG2 proteins and occurs in two steps. Cell 83:387-95. McClintock B (1953) Controlling elements and the gene. Cold Spring Harbor Symp Quant Biol 21:197-216. McClintock B (1987) The discovery and characterization of transposable elements: the collected papers of Barbare McClintock. Garland, New York, USA. Mellor J, Malim MH, Gull K, Tuite MF, McCready S, Dibbayawan T, et al. (1985). Reverse transcriptase activity and Ty RNA are associated with virus-like particles in yeast. Nature 318:583-6. Merkulov GV, Swiderek KM, Brachmann CB, Boeke JD (1996). A critical proteolytic cleavage site near the C terminus of the yeast retrotransposon Ty1 Gag protein. J Virol 70:5548-56. Mhammedi-Alaoui A, Pato M, Gama MJ, Toussaint A. (1994). A new component of bacteriophage Mu replicative transposition machinery: the Escherichia coli ClpX protein. Mol Microbiol 11:1109-16.
Milot E, Belmaaza A, Rassart E, Chartrand P (1994). Association of a host DNA structure with retroviral integration sites in chromosomal DNA. Virology 201:408-12. Mit’kina LN (2003). Transposition as a way of existence: phage Mu. Genetika 39:637-56. Mizuuchi K (1983). In vitro transposition of bacteriophage Mu: a biochemical approach to a novel replication reaction. Cell 35:789-94. Mizuuchi K (1992). Transpositional recombination: Mechanistic insights from studies of Mu and other elements. Annu Rev Biochem 61:1011-51. Mizuuchi K (1997). Polynucleotidyl transfer reactions in site-specific DNA recombination. Genes Cells 2:1-12. Mizuuchi K, Adzuma K (1991). Inversion of the phosphate chirality at the target site of Mu DNA strand transfer: evidence for a one-step transesterification mechanism. Cell 66:12940. Mizuuchi K, Baker TA (2002). Chemical mechanisms for mobilizing DNA. In Craig NL, Craigie R, Gellert M, Lambowitz AM (eds.), Mobile DNA II. American Society for Microbiology, Washington DC, pp. 12-23. Mizuuchi M, Mizuuchi K (1993). Target site selection in transposition of phage Mu. Cold Spring Harb Symp Quant Biol 58:515-23. Mizuuchi M, Mizuuchi K (1989). Efficient Mu transposition requires interaction of transposase with a DNA sequence at the Mu operator: implications for regulation. Cell 58:399-408. Mizuuchi M, Baker TA, Mizuuchi K (1991). DNase protection analysis of the stable synaptic complexes involved in Mu transposition. Proc Natl Acad Sci USA 88:9031-35. Mizuuchi M, Baker TA, Mizuuchi K (1992). Assembly of the active form of the transposaseMu DNA complex: a critical control point in Mu transposition. Cell 70:303-11. Mizuuchi M, Baker TA, Mizuuchi K (1995). Assembly of phage Mu transpososomes: cooperative transitions assisted by protein and DNA scaffolds. Cell 83:375-85. Moore SP, Garfinkel DJ (2000). Correct integration of model substrates by Ty1 integrase. J Virol 74:11522-30.
55
References
Moore SP, Powers M, Garfinkel DJ (1995). Substrate specificity of Ty1 integrase. J Virol 69:4683-92. Morgan JM, Hatfull GF, Casjens S, Hendrix RW (2001). Bacteriophage Mu genome sequence: Analysis and comparison with Mulike prophages in Haemophilus, Neisseria and Deinococcus. J Mol Biol 317:337-59. Naigamwalla DZ, Chaconas G (1997). A new set of Mu DNA transposition intermediates: alternate pathways of target capture preceding strand transfer. EMBO J 16:5227-34. Naigamwalla DZ, Coros CJ, Wu Z, Chaconas G (1998). Mutations in domain III alpha of the Mu transposase: evidence suggesting an active site component which interacts with the Muhost junction. J Mol Biol 282:265-74. Nakai H, Kruklitis R (1995). Disassembly of the bacteriophage Mu transposase for the initiation of Mu DNA replication. J Biol Chem 270:19591-8 Nakai H, Doseeva V, Jones JM (2001). Handoff from recombinase to replisome: insights from transposition. Proc Natl Acad Sci USA 98:8247-54. Nakayama C, Treplow DB, Harshey RM (1987). Structural domains in phage Mu transposase identification of the site-specific DNA-binding domain. Proc Natl Acad Sci USA 80:1809-13. Namgoong SY, Harshey RM (1998). The same two monomers within a MuA tetramer provide the DDE domains for the strand cleavage and strand transfer steps of transposition. EMBO J 17:3775-85. Namgoong SY, Sankaralingam S, Harshey RM (1998b). Altering the DNA-binding specificity of Mu transposase in vitro. Nucleic Acids Res 26:3521-7. Namgoong SY, Jayaram M, Kim K, Harshey RM (1994). DNA-protein cooperativity in the assembly and stabilization of mu strand transfer complex. Relevance of DNA phasing and att site cleavage. J Mol Biol 238:514-27. Namgoong SY, Kim K, Saxena P, Yang JY, Jayaram M, Giedroc DP, Harshey RM (1998a). Mutational analysis of domain II beta of bacteriophage Mu transposase: domains II alpha and II beta belong to different catalytic
56
complementation groups. J Mol Biol 275:22132. Naumann TA, Reznikoff WS (2000). Trans catalysis in Tn5 transposition. Proc Natl Acad Sci USA 97:8944-9. Neumann P, Pozarkova D, Macas J (2003). Highly abundant pea LTR retrotransposon Ogre is constitutively transcribed and partially spliced. Plant Mol Biol 53:399-410. Noma K, Nakajima R, Ohtsubo H, Ohtsubo E (1997). RIRE1, a retrotransposon from wild rice Oryza australiensis. Genes Genet Syst 72:131-40. Palmer KJ, Tichelaar W, Myers N, Burns NR, Butcher SJ, Kingsman AJ, et al. (1997). Cryo-electron microscopy structure of yeast Ty retrotransposon virus-like particles. J Virol 71:6863-8. Pato ML (1994). Central location of the Mu strong gyrase binding site is obligatory for optimal rates of replicative transposition. Proc Natl Acad Sci USA 91:7056-60. Pato ML, Banerjee M (1996). The Mu strong gyrase-binding site promotes efficient synapsis of the prophage termini. Mol Microbiol 22:28392. Pato ML, Banerjee M (1999). Replacement of the bacteriophage Mu strong gyrase site and effect on Mu DNA replication. J Bacteriol 181:5783-9. Pato ML, Howe MM, Higgins NP (1990). A DNA gyrase-binding site at the center of the bacteriophage Mu genome is required for efficient replicative transposition. Proc Natl Acad Sci USA 87:8716-20. Pato ML, Karlok M, Wall C, Higgins NP (1995). Characterization of Mu prophage lacking the central strong gyrase binding site: localization of the block in replication. J Bacteriol 177:5937-42. Pearce SR, Harrison G, Heslop-Harrison PJ, Flavell AJ, Kumar A (1997). Characterization and genomic organization of Ty1-copia group retrotransposons in rye (Secale cereale). Genome 40:617-25. Peterson-Burch BD, Wright DA, Laten HM, Voytas DF (2000). Retroviruses in plants? Trends Genet 16:151-2.
References
Polard P, Chandler M (1995). Bacterial transposases and retroviral integrases. Mol Microbiol 15:13-23. Polard P, Prere MF, Fayet O, Chandler M (1992). Transposase-induced excision and circularization of the bacterial insertion sequence IS911. EMBO J 11:5079-90. Pouteau S, Grandbastien M-A, Boccara M (1994). Microbial elictors of plant defence responses activate transcription of a retrotransposon. Plant J 5:535-42. Pouteau S, Huttner E, Grandbastien MA, Caboche M (1991). Specific expression of the tobacco Tnt1 retrotransposon in protoplasts. EMBO J 10:1911-8 Rao JE, Miller PS, Craig NL (2000). Recognition of triple-helical DNA structures by transposon Tn7. Proc Natl Acad Sci USA 97:3936-41. Reznikoff WS (2002). Tn5 transposition. In Craig, NL, Craigie R, Gellert M, Lambowitz AM (eds.), Mobile DNA II. American Society for Microbiology, Washington DC, pp. 403422. Reznikoff WS (2003). Tn5 as a model for understanding DNA transposition. Mol Microbiol 47:1199-206. Rezsohazy R, Hallet B, Delcour J, Mahillon J (1993). The IS4 family of insertion sequences: evidence for a conserved transposase motif. Mol Microbiol 9:1283-95. Rice P (2005) Visualizing Mu transposition: assembling the puzzle pieces. Genes & Dev 19:773-5. Rice PA, Baker, TA (2001). Comparative architecture of transposase and integrase complexes. Nat Struct Biol 8:302-7. Rice P, Mizuuchi K (1995). Structure of the bacteriophage Mu transposase core: A common structural motif for DNA transposition. Cell 82:209-220. Rice P, Craigie R, Davies DR (1996). Retroviral integrases and their cousins. Curr Opin Struct Biol 6: 76-83. Richardson JM, Dawson A, O’hagan N, Taylor P, Finnegan DJ, Walkinshaw MD (2006). Mechanism of Mos1 transposition:
insights from structural analysis. EMBO J, 25:1324-34. Roldan LA, Baker TA (2001). Differential role of the Mu B protein in phage Mu integration vs. replication: mechanistic insights into two transposition pathways. Mol Microbiol 40:14155. Roth JF (2000). The yeast Ty virus-like particles. Yeast 16:785-95. Royo J, Nass N, Matton DP, Okamoto S, Clarke AE, Newbigin E (1996). A retrotransposon-like sequence linked to the S-locus of Nicotiana alata is expressed in styles in response to touch. Mol Gen Genet 250:180-8. Rousseau P, Normand C, Loot C, Turlan C, Alazard R, Duval-Valentin G, Chandler M (2002). Transposition of IS911. In Craig NL, Craigie R, Gellert M, Lambowitz AM (eds.), Mobile DNA II. American Society for Microbiology, Washington DC, pp. 367-383. Saedler H, Gierl A (1996). Transposable elements. Curr Topics Microbiol Immunol 204. Sakai J, Kleckner N (1997). The Tn10 synaptic complex can capture a target DNA only after transposon excision. Cell 89:205-14. Sandmayer SB, Aye M, Menees T (2002). Ty3, a position specific, gypsy-like element in Saccharomyces cerevisiae. In Craig NL, Craigie R, Gellert M, Lambowitz AM (eds.), Mobile DNA II. American Society for Microbiology, Washington DC, pp. 663-683. Sarnovsky RJ, May EW, Craig NL (1996). The Tn7 transposase is a heteromeric complex in which DNA breakage and joining activities are distributed between different gene products. EMBO J 15:6348-61. Savilahti H, Mizuuchi K. (1996): Mu transpositional recombination: donor DNA cleavage and strand transfer in trans by the Mu transposase. Cell 85:271-280. Savilahti H, Rice PA, Mizuuchi K (1995). The phage Mu transpososome core: DNA requirements for assembly and function. EMBO J 14:4893-903. Schmidt T (1999). LINEs, SINEs and repetitive DNA: non-LTR retrotransposons in plant genomes. Plant Mol Biol 40:903-10.
57
References
Schulman AH, Kalendar R (2005). A movable feast: diverse retrotransposons and their contribution to barley genome dynamics. Cytogenet Genome Res 110:598-605. Schumacher S, Clubb RT, Cai M, Mizuuchi K, Clore GM, Gronenborn AM (1997). Solution structure of the Mu end DNA-binding I beta subdomain of phage Mu transposase: modular DNA recognition by two tethered domains. EMBO J 6:7532-41. Shapiro JA (1979). Molecular model for the transposition and replication of bacteriophage Mu and other transposable elements. Proc Natl Acad Sci USA 76:1933-7. Shcherban’ AB, Vershinin AV (1997). BAREID, a representative of a family of BARElike elements of the barley genome. Genetica 100:231-40. Sherrat DJ (1995). Mobile genetic elements. Oxford University Press, Oxford, UK. Shiba T, Saigo K (1983). Retrovirus-like particles containing RNA homologous to the transposable element copia in Drosophila melanogaster. Nature 302:119-24. Shirasu K, Schulman AH, Lahaye T, SchulzeLefert P (2000). A contiguous 66-kb barley DNA sequence provides evidence for reversible genome expansion. Genome Res 10:908-15. Shockett PE, Schatz DG (1999). DNA hairpin opening mediated by the RAG1 and RAG2 proteins. Mol Cell Biol 19:4159-66. Sokolsky TD, Baker TA. (2003). DNA gyrase requirements distinguish the alternate pathways of Mu transposition. Mol Microbiol 47:397-409. Song SU, Gerasimova, T, Kurkulos M, Boeke JD, Corces VG (1994). An env-like protein encoded by a Drosophila retroelement: evidence that gypsy is an infectious retrovirus. Genes Dev 8:2046-57. Summer EJ, Gonzalez CF, Carlisle T, Mebane LM, Cass AM, Savva CG et al. (2004). Burkholderia cenocepacia phage BcepMu and a family of Mu-like phages encoding potential pathogenesis factors. J Mol Biol 340:49-65. Suoniemi A, Narvanto A, Schulman AH (1996a). The BARE-1 retrotransposon is
58
transcribed in barley from an LTR promoter active in transient assays. Plant Mol Biol 31:295-306. Suoniemi A, Anamthawat-Jonsson K, Arna T Schulman AH (1996b). Retrotransposon BARE-1 is a major, dispersed component of the barley (Hordeum vulgare L.) genome. Plant Mol Biol 30:1321-9. Suoniemi A, Schmidt D, Schulman AH (1997). BARE-1 insertion site preferences and evolutionary conservation of RNA and cDNA processing sites. Genetica 100:219-30. Suoniemi A, Tanskanen J, Schulman AH (1998a). Gypsy-like retrotransposons are widespread in the plant kingdom. Plant J 13:699-705. Suoniemi A, Tanskanen, J, Pentikäinen O, Johnson MS, Schulman AH (1998b). The core domain of retrotransposon integrase in Hordeum: predicted structure and evolution. Mol Biol Evol 15:1135-44. Surette MG, Chaconas G (1991). Stimulation of the Mu DNA strand cleavage and intramolecular strand transfer reactions by the Mu B protein is independent of stable binding of the Mu B protein to DNA. J Biol Chem 266:17306-13. Surette MG, Chaconas G (1992). The Mu transpositional enhancer can function in trans: requirement of the enhancer for synapsis but not strand cleavage. Cell 68:1101-8. Surette MG, Buch SJ, Chaconas G (1987). Transpososomes: stable protein-DNA complexes involved in the in vitro transposition of bacteriophage Mu DNA. Cell 49:253-62. Surette MG, Harkness T, Chaconas G (1991). Stimulation of the Mu A protein-mediated strand cleavage reaction by the Mu B protein, and the requirement of DNA nicking for stable type 1 transpososome formation. In vitro transposition characteristics of mini-Mu plasmids carrying terminal base pair mutations. J Biol Chem 266:3118-24. Surette MG, Lavoie BD, Chaconas G (1989). Action at a distance in Mu DNA transposition: an enhancer-like element is the site of action of supercoiling relief activity by integration host factor (IHF). EMBO J 8:3483-9.
References
Swanstrom R, Wills JW (1997). Synthesis, assembly, and processing of viral proteins. In Coffin, J.F., Hughes S.H and Varmus H.E. (eds.), Retroviruses, Cold Spring Harbor Laboratory Press, New York, USA.
Vicient CM, Kalendar R, AnamthawatJonsson K, Schulman AH (1999a). Structure, functionality, and evolution of the BARE-1 retrotransposon of barley. Genetica 107:5363.
Symonds N, Touissaint A, Van de Putte P, Howe MM (1987). Phage Mu. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
Vicient CM, Suoniemi A, AnamthawatJonsson K, Tanskanen J, Beharav A, Nevo E, Schulman AH (1999b). Retrotransposon BARE-1 and Its Role in Genome Evolution in the Genus Hordeum. Plant Cell 11:1769-84.
Syomin BV, Kandror KV, Semakin AB, Tsuprun VL, Stepanov AS (1993). Presence of the gypsy (MDG4) retrotransposon in extracellular virus-like particles. FEBS Lett, 323:285-8.
Voytas DF, Boeke JD (1993). Yeast retrotransposons and tRNAs. Trends Genet 9:421-7.
Takeda S, Sugimoto K, Kakutani T, Hirochika H (2001). Linear DNA intermediates of the Tto1 retrotransposon in Gag particles accumulated in stressed tobacco and Arabidopsis thaliana. Plant J 28:307-17.
Voytas DF, Boeke J (2002). Ty1 and Ty5 of Saccharomyces cerevisiae. In Craig NL, Craigie R, Gellert M, Lambowitz AM (eds.), Mobile DNA II. American Society for Microbiology, Washington DC, pp. 631-662.
Tavakoli NP, Derbyshire KM (2001). Tipping the balance between replicative and simple transposition. EMBO J 20:2923-30.
Voytas DF, Cummings M, Koniczny A, Ausubel FM, Rodermel SR (1992). Copia-like retrotransposons are ubiquitous among plants. Proc Natl Acad Sci USA 89:7124-8.
Taylor L (1963). Bacteriophage-induced mutation in E. coli. Proc Natl Acad Sci USA 50:1043-51. Tsai CL, Chatterji M, Schatz DG (2003). DNA mismatches and GC-rich motifs target transposition by the RAG1/RAG2 transposase. Nucleic Acids Res 31:6180-90. Turlan C, Chandler M (2000). Playing second fiddle: second-strand processing and liberation of transposable elements from donor DNA. Trends Microbiol 8:268-74. van Luenen HG, Plasterk RH (1994). Target site choice of the related transposable elements Tc1 and Tc3 of Caenorhabditis elegans. Nucleic Acids Res 22:262-9. Vicient CM, Kalendar R, Schulman AH (2001b). Envelope-class retrovirus-like elements are widespread, transcribed and spliced, and insertionally polymorphic in plants. Genome Res 11:2041-9. Vicient CM, Kalendar R, Schulman (2005). Variability, recombination, and mosaic evolution of the barley BARE-1 retrotransposon. J Mol Evol 61:275-91. Vicient CM, Jääskeläinen MJ, Kalendar R, Schulman AH. (2001a). Active retrotransposons are a common feature of grass genomes. Plant Physiol 125:1283-92.
Waddell CS, Craig NL (1988). Tn7 transposition: two transposition pathways directed by five Tn7-encoded genes. Genes Dev 2:137-49. Wang Z, Harshey RM (1994). Crucial role for DNA supercoiling in Mu transposition: a kinetic study. Proc. Natl Acad Sci USA 91:699703. Wang PW, Chu L, Guttman DS (2004). Complete sequence and evolutionary genomic analysis of the Pseudomonas aeruginosa transposable bacteriophage D3112. J Bacteriol 186:400-10. Wang JY, Ling H, Yang W, Craigie R (2001). Structure of a two-domain fragment of HIV-1 integrase: implications for domain organization in the intact protein. EMBO J 20:7333-43. Wang Z, Namgoong SY, Zhang X, Harshey RM (1996). Kinetic and structural probing of the precleavage synaptic complex (type 0) formed during phage Mu transposition. Action of metal ions and reagents specific to singlestranded DNA. J Biol Chem 271:9619-26. Watson MA, Chaconas G (1996). Three-site synapsis during Mu DNA transposition: a critical intermediate preceding engagement of the active site. Cell 85:435-45.
59
References
Waugh R, McLean K, Flavell AJ, Pearce SR, Kumar A, Thomas BB, Powell W (1997). Genetic distribution of BARE-1like retrotransposable elements in the barley genome revealed by sequence-specific amplification polymorphisms (S-SAP). Mol Gen Genet 253:687-94. Weinert TA, Derbyshire KM, Hughson FM, Grindley ND (1984). Replicative and conservative transpositional recombination of insertion sequences. Cold Spring Harb Symp Quant Biol 49:251-60. Wellink J, van Kammen A (1988). Proteases involved in the processing of viral polyproteins. Brief review. Arch Virol 98:1-26. Wessler SR (1996). Turned on by stress. Plant retrotransposons. Curr Biol 6:959-61. Wessler SR, Bureau TE, White SE (1995). LTR-retrotransposons and MITEs: important players in the evolution of plant genomes. Curr Opin Genet Dev 5:814-21. Williams TL, Jackson EL, Carritte A, Baker TA (1999). Organization and dynamics of the Mu transpososome: recombination by communication between two active sites. Genes Dev 13:2725-37. Wright DA, Voytas DF (2002). Athila4 of Arabidopsis and Calypso of soybean define a lineage of endogenous plant retroviruses. Genome Res 12:122-31.
Xiong Y, Eickbush TH (1990). Origin and evolution of retroelements based upon their reverse transcriptase sequences. EMBO J 9:3353-62. Yamauchi M, Baker TA (1998). An ATP-ADP switch in MuB controls progression of the Mu transposition pathway. EMBO J 17:5509-18. Yanagihara K, Mizuuchi K (2002). Mismatchtargeted transposition of Mu: a new strategy to map genetic polymorphism. Proc Natl Acad Sci USA 99:11317-21. Yanagihara K, Mizuuchi K (2003). Progressive structural transitions within Mu transpositional complexes. Mol Cell 11:215-24. Yang JY, Kim K, Jayaram M, Harshey RM (1995). A domain sharing model for active site assembly within the Mu A tetramer during transposition: the enhancer may specify domain contributions. EMBO J 14:2374-84. Erratum in: EMBO J 14:3596. Yang ZN, Mueser TC, Bushman FD, Hyde CC (2000). Crystal structure of an active two-domain derivative of Rous sarcoma virus integrase. J Mol Biol 296:535-48. Yoshioka, K, Honma H, Zushi M, Kondo S, Togashi S, Miyake T, Shiba T (1990). Viruslike particle formation of Drosophila copia through autocatalytic processing. EMBO J 9:535-41.
Wu Z, Chaconas G (1992). Flanking host sequences can exert an inhibitory effect on the cleavage step of the in vitro Mu DNA strand transfer reaction. J Biol Chem 267:9552-8.
Yuan JF, Beniac DR, Chaconas G, Ottensmeyer FP (2005). 3D reconstruction of the Mu transposase and the Type 1 transpososome: a structural framework for Mu DNA transposition. Genes Dev 19:840-52.
Wu Z, Chaconas G (1994). Characterization of a region in phage Mu transposase that is involved in interaction with the Mu B protein. J Biol Chem 269:28829-33.
Zhou L, Mitra R, Atkinson PW, Hickman AB, Dyda F, Craig NL (2004). Transposition of hAT elements links transposable elements and V(D)J recombination. Nature 432:995-1001.
Wu Z, Chaconas G (1995). A novel DNA binding and nuclease activity in domain III of Mu transposase: evidence for a catalytic region involved in donor cleavage. EMBO J 14:3835-43.
Zou AH, Leung PC, Harshey RM (1991). Transposase contacts with mu DNA ends. J Biol Chem 266:20476-82.
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