recombination can be explained in a few discrete steps and these are (figure ... protein-Sae2 which together remove the 5' end on both the strands (on ... 3'overhang which was not part of the strand invasion forms a Holliday junction; thus ... filling through DNA synthesis and finally ligation to produce non-crossover product.
UNIT III: DNA Replication, Recombination and Repair (Sandip Das)
Module 15
3.1 Recombination 3.1.1 Homologous recombination 3.1.2 Non-homologous recombination or Non-homologous end Joining (NHEJ) 3.1.3Synaptonemal complexes 3.1.4 Site-specific recombination
3.2 Recombination repair system
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3.1 Recombination 3.1.1 Double strand-break andHomologous recombination: As the term suggests, homologous recombination or HR refers to a process of exchange of genetic material between “similar or identical” genetic material such as non-sister chromatids in a cell. The process is used to carry out repair of breaks (such as double stranded breaks) in somatic cells, and generate newer combination of alleles in gametes during meiosis in germ cells (meiotic recombination). Prokaryotic organisms (bacteria and viruses) use mechanism of HR to exchange genetic material through horizontal gene transfer. The process of HR is also employed by the cellular machinery to conserve integrity of DNA replication fork, telomere maintenance and segregation of chromosome during meiosis. Within the cell cycle, HR occurs before the cell enters into the mitotic phase i.e. in the S and G2 phases. HR operates mainly through repair of double stranded breaks (DSBs) that are created by ionizing radiations and other mutagens in somatic cells; whereas in germ cells undergoing
meiosis,
DSBs
are
created
enzymatically
by
Spo11.Two
separate
models/pathways have been proposed to explain the mechanism of HR and these are termed as Double stand break repair pathway (DSBR) or Double Holliday junction model,Synthesis dependent pathway (SDSA) and Break-induced Repair (BIR) model. HR pathway is dependent on two recombinases, Rad51 and Dmc1(helped by a number of other accessory factors and proteins/ enzymes)that catalyzes both pairing and recombination events via formation of a pre-synaptic filament (a single stranded filamentous DNA intermediate). The process of homologous recombination can be explained in a few discrete steps and these are (figure 1): i. Formation of a DSB and initiation of HR: Once a DSB has been made, a protein complex termed MRX complex or MRN complex (humans) binds to DNA present on either side of the double stranded break. ii.Resection reaction leading to formation of a single stranded tail with 3’-OH end. Resection reaction refers to removal of the 5’end of a DSB such that overhanging 3’ends are generated. Resection reaction is a two-step process in which MRX/MRN complex recruits another protein-Sae2 which together remove the 5’ end on both the strands (on either side of DSB) to generate short 3’overhangs. Subsequently, another group of enzymes join- Sgs1 (helicase) and two nucleases-
Exo1 and Dna2. While Sgs1 performs its helicase activity and unwinds the dsDNA, the two nucleases, Exo1 and Dna2 remove the ssDNA generated by the unwinding action of Sgs1. iii. a recombination filament is formed at the single stranded 3’ends: The exposed single stranded region is bound or coated by Replication protein A (RPA) and then several other proteins including Rad51 (in mitosis) and Dmc1 (in meiosis) along with RPA form a nucleoprotein filament. iv.extension of the 3’end of the invading strand by DNA polymerase and formation of a D-loop intermediate structure (see module 13): The single stranded filament acts as an invading strand and attempts to locate homologous sequences; once a homologous sequence is identified, the ssDNA filament and the homologous sequence forms a D-loop structure. The homologous sequence in case of mitosis is identical and generally provided by sister chromatid. In meiosis, the homologous sequence is similar, and may not be identical and generally derived from non-sister chromatid. The homologous sequence acts as template for DNA repair v. annealing of the second DSB to the extended D-loop and formation of Holliday junction: Once the D-loop has been established, DNA polymerase extends the 3’ end of the invading DNA filament and converts the D-loop to a Holliday junction vi. generation of cross-over or non-cross-over products and resolution of Holliday junction: Once a Holliday junction has been formed, DNA synthesis continues on the invading strand based on the template provided by the homologous region and eventually restores the entire stretch of damaged DNA, and resolves the Holliday junction
A few initial steps are common between the two models that have been proposed to explain HR- namely DSBR and SDSA model. Double strand-break repair pathway (DSBR pathway):In this model, even the 3’overhang which was not part of the strand invasion forms a Holliday junction; thus two Holliday junctions are formed or what can be termed as double Holliday junction (dHJ). The action of nicking endonucleases which cuts only one strands of a
dsDNA converts Holliday junctions into recombination products or cross-over products.Thesite of recombination activity on the Holliday junction determines the type of products i.e. if both nicks are made on the strands taking part in crossing over, then non-cross over products are formed; in contrast if one cut is made on the crossing strand and the other on a non-crossing strand then a cross-over product is formed.
Synthesis dependent pathway (SDSA pathway): The formation of non-cross over products can be explained by another pathway termed as Synthesis dependent pathway (SDSA). In contrast to DSBR pathway, repair occurs without the formation of two Holliday junctions. Infact, as has been found in yeast, once a D-loop is formed by strand invasion using Rad51 and Rad52, the D-loop is acted upon by a helicase Srs2 which prevents the formation of double Holliday junction formation.DNA polymerase extends the invading DNA strand based on the template provided by the homologous DNA in 5’-3’ direction and thus physically dislocates the D-loop. This process is refered to as a branch migration DNA synthesis.As a result of this the Holliday junction also shifts in the same direction through a process termed as branch migration which displaces the extended strand from template strand. The displaced strand forms a 3’ overhang in the original DSB duplex which can then pair withthe opposite end of the DSB in a complementary manner. This is followed by gap filling through DNA synthesis and finally ligation to produce non-crossover product. SDSA mechanism is often associated with repair during mitosis whereas DSBR is preferred during meiosis leading to generation of cross-over products.
A third model has also been proposed and is termed as break-induced replication (BIR) which is invoked when only one end of DNA is available, for example in telomerase deficient cells during lengthening of telomeres. Such a situation can also occur in a hemizygous state or in haploid cell where no homologous regions are present. In this model, the D-loop intermediate can initiate DNA replication by acting as replication fork (Krejci et al.. 2012). It has been shown that BIR requires all the components and stages needed for DNA replication except for pre-RC (pre-replicative fork) assembly (Lydeardet cl. 2010).
DSBR and SDSA are Rad51 dependent whereas BIR is Rad51 independent but Rad52 dependent.
Table 1: Selected protein components of HR S.No Protein
Function
1
Repair of DSB; homologous to bacterial RecA; interacts with
Rad51
RPA, BRCA2, PALB2 and Rad52 2
3
MRN complex/ DNA binding and nuclease activity ; involved in DNA damage MRX complex
checkpoint and DSB end resection ; functions with Rad51
Rad52
ssDNA binding and annealing activity ; interacts with Rad51 and RPA ; recombination mediator
4
BRCA2
ssDNA binding and annealing activity ; interacts with Rad51 and RPA, Dmc2, PALB2 and DSS1; recombination mediator
5
Rad54
ATP-dependent dsDNAtranslocase and induces superhelical stress ; stimulates D-loop reaction
6
Hop2-Mnd1
Stimulates D-loop reaction ; stabilized pre-synaptic filament and promotes duplex capture ; functions with both Rad51 and Dmc1
3.1.2 Non-homologous recombination or Non-homologous end joining (NHEJ) : NHEJ is involved in a whole range of cellular processes including DNA repair, telomere maintenance and even insertion of repeats, transposon and viruses into host genome. NHEJ initiates after the DNA has undergone a double stranded break (DSB) and again for the sake of our understanding can be divided into three discrete steps. i.
DNA end-binding and bridging:A heterodimeric protein Ku (composed on Ku70 and Ku80), which is part of a DNA-binding component of DNA binding protein kinase (DNA-PK)encircles two full turns of the duplex DNA and forms bridge between the broken ends of the DNA. Ku70/80 forms a basket like structure that binds to DNA via its central domain (beta-barrel domain). This activity of Ku to form the DNA-PK complex serves to provide a scaffold to the DNA, align the broken ends of DNA, prevents degradation of broken ends and promiscuous binding, and finally allows enzymes such as DNA polymerases, nucleases and ligases to act perform end joining. The formation of DNA-PK complex further recruits a serine/threonine protein kinase which is the catalytic subunit of PK and termed as DNA-PKcs (cs for catalytic subunit). DNA-PKcs carry out phosphorylation of several proteins/enzymes that participate in the NHEJ such as DNA ligase IV and XRCC4.
ii.
Terminal end processing:In case the DSB has generated overhangs, these need to be processed such that blunt ends are generated for NHEJ. Polishing of ends can be catalysed by either DNA polymerase as fill-in of overhangs or via nuclease activity of enzymes such as Artemis. The activity of Artemis is dependent on its interaction and binding with the DNA-PKcs complex formed as mentioned earlier.
iii.
Ligation: Once blunt ends have been created by either the action of DNA polymerase or by Artemis (nuclease), the ends are joined by DNA-ligase IVXRCC4 complex.
NHEJ is critical not only for joining of ends as repair process but significantly joining of ends created as a result of Variable (Diversity) Joining Recombination [V(D)J] and Class Switch Recombination (CSR). Therefore individuals lacking NHEJ machinery are severely immune-compromised.
Table 2: list of proteins involved in NHEJ S.No Protein
Function/role
1
Binds to DSB ends; heteromer and composed of Ku70 and
Ku70/80
Ku 80 2
DNA-PKcs
DNA dependent protein kinase-catalytic subunit of nuclear DNA dependent serine/threonine protein kinase; interacts with Ku80, p53, RPA2
3
DNA ligase IV
Encoded by LIG4 in humans; ATP dependent DNA ligase; forms a complex wit XRCC4, and further with DNA-PK
4
XRCC4
X-ray cross-complementing protein 4; interacts with DNA ligase IV; performs the crucial role of bringing and bridging DNA with DNA Ligase IV. XRCC4 is anchored to ends of DNA (in DSB with DNAPKcs and Ku70/80), and is in complex with DNA Ligase IV.
5
XLF/ Cernunnos
XRCC4-like factor; interacts with XRCC4 and Ku70/80; XRCC4-XLF filaments may be involved in forming the crucial bridge between DNA ends of DSB and Ligase during end-joining step
6
Artemis
Encoded
by
DNA
cross-linking
repair
1C
gene
(DCLRE1C); nuclear protein with endonuclease property on single stranded overhangs (both 5’ and 3’) 7
DNA Polymerase λ
Resynthesizes missing nucleotide during NHEJ; also participates in base excision repair (BER) and provides redundant/backup role to DNA pol β; has lyase activity which can be used to remove 5’ deoxyribose phosphate group from ends of strand break; interacts with PCNA; Structures available at 1NZP, 1RZT, 1XSL, 1XSN, 1XSP, 2BCQ, 2BCR, 2BCS, 2BCU, 2BCV, 2GWS, 2JW5, 2PFN, 2PFO, 2PFP, 2PFQ, 3C5F, 3C5G, 3HW8, 3HWT, 3HX0, 3MDA, 3MDC, 3MGH, 3MGI, 3PML, 3PMN, 3PNC, 3UPQ, 3UQ0, 3UQ2, 4FO6
8
DNA polymerase µ
Carried out re-synthesis of damaged DNA; interacts with
Ku70/80 and DNA ligase IV; structurally and functionally related to DNA pol λ; has the unique property of adding nucleotide to a blunt end template by overhang on opposite end of DSB; structure available at 2DUN, 2HTF, 4LZD, 4LZG, 4M04, 4M0A 9
PNKP
Has polynucleotode phosphatase and kinase property (bifunctional); structures available at 2BRF, 2W3O
3.1.3 SynaptonemalComplex: An important structure that is formed between homologous chromosomes that facilitates recombination is the Synaptonemal Complex (SC). Initial understanding of synaptonemal function was that it is responsible for homologous recombination by bringing homologous regions in close proximity; but current understanding reveals a much wider role of synaptonemal complexes that includes number and position of cross-over structures and conversion of these cross-over structures into functional chiasmata. Indeed the current understanding is that SC is not required for genetic recombination but primarily acts as a scaffold to allow interacting chromatids to complete cross-over activities. This view comes after observations that mutants of yeast defective in SC can still exchange genetic material, and that SC is formed after genetic recombination has initiated. Synaptonemal complex is multi-proteinaceous complex that is found at the junction of homologous chromosomes. Formation of Synaptonemal complex during meiosis initiates with DSB and Spo11protein during early prophase. The synaptonemal complex has three major proteins-SYCP1, SYCP2 and SYCP3 or Synaptonemal complex Protein 1, 2 and 3. SC is a zipper-like structure that form a single proteinaceous axis, or axial element or axial core along the two sister chromatids and these axial cores are connected by transverse filaments along the length to produce a complete SC. The axial core / axial filament consist of longitudinal element and two lateral elements and together are also referred to as central element of SC.The single linear axis can also be referred to as Cohesin. Cohesins are composed of proteins such as Smc3 and Rec8. Lateral elements are attached to the chromatin, and along the central element are present “recombination modules” that are believed to facilitate cross-over or recombination. The SC initiates at sites where recombination / cross-over structures are likely to form. The process begins as DSB followed by meiotic recombination repair wherein SC play the role of holding the two partner homologous chromosomes together. DSB is induced by a topoisomerase I like enzyme Spo11 which links to 5’ end of the DNA. Subsequently, Dmc1 protein (equivalent of Rad51) along with Mei5 and Sae3 protein generates Dmc1-filament. This promotes association of Red1 (similar to SYCP3 and SYCP2) and Hop1proteins that are the major components of the lateral element and SYCP1 / Zip1 protein which is the major component of the Central element. Zip1 proteins interact with each other via Nterminal to form the central element, and interact with C-terminal with the lateral elements. The axial or central elements elongate in both directions such that the two homologous
regions are completely synapsed. SYCP2 and SYCP3proteins make up the structural components oflateral elements of the SC. SYCP1 protein is ca. 125 KDa with a characteristic central α-helal domain with globular domains at the N- and C- terminal. The protein gives rise to a coiled-coil structure. Simlalrly, SYCP3 (33 KDa) also has a central α-helical domain and gives rise to a coiled-coil structure. SYCP2 is the longest protein with ca, 1500 amino acids (nearly 175 KDa) and has a short α-helical domain at its N-terminal (Fraune et al. 2012). Thus the SC formation proceeds in a stepwise manner with appearance of DSB followed by individual pairing reactions, assembly of proteins that form central and lateral elements, and finally formation of recombination structures. Assembly of Axial elements begins at Leptotene, the SC complex forms by Zygotene and finally SC dissociates by early Diplotene.
3.1.4 Site-Specific Recombination (SSR): A form of genetic recombination that is dependent on only short stretches of sequence homology, and is catalysed by site-specific recombinases.The process involves binding of enzymes to short regions of sequence homology, cleaving the DNA backbone, exchange of genetic material and finally ligation. This can be catalysed by a single enzyme or by a number of enzymes and generally clubbed as recombinases. Some well-known examples of such enzyme are Cre from Phage P1, FLP recombinases from yeast, and Int from Phage lambda.The sites that mediate SSR in phages and bacteria are termed as attachment (att) sites. We will illustrate the SSR mechanism using the example of integration of phage into bacterial genome. The two genomes (phage and bacteria) share a short stretch of sequence termed as core sequence (O). In bacterial the core sequence is flanked on either side by B and B’ to form BOB’ to form attB; in phage, the O (=core) is flanked by P and P’ to form POP’and resides in attP. The recombination reaction between E.coli and the phage is a reversible
reaction
termed
as
integration
and
excision.
Integration
requires
Integrase(product of Int) and Integration host factor (IHF); Excision in addition requires another enzyme Xis, apart from Integraseand IHB.Two models have been proposed to explain the mechanism of recombination between bacterial and phage genome- i) whether the two genomes undergo simultaneous cleavage followed by exchange (concerted exchange); or ii) exchange occurring between the two strands as one-at-a-time with the first strand forming a Holliday junction and the second cycle of nicking and ligation completing the exchange (sequential exchange). What is known that the attB and attP sites undergo staggered cleavage such that the ends generated are compatible / complimentary thus facilitating ligation / re-union reaction.The generation of staggered ends follows essentially a pathway that is similar to DSB to generate recombinants. Integraseuse a strategy used by Topoisomerase Iand creates a break in one strand at a time, manipulates the ends and ligates the compatible ends. One molecule each of Integrase is used to catalyse the reaction on each of the four strands of homologous pair. Before we understand the reaction mechanism, let us look at the recombinases. There are two categories of site-specific recombinases based on the amino acid sequence and these are – tyrosinerecombinase family and serine recombinase family.The members of these two families use tyrosine and serine, respectively to carry out the nucleophilic attack on the target DNA and for covalent linkage with DNA. Integrase, Cre, and FLPare examples of Tyrosine recombinasewhereas gamma-delta resolvase, Tn3 resolvases are examples of serine recombinases. The catalytic site amino acids (tyrosine, for example) may be provided by the enzyme subunit that is bound on the DNA undergoing cleavage (ciscleavage) as in Intor by the subunit bound on the DNA not taking part in cleavage –reunion cycle (trans cleavage) as in FLP. Apart from Int (Integrase), the phage-bacteria site-specific recombination requires IHF protein which is composed of two different subunits. IHF is required for both integration and excision reaction and binds to a sequence that is adjacent to Int binding region on attP. Xisis additionally required for excision reaction and together the three proteins-Int, IHF and Xis cover the entire attP. The binding of Int and IHF on attP creates a structure such that all the binding sites aggregate on the surface of this protein-complex and is termed as Intasome. This Intasome is used to interact with the bacterial sequence-attB. The recombinases recognize the sites of site-specific recombination; two units of enzyme bind to each duplex DNA (i.e. a total of four units of enzymes binding on four strands). This is followed by synapsis and each duplex is cleaved only on one strand. The
cleavage generates 6-8 bp staggered ends. This synaptic complex is the site for strand exchange which proceeds via a Holliday junction like intermediate structure. The process catalysed by Tyrosine recombinasesis thus sequential exchangeofbreakage-reunion. In contrast, Serine recombinases cleave both the strands of the homologous region simultaneously and generate 2-bp overlap and thus follow concerted exchange.
3.2 Recombination repair system Recombination repair system can summarized as a collection of various strategies that the cellular machinery uses to repair its genetic material-DNA. Damage to DNA can occur due to various reasons including chemical, environmental and due to other endogenous reasons. Some such examples of DNA damaging agents are UV- rays, X-rays, free radical, Reactive oxygen species, chemical mutagens, viruses, toxins etc. DNA damage can be exemplified by thymine dimers, depurination, alkylation and oxidation of bases, loss of a base, tautomeric shift etc. Often the cellular system uses recombination-repair based strategy and as these occur after replication, they are also referred to as post-replication repair. Errors that have occurred as a result of replication are repaired by Mismatch repair system. Translesion synthesis (TLS) allows DNA replication machinery to bypass structural hindrances such as thymine dimers. Pyrimidine dimers are repaired using photo-reactivation mechanism using Photolyase enzyme; single strand damage is repaired via base-excision repair using sequential action of DNA glycolyase, AP endonuclease,DNA polymerase and DNA ligase.Nucleotide excision repair (including transcription coupled NER) similarly is used to repair helix distorting damages such as pyrimidine dimers, 6-4 photoproducts. In contrast to errors or damages that have occurred on one strands, in case both strands of DNA are damaged such as during double stranded breaks (DSB), homologous recombination and Non-homologous end joining (NHEJ) mechanisms are employed. Most of these pathways have been elaborated elsewhere, and therefore are not explained again (see module 16).
