knowiedge. Albert Einstein ...... W. Dond, W. M. Gallatin, T. St. Jobn, H. T. Smith, V. A. Fried nnd I. L. ... St. John, T., W. M. Gallatin, M. Siegelman, 11. T. Smith ...
ISOLATION OF HUMAN DNA REPAIR GENES BASEDON NUCLEOTIDE SEQUENCE CONSERVATION
Marcel Koken
ISOLATION OF HUMAN DNA REPAIR GENES BASED ON NUCLEOTIDE SEQUENCE CONSERVATION
ISOLATIE VAN HUMANE DNA HERSTEL GENEN OP BASIS VAN NUCLEOTIDE SEQUENTIE CONSERVATIE
PROEFSCHRIFT
TER VERKRIJGING VAN DE GRAAD VAN DOCTOR AAN DE ERASMUS UNIVERSITEIT ROTTERDAM OP GEZAG VAN DE RECTOR MAGNIFICUS PROF. DR. P. W. C. AKKERMANS, M.A. EN VOLGENS BESLUIT VAN HET COLLEGE VOOR PROMOTIES
DE OPENBARE VERDEDIGING ZAL PLAATSVINDEN OP WOENSDAG 23 OKTOBER 1996 OM 13 UUR 45
DOOR MARCEL HUBERT MARIE KOKEN GEBOREN TE KERKRADE
PROMOTIE·COMMIssm PROMOTOREN:
Prof. dr. D. Bootsma Prof. dr. J.H.J. Hoeijmakers
OVERIGE LEDEN:
Prof. dr. J.A. Grootegoed Prof. dr. ir. P. van de Putte Dr. E.C. Zwarthoff
Dit proefschrift werd bewerkt binnen de vakgroep Celbiologie en Genetica van de Faculteit der Geneeskunde en Gezondheidswetenschappen van de Erasmus Universiteit Rotterdam. De vakgroep
maakt deel uit van het Medisch Genetisch Centrum Zuid-West Nederland. Het onderzoek en deze uitgave werden financieel gesteund door de Nederlandse Kankerbestrijding - Koningin Wilhelmina
Fonds.
/~
Gedrukt door: Drukkerij Haveka B.V.• Alblasserdam
Omslag: Lcu Wouters
Dans Ie champ de l' expérimentation Ie hasard ne favorise que l' esprit préparé.
Louis Pasteur
Imagination is more important than knowiedge. Albert Einstein
Voor mijn moeder die nooit ophield
mij te stimuleren en te helpen, en voor Manon die voortdurend probeerde dit boekje (letterlijk) teniet te doen.
CONTENTS Chapter I. 8
Aim of the Thesis General Introduction & Conclusions
DNA repair: -Generalintroduction to DNA repair -Cloned human DNA excision [epair genes Ubiquitin:
8
9
-Cloning methods for DNA repair genes
11
-General introduction to ubiquitin -Ubiquitin conjugation pathway
IS IS
-Involvement ofubiquitin in ceHular processes
16
-A choice for or against degradation: linkage types
17
-Ubiquitin genes & Fusion proteins
17
-Ubiquitin-like proteins
18
-Ubiquitin-specific proteases
19
-Ubiquitin in protein degradation -Ubiquitin degradation signaIs:
20
N-rule system and RAD6
20
Ubiquitin Fusion Degradation
25
"2nd Codon rule" and Destruction box
25
-Ubiquitin in anti-proteolysis, protein structure and folding
26
-DNA repair and ubiquitination
27
-HHR23B, DOA4 and p53
27
-RAD6 mutant, gene and protein
28
-RAD6 and histones
29
-RAD6 targets
32
References
34
Chapter H.
51
The rhp6+ gene of Schizosacclzaromyces pombe: a structural aod functional homologue of the RAD6 gene from the distantly related yeast Saccharolllyces cerevÎsÎae. (1990) EMBO J. 9: 1423-1430.
Chapter Hl. Dhr6, a Drosophila homologue of the yeast DNA repair gene RAD6. (1991) Proc.Natl.Acad.Sci. USA 88: 3832-3836.
60
ChapterIV. Structural and functional conservation of two human homologues of the yeast DNA repair gene RAD6. (1991) Proc.Nafl.Acad.Sci.USA 88: 8865-8869.
66
ChapterV.
72
Localization of two human homologues. HHR6A and HHR6B, of the yeast DNA repair gene RAD6 to cbromasomes Xq24-25 and 5q23-31. (1991) Gel/omics 12: 447-453.
ChapterVI.
80
Expression of the human ubiquitin-conjugating DNA repair enzymes HHR6A and 6B suggests a role in spermatogenesis and cbromatin modification. (1996)
Dev.Biol. 173: 119-132.
Chapter VII.
95
Inactivation of the HR6B ubiquitin-conjugating DNA repair enzyme in miee causes male sterility associated with cbromatin modification. (1996) eell 86: 799-810.
Summary
108
Samenvatting
111
Curriculum vitae List of PublicatIons Nawoord
117
115 121
I I I I I I
I I I I I I
I I I I I I
AlM Thc aim of thc work described in this thesis was thc development (and subsequent application) of a genera! method for the isolation of human (DNA repair) genes using probes from already cloned homologous counterparts in athef organisms. After many initial problems developing and optimising this method, it was used for thc isolation of two groups of genes: thc Drosoplzila melallogaster l • SchizosaccJzarol1lyces pombe (unpublished) and Saccharomyces cerevisiae 2 homologues of the TFIIH component XPBCIERCC3, and an Ss.pombe, a Drosophila and two human homologues of the yeast post-replication repair gene RAD6. Only the characterisation of the RAD6 homologous genes and polypeptides,
implicated in the ubiquitin pathway, will be described in this thesis, Chapters II to VII. Publications related to the other part of the work conducted in the context of this PhD thesis can be found in the list of publications at page 117
GENERAL INTRODUCTION & CONCLUSIONS General inlroduclion 10 DNA rep air For all organisms it is of vital importance to secure reliability of genetic information. The gen ome of the cell is constanUy under attack by a plethora of DNA damaging agents (e.g. the UV component of the sunlight). Therefore all living beings had to develop effieient systems to recognise and rernove DNA injury. Lesions in DNA -if unrepaired- have inunediate deleterious effects on transcription and replication, or after fixation into permanent mutations, they cao change the coding potentialof genes. This last feature imp lies also that correct removal of lesions from the DNA is of utmost importance for the prevention of cancer or congenital aberratiolls in higher organisms. Since numerous genotoxic compounds exist, each of which can induce a wide spectrum of lesions, it is not surprisillg that most organisms acquired a network of partially overlapping repair pathways to recognise and remove these different adducts from their DNA. In the bacterium Esclzericlzia eaU several of the biochemical pathways leadillg to the elimination of DNA damage are rather well understood. The work described in this thesis concerns mainly two major repaÎr mechanisms: post-replication repair (PRR) and nucleotide excision repair
(NER) (For reviews on both subjects see3'IO). Post-replication repair, a poorly understood error-prone system, is thought to pennit the replication machinery to bypass lesions in the DNA strands. This, may occur either via mutagenic trans-iesioll DNA synthesis or by rein1tiation of DNA replication behind the lesiou, in which case the single-stranded gap
-opposite of the damage- is filled in using the newly synthesised complementary daughter strand as template. In both models the lesioll is not removed but only tolerated, which implies that - if not repaired - it still eau cause mutations. Therefore, PRR, which is also known as "daughter-strand gap repair" has to be considered mainly as a damage-tolerance process. As in contrast to the NER pathway, only very liWe is known about the molecular mechanism of PRR, this part of the introduction wiII essentially focus on the DNA excision repair system. Nucleotide excision repair in E.co/i is a process in which a minimum of 6 proteins participates in the elimination of a wide range of structurally diverse DNA lesions (For
8
review see ll ). A complex consisting of two molecules UvrA and one molecule UvrB is thought to scan the DNA for loc al distortions caused by the damage. After tracing DNA injury, the UvrA2B complex unwinds partially the DNA around the lesion l2 . The two UvrA
proteins leave the complex, and UvrB attaches more tightly inducing astrong eonformational change in the double helix around the damage. The bound UvrB molecule and the frozen DNA structure serve as tag for a tbird polypeptide, UvrC. The UvrBC complex makes incisions in the damaged strand; UvrC at the eighth phosphodiester bond 5', and it is unknown whether UvrC, UvrB or both are responsible for cleaving at the fifth phosphodiester bond 3' of the lesion l3 . A second DNA-helicase, UvrD, subsequently removes the damaged part from the DNA backbone, and DNA polymerase and ligase fill in and close the gap. The E.coli system is relatively simple when compared to the process in eukaryotes, where in yeast and man already more than fifteen different NER genes are identified, and in part cloned (For details on the different genes and cloning methods, see below). Many of the cloned human NER genes were isolated by DNA transfection of normal genomic or cDNA
into repair-deficient mutant eells. These were either laboratory-derived rodent cells or eell lînes from patients with one of several rare repair disorders. Individuals with the autosomal reeessive disease xeroderma pigmentosum (XP), characterised by hypersensitivity to sunlight (UV), pigmentation abnormalities and a high incidence of skin tumours in the sun-exposed areas, led Cleaver in 1968 to think that this illness could be a DNA repair disorder (For review see I4). Cell fusion experiments showed th at the XP (as well as above mentioned rodent mutant) cell lines could be divided into at least eight (and eleven) complementation
groups. Seven of the XP groups are severely disturbed in the incision step of the nucleotide excision repair pathway, whereas cells of the eighth group (the variants) have problems in
post-replication repair l5 . These XP variants were of special interest as the second part of this thesis describes the isolation of homologous RAD6 genes which are implicated in postreplication repair. Since the deseription of XP as a NER disorder in the late seventies, many reports appeared in the literature associating diverse diseases with one of the repair pathways. Although most of these links are still rather uncertain, some well-documented genetic instability syndromes have been found. Two of them, Cockayne's syndrome (CS)6,16 and trichothiodystrophy (TTD)17, were shown to represent different forms of the XP-NER
syndrome (see below), which, however, unlike XP and other putative DNA repair disorders as Bloomls syndrome 18 , Faneoni's anaemia 19 ,20, or ataxia telangieetasia21 -25 , were not found to be associated with a high cancer incidence (For an extensive discus sion on this subject, see IO,26).
Cloned human DNA excision repair genes A short description of the DNA excision repair genes isolated thus far, mostly by DNA
transfections to above-mentioned mutant celllines, is given below to illustrate the high level of evolutionary conservation which exists in the DNA excision repair pathway. The first human NER gene isolated, ERCCI, was cloned27 via the correction of the UVsensitive, DNA excision repair-deficient, Chinese hamster mutant eell lines of rodent complementation group 1 (For review see28 ). The 32 kD protein is homologous to the yeast
9
NER protein RAD 10, and shares at its C-terminus additional regions of similarity with parts of the prokaryotie E.coli UvrA and UvrC polypeptides29 . Recently ERCCI was shown to be complexed with the correcting activities of ERCC4, ERCCII and XP-F celllines 30,3l. The recent cloning of ERCC4 32 .33 showed it to be partially homologous to yeast RAD!. Moreover, the same gene corrects by DNA transfeetion or microneedie injection the repair defect of the ERCC4, ERCCI! and XPF mutants, and several causative mutations from these cell lines have been characterised. In analogy with the situation in yeast, where RAD 10 interacts with RADI34, ERCCI forms a tight complex with ERCC4 and induces an endonucleolytic cleavage at the transition of a single-stranded to a double-stranded DNA region, only in the strand carrying the 3' single stranded end 33 ,35,36. This is consistent with the idea that this complex is implicated in making the 5' incision of the NER process. ERCC5 37 , isolated by transfections of CHO complementation group 5 cells, is apparently identieal to the XPG correcting factor which was cloned, in a way by accident, using a systemic lupus erythematosus autoimmune serum38 . Thc gene was also isolated using PCR amplification with degenerated primers designed from the homologOlIs Schizosaccharoll/yces pOli/be RAD2 and RAD13 genes39 ,40. The cDNA encodes an acidic helix-loop-helix protein partially resembling the yeast RAD2 protein. In analogy with RAD2, and the related FEN-I (implicated in the joining of Okasaki fragments), XPG may display structure-specific ss-endonuclease activity4l.43 which, like ERCC!/ERCC4, might be required for the incision step of NER. ERCC644 , which corrects CHO complementation group 6, was shown to be affected in Cockayne's syndrome (CS) patients of CS-complementation group B. CS patients are characterised by a small stature, wizened appearance, sUllwsellsitivity, and often mental and physical retardation, but no elevated risk for callcers (For review, see4 5). The proteill, of which the yeast homologue (RAD26) has recently been isolated46, represents a putative DNA helicase implicated in preferential repair l6,47. This process couples DNA excision repair to transcription, assufÎllg the preferential reparatioll of the coding DNA strand in transcriptionally active genes 48A9 , Thc XPA gene isolated after tedious transfeetions of mause DNA inta XPA cells 5o , encodes a Zn 2+finger protein which is likely to be directly involved in recognition of thymidine dimers by the excisian repair system. Shawn ta be very weIl canserved during evolution 5l , it was not unexpected that the yeast RADI4 NER protein was found to be its yeast homologue 52 . XPC was cloned twiee. A partial cDNA was isolated by DNA transfection of an XPC cellline with a cDNA library cloned into an extrachromosomally replicating EBNA-vector5 3. The encaded hydraphilic pratein is related in its C-terminal region ta, but not necessarily the homologue of, yeast RAD4 54 . Two years later Masutani et al. cloned the gene again 55 . Using an in vitro repair assay, they isalated a protein fraction whieh complemented XPC eell extracts. After determination of the N-terminal amino acid sequence of the two proteins. p 125 and p58, present in the correcting fraction, now a fulliength XPC cDNA was isolated. The second protein (p58), necessary for complete correction and forming a complex with XPC, appeared to be an ancient ubiquitin-fusion protein, HHR23B (see below). A homologue of the
10
protein, HHR23A was also reported, but apparently not involved in the XPC-HHR23B protein complex55 ERCC2 56, and in ref.57 and ERCe3 58. isolated by corrcction of rodent complemcntation group 2 and 3 mutants, appear to be involved in xeroderma pigmentosum, complementation groups D (ERCC2) and B (ERCC3). Mutations in both genes are underlying also two other hereditary diseases, Trichothiodystrophy (ITD) and Cockayne's syndrome, whieh eo-oecm also in some of the XPD patients (in ref.'7), and in the three XPB (2xCS, IxITD) patients described to date (ref. 59 and uupublished data). A substantial fraction of TTD patients, comprising tlrree complementation groups, display a repair-defective phenotype 17 , They were originally described as having a problem with their sulphur metabolism, leadiug to sulphurdeficient brittie hair (For review seo6o). Both ERCC2 and ERCC3 (as weil as ERCC6) are members of a recently defined group of DNAIRNA helicases, as they share seven consecutive amino acid domains characterising this famil y61,62, ERCC2 was shown to be the human homologue of yeast RAD3. We and others demonstrated that ERCC3 is also weil conserved in evolution 1,2,63, 64 1eading to the isolation of a thus far unknown yeast mutant, RAD2S 2, In view of receut data this conservation is not surprising as ERCC2 (RAD3), ERCC3 (SSL2/RAD25), SSLI, TFBI, 2 and 3, CCL! aud KIN28, together with one or more as yet uncharacterised proteins, were shown to constitute the general basal transcript ion factor
TFIIH 65 ,66 (For reviews seo67.6"). Apparently, these proteins (and perhaps also ERCC6) have all a primary task in transcription llext to their repair functions 7o . The protein complex can explain why mutations in different proteins give very similar diseases (XPB and D), as a mutated component deregulates apparently the total complex. Moreover, it can also explain
how, perhaps depending on the type ofmutation, different illnesses like XP, CS and ITD can originate lO,71,n. As almost all constituents of the general transeription maehinery are very well conserved in the course of evolution 73 , it is also probably that this eould be a general phenomenon for DNA repair enzymes as the listing above may indicate. The work described in this thesis was initiated, when only ERCCI was isolated, to prove the overall eonservation of repair genes and to use thls conservation for the isolation of additional human DNA repair genes.
Cloning methods
rOl'
DNA repair genes
The classical method for the isolation of marmnalian repair genes (explained in detail in ref. 27 ) uses the transfeetion of normal hunmn or mouse gel10mie DNA into Chinese hamster or human mutant eeH lines, respectively. After selection of clones resistant to the DNA damaging agent, genomic DNA is isolated for a consecutive round of transfection which reduces considerably the amount of co-incorporated irrelevant human or mause sequences. Finally, the correcting human (or mouse) DNA is isolated from the hamster (or hu man) background by standard molecular biological techniques. Although this method has important
pitfalls (e.g. the amollnt and length of intact genomie DNA whieh is stably taken lip by the transfected cells 74), it has thus far sllceessfully been used for the isolation of most of the DNA excision repair genes, i.e. ERCC], ERCC21XPD, ERCC3IXPB, ERCC4, ERCC5IXPG, ERCC6/CSB ,XPA and XPG. 11
A second methad - aften tried but thus far not very fruitful in the repair field, probably due ta the low expression levels of repair proteins - consists in purification of the correcting proteins, after whlch the correspollding genes have to be isolated by molecular biological
techniques. In this approach micro-injection of proteill extracts combined with the Unscheduled DNA Synthesis assay75 or lil vitro repair systems are the essential screening methods 55 ,76-78, This approach has led thus far to the isolation of two human repair genes: tbe XPC gene, isolated as part of a complex with HHR23B by Masutani et al. (see also above), and the DNA ligase I gene, which was previously thought to be implicated in the repair disorder Bloom's syndrome79 ,80. However, recent evidence contradicted this finding but showed that the gene is disturbed in a cellline derived from a unique patient, 46BR 81 - 83 ,
Moreover, recently the real Blaam's syndrome gene was isolated and appears to exhibit homology with the RecQ helicases, a subfamily of DExH box-containing DNA/RNA helicases 18 Finally, as in the yeasts SaccJzaromyces cerevisiae (baker's yeast)84 and ScJzizosaccharom)'ces pombe85 a large number of DNA repair mutants had been isolated, it would be very convenient to utilise the many correcting yeast genes c10ned for the isolation of human counterparts. Also in view of the limited number of human and hamster repair mutants (and the tedious transfection experiments) it was tempting to try to develop as first part of this thesis a general method for isolation of homologues of genes already cloned from other species. From the lessons learned in the course of this work it becarne obvious that, when trying to isolate similar genes in other organisms, it is essential to take the, evolutionary direction which is followed into account. Descending the evolutionary ladder (e.g. from man to Drosophila) via low stringency hybridisations using standardised methods86 is relatively easy, as the isolation starts from a complex genome (3x10 6kb) with large introns and thus many possibilities for accidental homology to the relatively simple genome of the fly (IxlOSkb). However, when cloning a human homologue of a yeast gene (genome size: !x104kb) the whole complexity of the human genome is encountered. Therefore, the cloning strategy had to be adapted several times, otherwise many small regions with fortuitous homology would have been isolated. The small protein domain shown in Fig.1 (Ec09), for instance, was isolated by screening DrosopJzila cDNA libraries with the total yeast RADl gene.
r
Ad. 2 minor coat protein V
K E
D.lllelal/ogaster cDNA Ec09
Region of nucleotide identity between Ec09 and RAD!:
S.cerevisiae RAD!
15' GGG AAG GAC GAC GAC GAT 3'1
Figure 1. Representation of the dOll13inal homology found between a Drosophila cDNA clone (Ec09), RADi, and the adellovirus minor coat protein V.
12
The nucleotide sequence identity between Ec09 and RAD1 did not exceed 18 base pairs. However, on the protein level a more extended similarity existed between these proteins. Remarkably t the same domain is also found in adenovirus 2 minor care protein V. Unfortunately, no il1dication exists for the function of thls protein region. As we show here, small domainal homology cao be very illustrative and sometimes lead ta identification of protein sequence motifs. However, with the exception of several rare examples where it is the only conserved part of a gene, like for instanee in the case of same of the important. but lowly conserved interleukin genes 87-89 • sueh short sequences hardly ever represent a homologous counterpart. Ta avoid isolation and sequencing of mauy clones with fortuitous sequence homology, hereafter two flanking cDNA probes were used. In thls "junction probe" strategy (see Fig. 2) it is assumed that only homology spread over a long area of DNA is of interest. When an extended area of nucleotide sequence similarity exists in the organism under investigation, and the genomic DNA is digested witlt several different restriction enzymes, a reasonabIe chance exists that the 5' as weil as the 3' cDNA probes hybridise to the same genomic fragment; the junction fragment. It should be noted that instead of 5' and 3' probes also genes from distantly related species, for instance S.cerevisiae RAD6 and Ss.pombe rhp6+ (as done in Chapter H), can be used, at least if the two genes do not display large areas of nucleotide sequence conservation.
5' 3' ~--------IJ--
- -
Flanking cDNA probes
~ of species Y
t I t + I _.t+__I:C?::::Io_!,-...t==::::r. __.I'_I:CDIo..c._.!I.....tC:Ioo_,-__=t:::::t.'I.._"';' 5'
3'
}
Homologous
genOlntc gene of species X
Hybridising fragments
Junction fragment
Figure 2. Junction Probe Principle. Open boxes in the genomic gene represent exons and the small arrows indicate restriction enzyme sites. The large arrow points to the junction fragment recagnised by bath the 5' and 3' probes. However, also thls methad is not a 100% guarantee for the isolation of the correct gene. Again, in the case of the Ec09 gene, for instanee, it appeared that the above described domail1 was repeated twice in a partial cDNA. Moreover, when digestl-: of phage lambda clones harbouring the genomic Eco9 gene were hybridised with the conserved nucleotide stretch, the domain appeared to be repeated at least five times in the Ec09 gene. A gene with intel1lal repetitions like Ec09 can easily lead to the isolation of sequences with fortuitous homology, if
each of the junction probes contains one of the repeated domains, as tltis eliminates the advantage of the junction probes. Also under other circumstances repetition can lead to 13
problems. In the case of the RAD6 polypeptide, described below, the yeast protein contains a stretch of acidic amino acids. As thc codons for these amino acids are rather unifonn (GAg, QAa, GAt and GAc) repeated structures appear readily, leading in th is approach to the isolation of onc of the numerous proteins harbouring acid ie regions90 • Therefore, it was always tricd to avoid thc presence of repeated areas in thc junction probes. (Each cloning attempt should be preceded by a self~cornparison of thc genes and proteins. a hybridisation of the 5' and 3' probes with each ather, as well as an extensive computer-library screen to determine whether common or repeated motifs/protein regions are cncoded by the probes). When we developed the method it also appeared that accidentally cloned plasmid contaminatiolls present in many lambda cDNA or genomlc libraries show up by thc low stringency hybridisations. due to thc vector-DNA contamination of the gene probes used. This problcm we circumvented by the use of PCR-generated probes or plasmid free libraries. To avoid a large part of the accidental homology one could arguc not to use genomic DNA libraries for the approach but cDNA libraries. The expression levels of the repair genes isolated thus far, however, are so low that large and high complexity librarics are needed. Moreover, because a gene is not by definition conserved over its entire length, astrong need for full-length clones is obvious. Taken these facts together, genomic libraries are normally preferabie over cDNA libraries when studying gene conservation in this way. However, it should be noted that especially in the higher eukaryotes the sometimes extremely large genes and distantly located small exons still may oblige to the use of cDNA libraries. Although the above described procedure has many advantages over classic al hybridisation methods, it is only applicable if several relatively extended regions of homology exist. When similarity between DNAs is toa smalt or only found in a single small region of a gene, other methads have to be applied, which all but one have in common that brute sequencing force has to be used, as na early eonclusive indications exist that the correct gene has been isolated. (In our method a double positivity with 5' and 3' probes presents good evidence for the correct gene). A quick and easy method was recently described as "computer cloning"91. An optimal computer search should certainly precede every attempt to clone homologous counterparts of known genes. In contrast, two brute force methods are, for instance, the enrichment for small regions of high homology using RecA protein 92 combined with low stringency hybridisations for the isolation of small domainal homology, or PCR appIications with degenerated primers for the cloning of lowly conserved genes. In these cases it is worthwhile to consider other methods based on protein-sequence similarity, which is normally much higher than nucleotide homology. Antibody screening of bacterial protein expression libraries (e.g. Àgtl1), is very dependent on specific high affinity antisera to prevent cross-reaetions. The method has been used for homolagy searches (far instanee, RecA), but thus far not very successfully. Finally, also functional cloning, relatively quick and easy, but not yet really widely used, should perhaps be tried. The human CDC2 and CDC34 genes (see belaw), for instanee, were isolated by transfection of a human eDNA library driven by an SV40-promoter93 into a Ss.pombe cdc2ts 94 or a S.cerevisiae meel mutant9 5, respectively. Several of the photoreactivating enzyme homologues were isalated by correcting a phrwdefective E.coli mutant96 , and rer. therein and the isolation of yeast 14
topoisomerase II with Drosophila topoll was feasible with the sectoring/selection-method described by Kranz et 01. 97 • In our case the junction-probe strategy resulted in the isolation of two groups of genes; the Dl'osophila l , Ss.polI/be (unpublished) and S.cel'evisiae 2 hamalagues of ERCC3, and the Ss.polI/be 9s , Drosophila99 and humanloo hamalagues of RAD6/UBC2. These latter genes are the subject of this thesis, and as RAD6 has been shawn ta play a role in the nbiqnitin pathway, this system wiII be reviewed in the following part of the introduction,
General illiroductiall 10 ubiquitill "Ubiquitin is too small and too abundant to be important; you should change your research subject!" (Tald ta A. Haas abaut eleven years aga). To state this about ubiquitin, one of the proteins most conserved in evolution, is nowadays impossible in view of the plethora of processes in which this "giant dwarf' plays a major role, Because it is impossible to cover the ubiquitin field within the Iimits of this introduction, the major topics wiII be highlighted, especially thase in relatian with DNA repair (For a bevy of recent reviews see lOl - lJ I), The -6x 107 malecnles of this 76 amina acid protein faund in each cell of aur body make it one of the most abundant polypeptides 106 , Ubiquitin has been detected in a wide variety of organisms ranging from archaebacteriae Il2-114 to man, and rccently even in a eubacterium, the cyanobactcrium Anabaella variabilis 115, Considered as the slowest evol\'ing proteÎIl known 116, it allowed in the 1.2 billion years of evolution which separate yeast from man only three amino acid changes to occur l14 ,
Ubiquitill cOlljugatioll patbway In a cell the majority of the ubiquitin molecules are not found as free protein, but conjugated to other polypeptides lO6 , The linkage reaction and the proteins performing it, are apparently almast as canserved as ubiquitin itselfIOO.117.121(Fig.3). Conjugation commences when the C-tenninal glycine residue of a ubiquitin molecule is activated by one of the ubiquitin-activating cnzymes (referred to as Uba or El), which uses ATP to form a high energy thiol ester intermediate, that is covalently lillked to an internal cysteine residue of the EI-molecule 120, This protein-complcx is able to donate the 76 amino acids proteill to ane of a growing family of ubiquitin-conjugating enzymes (Ubc or E2)(e.g. RAD6/UBC2). The E2-ubiquitin complex links the ubiquitill maiety via its C-tennillal glycine residue to the e-NH2 group of a lysine residue in the target protein, with or without the help of a member of a family of ubiquitin ligases (Ubr or E3). The question whether (all) the E3-proteins are only docking proteins or bind ubiquitin to themselves, and thus actually perform an enzymatic activity, rcmains to be resolved l22 , Originally, ubiquitination was shown to be involved in specific (extra-lysosomal) targeted degradation of the bulk of mislacalised, improperly processed, fareign or damaged 15
proteins 106, as weil as of undamaged polypeptides whieh are naturolly short-lived l23 or which have to be maturated. The ubiquitination finally leads to ATP-dependent degradation of the targeted proteins by the complex multicatalytic 26S protease or proteasome, and release of free or branched ubiquitin (sec below), which can be re-utilised. The proteasome is the major extralysosomal proteolytic system known. Present in the cytoplaSlll as weIl as in the nucleus, it is involved in both ubiquitin dependent and independent 124 degradation (por reviews on thls issue not dealt with in this introduction, see 107 ,11O,125-131). Note, however, that a180 several links of the ubiquitin system with the lysosomal system (or the yeast vacuole) have been established, and th at degradation of tagged proteins (especially of the ubiquitinated surface receptors) is therefore not obligatorily executed by the proteasome 132 -144 • Moreover, a protein can be degraded by different collaborating degradation systClnsI45.147. [Thc enormous literature conceming ubiquitill 148 (and ubiquitin-system proteins l49 ) as marker for autoimmune and neuro-degenerative diseases is not considered in this review, as at present it is unknown whether the antibodies or changes in ubiquitin expression-Ievels are cause or consequence of the disease. For reviews, see 150-156].
-
........ ~ Degradation
AlP
- -.... ~ Stabilisation Figure 3_ The ubiquitîn conjugation pathway (simplified).
Involvement ofubiquitin in cellular processes Thc role of ubiquitin in degradation implicates the sm all protein in a deluge of regulatory processes within the cell, inc1uding regulation of geile expressioll via (limited) degradation or posttranslational processing (NFkBI57, p53158.159, c_fos l60, c_jun I61 , c_mos I62 164, c_myc/c_fosI60,165), eell cyc/e eontroI166-171. DNA repairl72.173. recombillatiOll170.174, ligand-indlleed degradatioll of eell smface reeeptorsl75.176, cel/ular stress respoJlse142.177.178, alltige1l, processi1lg mld preselltation179-18l, apoptosisl82-184, s)'naptic eOllIleetivity l85-188, perhaps subcellular compartme1ltalizatÎoJl (import in mitochondria, uptake in synaptosomes, peroxisome biogcnesis)174,189,190, and indirect indications exist even for aIl implication in nucleoside transport l91 . Besides its degrading function the polypeptide is also illvolved in assuring correct protein synthcsis and protein conformatioIl, as it has been purported to be directly concerned 16
in the (re?)folding of (damaged?) proteins. The protein also seems to have anti-degrading functions, maybe due ta its lnvolvement in folding, as in certain circumstances ubiquitin protects against breakdown (see bel ow).
A choice fol' or against degradation: Iinkage types As far as \Vltat is known, the choice for or against degradation of ubiquitinated targets depends on two facts. First, whether the polypeptide is mono- or poly-ubiquitinated, and second, where in the target protein-backbone ubiquitin is attached. It was found that proteins ean contaill either single ubiquitin molecules (mono-ubiquitination) or tree structures of branched ubiquitin (poly-ubiquitination). These Iatter structures, whose formation is often dependent on the presence of an E3 enzyme, consist of ubiquitin molecules which are linked via their C-terminal glycine to specific intemallysine residues of another ubiquitin molecule. n is generally assumed that at least the lysine 48 (KA8) poly-ubiquitination leads to breakdown l92 ,193. The existence of K-6, K-11, K-29 and K-63 poly-ubiquitination has also recently been describedl73,194-196. And altllOugh RAD6 can make K-6 tree stmctmes on histone H2B in the absence of an E3 protein, the function of this linkage type remains unknown l96 . The human E2 enzyme EPF149.197 is making K-ll linkages, which like K-29 and K-48 poly-ubiquitination, are involved in protein breakdown 198 . Finally K-63 polyubiquitination, performed by RAD6 in an E3-dependent mannef, is apparently a poor iuduccr of degradation. This conjugation-type has recently been implicated in DNA repair, perhaps with a regulatory function. A yeast mutant whieh is unable to perform the K-63 linkage shows a phenotype which in part is comparable to that of a rad6 deletion mutaIlt 173 (see below). Mono-ubiquitination is llonnally not involved in breakdown but in the stabilising/folding functions (see below), although, in the case of the artificially-made ubiquitin-proline-p-galactosidase 199 , and in the case of o:_globin2OO,201 1t may be sufficient for degradation. In broader tcrms, all these results suggest that ubiquitin is a versatile signal in which different ubiquitin chain configurations are used for different funetions. A single ubiquitin conjugating enzyme is apparently able to perform different linkages (for RAD6: K6, K-48 and K-63) dependent on the target and the E3 involved l96 .
Ubiquitin genes & Fusion proteins When the first ubiquitin genes were cloned, it appeared that all organisms harboured many functional copies as weIl as many pseudogenes 202 . Moreover, always at least one of the genes was a poly-ubiquitin gene, harboudng a highly varia bie number of ubiquitin coding elements in a head-to-tail arrangement, and thus encodillg a poly-ubiquitin precursor protein [e.g. 3 to 9 copies (man)203,204, 14 (Arabidopsis)205, 11 (Caellol'habditis), 2 to more then 40 (trypanosomatidae), 18 (Dl'osophila), 7 (maize), 6 (sunflower), 5 (yeast)106]. The polyubiquitin genes are in general inducible in the stress response (e.g. heat shock)178,206, in contrast to the mono-ubiquitin gen es. The mono-ubiquitin gelles are often fusion genes as they encode the ubiquitin moiety in frame with a C-temlÎnal extension peptide (CEP)207. The CEPs were found to represent two types of small very conserved ribosomal polypeptides, implicating ubiquitin in ribosome biosynthesis; the CEP80 proteins (with a variabie length of
17
76, 80 or 81 amino acid residues) are found to be identical to ribosomal protein S27a which is part of the 40S particle, and the ribosomal CEP52 proteins whieh represent the IAO peptide, a constituent of the 60S or large ribosomal subunit20S-211. This fundamental finding led to quite a few publications deseribing the same phenomenon in other organisms [CEP52: Chlamydomollas 212 , Arabidopsis213 , Tetrahymella (CEP53)214, Dictyostelium21S-217, Drosophila 21S , ehieken219 , and man220 , CEP80: Neurospora(78)221 , Dietyostetium (78)217, maize(79)222, Arabidopsis(80)213, and Drosophila223 ], illustrating the evolutionaty eonservation of the C-terminal fusion partners.
Ubiquitin-like proteins The search for C-terminal extcnsion protcins led furthermore to the discovery of ncw
types of rlbosomal fusion proteins, like for instanee the Nicotiana tabacum CEP72 protein224 whieh is related to the CEP52 proteins. In addition the family of ubiquitin-like sequenees (Uhl) was expanded as several authors in their search for ubiquitin fusions, identified ubiquitin-like proteins with C-tenninal extensions22S-228 or proteins with a C-terminal (I) ubiquitin-like extension("NEPs")229. The family ofubi-like protein sequenees is eonstanUy growing (Tabie I). Table I: Ubiquitin-like proteins, Ubl-CEP fnsjQus:
1. Caenorhabditis CEP93225 2. Rat ribosomal protein S30 (also known as Fau protein or lymphokine MNSp226-228) NEP· Ubl fnsfons: 3. Tbc Ub! moiety fused to the C-terminal end of mammalian splicing factor SF3a120 and its yeast
homologue, PRP21 p229 Normal UbJs!
4. Xenopus Ania and b proteins fused to a Zn2+-finger protein 230 5. 15kD interferon-induced ISG15 gene product UCRP23 1-235
6. NEDD8 protein236.237 7 8. 9. 10. 11. 12. 13. 14.
Chinese hamster (and mouse) CHUB2 gene238 Earthwonn EiselliafetMa Andrei Ub1 239 X-chromosomal GdX protein 240 BAT3 polypeptide241 Baculoviral v-ubi protein242 DNA excision repair proteins HHR23A and B55 Positive regulator subunit pl8 of the sm general transcriptionfelongation factor 243 A whole group ofnon-expressed Ubl-pseudogenes in Arabidopsis 205
The idea that these Ub!'s ean replaee normal ubiquitin in its funetions, is thus far only founded on the detaHed analysis of UCRP. This di-ubiquitin-like protein is weakly homologous to normal ubiquitin and was shown to be conjugated to cellular proteins in vivo244 . The ubiquitin-like proteins in Table I should therefore probably be divided into two different classes. A first group of funetional "weil" eonserved (especially the C-tenninal glycine residues) ubiquitin-like proteins whieh have similar funetions as classical ubiquitin but are involved in parallel pathways (UCRP, NEDD8, Ubl-Fau, Ubl-CEP93, SF3aI20, v-ubi 18
and although less likely, Ubl-Anla and b). And a second class of ancient "normal ubiquitin"fusion proteins which lost the cleavage site between ubiquitin and the CRterminal extellsion (BAT3, GdX, sm pI8, CHUB2, sm pl8 and HHR23). As because of this most of the evolutionary pressure on the ubiquitin moiety was lost, the coding sequence slowly challged. The "stabiHsing function" (see below), however, was probably retained, and is apparently absoJutely required for correct function of the fused partner243 . With the identification of the ubiquitin-like molecules the complexity of the system is increasing even further. If narmal ubiquitin is already impIlcated in the plethora of processes specified in this introduction, \Vhat wiJl be the function of these ubiquitin homologues and why did they evolve? What wiII be the function ofrecently cloned EI-like proteins245.248 or of the different virus-encoded proteins: the ubiquitin(-like) proteins of baculovirus (vubi 242 ,249,250), bovine viral diarrhoea virus25 1,252, and Finkel-Biskis-Reilly murine sarcoma virus 226 ,227, and the E2 protein UBCvl (related to RADG) of African swine fever virus253-255? Do these vlruscs use the ubiquitin system for their benefit in a simllar way as Human Papilloma Virus 16 and 18, whose E6 protein interacts with a ceUular E3 protein, EG-AP, and forces it to recognise p53, leading to the poly-ubiquitination and degradation of th is antioncogene I59 ,17I,256,257? Or do they try to escape the attacks by the ceU's degradation systems by titrating the cellular ubiqnitin with non-conjugatable ubiquitin homologues or by mirnickillg their proper ubiquitination with their own E2's249?
Ubiquitiu speel/ic proteases Although ubiquitin carboxyl-terminal hydrolases or ubiquitin specific protcases (UBP's)
\Vere known ta exist. and ta be implicated in the production of mono-ubiquitin from the polyubiquitin precursors, the isolation and cloning of these hydrolases was also accelerated due ta the identification of the ubiquitin-CEP fusions. Same of them were shown ta remove smalt peptides or single amino acids from the Cterminal end of ubiquitin258 ,259, and ta be necessary for the maturation of the last ubiquitin moiety of a poly-ubiquitin protein. This last ubiquitin-copy of a poly-ubiquitin gene often contains some additional C-tenninal amino acids, probably to prevent the non-branched polyubiquitin molecules from partic1pating in the linkage reaetions. A second class is implicated in the production of single ubiquitin~moieties from the poly~ubiquitin precursors, or in the maturation of the C~terminal fusion proteins260 ,26l, Finally, the third group of ubiquitin Iyases, to which the human oncogene product Tre-2 or its yeast homologues DOA4 262 or UBP5 263 belong, releases andlor degrades poly-ubiquitin trees. Proteins from the last group are implicated in the rescue of faulty-targetted proteins or to recuperate free ubiquitin for reutilization after degradation of the tagged proteins264-266, Note, however, that degradation of tree-structures is not absolutely necessary as they also can be re-used directly, Moreover, free tree-structures can made by certain E2 enzymes independent of the presence of a target protein267-269,
With the identification of the UBPs the description of the ubiquitin system components is complete: single ubiquitin can be made from the fusion or poly~ubiquitin gene products; EI, E2 and E3 proteins can do their work; the proteasome degrades the targetted proteins; and finally the poly-ubiquitin trees can be recuperated to yield again free ubiquitin. 19
In the next paragraphs the implications of ubiquitin in degradation and in antiproteolysis, protein structure and folding wil! be briefly discussed. The chapter finishes with a summary of our CUlTent knowledge on the role of ubiquitin in DNA repair and chromatin structure, which is of course obligatorily linked to one of the E2-enzymes, RAD6.
Ubiquitin in protein degradation AH living cells have to reglIlate the content and composition of their resident proteins, but the mechanisms by which this is done are not weIl known. Intracellular protein degradation is important in determining steady state and fluctuations of protein concentrations as weIl as for the generation of protein fragments that act as hormones, antigens, or other effectors. Breakdown can be regulated by innate properties of the protein substrate (e.g. PEST270_ or KFERQ271-sequences), or by chemical modifications (e.g. ubiquitin) which mark them for breakdown, in other words which confer metabolic instability. The initial event leading to degradation mayor may not involve I) proteolysis, 2) non-proteolytic (covalent) modifications (e.g.oxidation of methionines, ubiquitin conjugation, AANDENYALAAtagging 272 [i.e. A COOH-terminal peptide-sequence, thus far only detected in E.coli, which is linked to a protein while it is being translated from an erroneous mRNA which does not encode a stopcodon. This tagged incomplete protein is subsequently degraded by tag-specilic proteases. The process involves a new RNA type (with both a transfer and messenger function (tmRNA)) and a switch ofthe translation machinery from the defective mRNA to the tmRNA. It represents a magnificent quality control mechanism for defective.mRNAs], 3) denaturation or unfolding of the protein, or 4) sequestration in cytoplasmic or nuclear "organelles". These processes, however, have to be selective as an enormous heterogeneity in degradation rates exists for the different proteins in the cell. Ubiquitination is one of the ways to achieve such a selectivity. Although the number of natural degradation-targets of the ubiquitin machinery273 starts to grow (see Table Il, pg. 21), the issue of what determines the specificity of the ubiquitin ligation system i.e. the degradation signals for commltment of certain proteins to degradation is not yet resolved.
Ubiquitin degradation signals The only general prerequisite for degradation of a protein via ubiquitination is the obligatory presence of a lysine residue to whose E-amino group the ubiquitin moiety will be finally attached31O . The other additional structural features of a substrate which are recognised by the ligation system are for the moment not weIl known, and rather non-uniform 31O (see below). To complicate the situation it even appeared that proteills which do not contain any degradatioll-sigllals themselves can be degraded by their interaction with other polypeptides or subunits which only serve as (undegradable) tag for the ubiquitin machinery (transrecognition) 199,280,286,311 .
N-rule system and RAD6 Varshavsky and co-workers identified the first of the ubiquitin-system degradationsignais; the presence of a free alpha-alllÎllo group (For extensive reviews and detailed explanations, seeI99.312). 20
T.ble IJ: Identified natm'al substr.tes of the ubiguitin-degradation system. 1. Plant photoreceptor chromoprotein: phytochrome274 2. Bovine photoreceptor G protein transducin 275 3. Sindbis virus RNA polymerase Uil vitro)276 4. Encephalomyocarditis Virus-3e Protease277 ,278 5. c-mos proto-oncogene product162.164 6. c-jull prota-oncogene product 161 7. c..Jos proto-oncogene product 160 8. c-cbl proto-oncogene product279 9. p53158.l65,257,280 10. N-myc, c-myc. c10s and E1A product (ill vitro)165 11. 12. 13. 14, 15. 16.
pl05-NF-KB (activation and processing via partial degradation) 157 NP-KB inhibitor 11
rad6L1
UbK63R
DNA rcvair nnd mutagenesis Sensitivity to:
+++ UV,4NQO Crosstinking agents (eg.8MOP + UV) +++ +++ Alkylating agents (eg.MMS) Xly-irradiation +++
Mutagenesis:
Spontaneous lnduced by damaging agents
Excision of dimers Post-repHcation repair Recombination Mitotic (spontaneous/induced) Meiotic Retrotrallsposition of Tv elements
Cell Growth Cell eyele Growth rate Sporulation -N-rulc degradation pathway
elevated 363
deficient nonnal
deficient* increased defective increased363-365
+++ ncl +++ wt \Vt deficicnt
ncl ncl nd nd nd
S-phase prolonged \Vt almost wt defective \Vt defective \Vt slow
*Defcct in reappcarance of high molecular weight DNA after replication of damagcd templates wt=wild type levels, nd= not done
RAD6 ond bistones While we \Vere executing our initial cloning attempts in Ss.pambe, Jentsch and coworkers cloned the already known S.cerevisiae RAD6 gene in their seareh for the ubiquitinconjugating enzymes of this fungus. The protein was shown to add ill vi/ra specifically a single ubiquitin-moiety to the C-terminallysine-119 of histone H2A or lysine-120 of histone H2B 172,375, but not to several other highly basic control proteins. This ubiquitin-conjugation activity was shown to be necessary for all RAD6 functions kllown, because a mutatioll of the ubiquitin-acceptor cysteine residue into a valine, alanine or sefÏlle residue leads to a RAD6deficient phenotype376.317 (see Table IV). In the presenee of the yeast E3 ubiquitin-ligase, VBRI, with whieh RAD6 interacts through its highly eonserved N-tenl1inus (Chapter IV), the histones can in vi/ra even be poly-ubiquitinated. This shows that RAD6, like for instanee CDC3495.166 (an E2 protein implieated in GI-S phase eell eycle transition) is a bifunetional 29
enzyme competent in both E3-independent and E3-dependent conjugation reactions l95 (see for
tbis bifunctionality also I97 ). This poly-ubiquitination of histones is dependent on the acidic tai! of yeast RAD6. Therefore sporulation, which is tai!-dependent, needs apparently polyubiquitination. whereas DNA repair and mutagenesis involve only mono-ubiqitination.
AIthough these ideas are generally accepted, the function of histone mono- and polyubiquitination by RAD6 in vivo and its implication in DNA repair remain a subject of debate.
--- Eukaryotic DNA is organised innucleosomes: a stretch of -146 base pairs of DNA is wound around a histone octamer which consists of two subunits of histones H2A( 14kD),
H2B(l4kD), H3(l5.3kD) and H4(l1.2kD) [(H2A:H2B12H32H42)]. The nucleosomes are connected by 50-100 base pair stretches of DNA to which, in (higher413 ) eukaryotes, a molecule of histone Hl(22kD) binds which stabilises the higher order chromatin stmcture resulting in the compact "30nm" fibers. The degree of local packing has to be tightly regulated, as it has been shown that the chromatin is highly condensed in regions containing quiescent genes and more accessible in regions of transcriptional activity. It is now generally
admitted that this regulation probably takes place through a variety of non-permanent posttranslational modifications; methylation, acetylation, phosphorylation, poly(ADP)ribosylation and ubiquitination of the flexible N- or C-terminal domains of the different nucleosome components. However, although extensive, often contradictory, literature exists on this subject, no really clear relationship between aspecific modification and its implication in transcription, replication, DNA repair, or spermiogenesis has been demonstrated, with the exception of lysine-acetylation and phosphorylatioll. (por an extensive review on the subject of histones and their modifications, see378 .)
Acetylation is found to arfect 5-10% of the N-terminal flexible domains of the core histones. These core histones are mainly present in transcriptionally active regions of the chromatin. Acetylation is thought to neutralise the net positive charge of the basic histone proteïns, and in that way it would contribute to opening up the chromatin.
Serine/threonine-phosphorylation of histones Hl and H3 is thought to counter-act acetylation thus favouring chromatin-condensation. Hl is moderately phosphorylated duriug S phase, but throughout 02 phosphorylation increases to reach a hyperphosphorylated state of all Hls at metaphase. Immediately upon nuclear division Hls are dephosphorylated to Sphase levels. Just before metaphase bistone H3 is also phosphorylated. Histones can be methylated irreversibly on lysine residues, a modification of which the
function is not known at present 379 . Poly(ADP)ribosylation is thought to cause loc al chromatin decondensation and is almost exclusively found upon illtroduction of DNA strand
breaks, and thus probably important for DNA repair380,381. FinaIly,
mono~ubiquitination
of the C-tenninal flexible domains of histones was shown
to occur principallyon histones H2A and H2B. 5-15% ofhistones H2A in higher eukaryotes alld .....2% of H2B are mono~ubiquitinated in vivo. Note, however, that these percentages vary enormously from cell to cell and organism to organism382.383. Ubiquitination is supposed to open up the chromatin, as it introduces a major structural perturbation due to the size of the 76 amillo acids proteill. However, na such structural changes are detected at present (by for
instance DNAse I footprinting)384.385. During the cell cycle uH2A and uH2B are present throughout S-phase and 02-phase up to prophase. From prophase to metaphase histones are 30
deubiquitinated, but immediately re~ubiquitinated in anaphase. The modification is important as for insta nee in the El~ts mutant eeIl Hne, ts85, it was shown th at with reduced
ubiquitination cells arrest close to the S/G2 boundary of the cell cycle, accompanied by a loss of uH2A. Mono-ubiquitinated histones are very stabIe and ubiquitin is thus apparently not involved in breakdown of these molecules. Some reports show an association of especially
uH2B with active DNA sequences387-391(and a higher affinity of uH2A for AT-rich DNA386), which is contradicted by others392.393. Thus although mono~ubiquitinated histones exist, and although they seem important, their precise function is still completely unknown. ~--
As outlined above RAD6 is able to mono- and poly-ubiquitinate histones ;11 vitro. However, the implication of RAD6 in the ubiquitination of histones in vivo remains a point
of debate, as may become clear from the following arguments. First, it seems thus far impossible to detect ubiquitinated histones in the yeast S.cerevisiae. This orgallism apparelltly contains very few, if any, ubiquitinated histones (less than the detection limit of 0.1 % of all histones). The C-terminal amino acids of H2A (and H2B), which in man harbour the unique ubiquitin-attachment site (Lysine-1l9/120), are very weIl conserved in evolution and shown to be essential for yeast viability. Swerdlow and coworkers wanted to test whether this same lysine residue is also used for ubiquitination in yeast. Therefore, the two normal H2A histone genes were replaced by a gene copy mutated in the (for yeast putative) ubiquitination site. This caused no detectable phenotypic change in growth (solidlliquid medium, different temperatmes, heat-killing, osmotic killing, use of alternative carbon sources), sporulation and 254nm UV radiation sensitivity383. Therefore, S.cerevisiae which contains mainly uncondensed/active chromatin, does either not need ubiquitination or ubiquitinates only a very smal1, undetectable, proportion of its histones at another site in the molecule. Second, histones are a general target protein used for in vi/ra testing of ubiquitinconjugating enzymes and many of these enzymes are able to add, mostly without high specificity, a single or multiple ubiquitin moieties to these basic molecules 166 ,254,375,394-398.
In the case of rabbit E214kD, the rabbit homologue of yeast RAD6, Haas et al. have shown that ill vitra this protein can weakly mono-ubiquitinate histones, but reaction kinetics and constants let these authors to consider the reaction as a~specific in an ill vivo situation375 . No specific poly-ubiquitination of histones can be detected with the rabbit protein375.397. It was however shown that poly-ubiquitination of histones can be performed by the yeast protein in vitro, and that it is dependent on the acidic tail 350. Without "acidic tail" yeast RAD6 can only mono-ubiquitinate histones. So the tail is important for poly-ubiquitination of histones and sporulation, but can be missed for mono-ubiquitination of histones (with
questionable specificity!) and thus for DNA repair and DNA mutagenesis. However, the Caenarhabditis elegans (which contains also an acidic tail sequence) and the Arabidopsis RAD6 homologues are apparently completely unable to ubiquitinate histones H2A or H2B ;1/ vitro 399 ,4oo. So, taken these results together, the proposition that histones are iJl vivo targets for the RAD6 or its homologues is unlikely. It is more plausible that RAD6, like many other E2's, ubiquitinates histones with low affinity and that these are not its real targets. The
phenomenon of histone-poly-ubiquitination by RAD6 alone (without E3-protein), an activity 31
which is only displayed by the yeast protein, probably represents "an artefact" as the acid ie tail could cause a higher but a-specific affinity of RAD6 for the basic histones. RAD6 targets What are the real targets for RAD6, especially in DNA repair and during late spermatogenesis? Histones would be ideal targets due to thcir requirement for DNA repair, meiosis or more broadly spennatogenesis. However, due to above-mentioncd results it seems at least unlikely that histones present one of the RAD6 targets in a normal cello Whether they are targetted in special ceH types, like for instanee spermatids, in thc prescnce of a specif1c E3 protein (which might target RAD6 or increase its affinity) remains to be established. (Preliminary experlments studying ubiquitination in mHR6B knockout mice could indicate that histones in spermatids might still be a target for HHR6B (W.M. Baarends & H. Roest, pers. comm.)). Also the interaction of RAD6 with the ssDNA-binding protein RAD 18 is in favaur of a function of RAD6 in close contact with the DNA402,403. The Prakash group provided indications that RADI8 can transport RAD6 to the DNA, where the protein then could perforrn its function. They al80 showed that the interaction site bctwecn RAD6 and RAD 18 is well conserved in evolution because the interaction of yeast RAD 18 is also possible with Ss.polI/be rhp6+ and human HHR6A aud 6B proteins402 . This is rather remarkable as the RAD18 protein, in contrast to RAD6, is not very weIl conserved in evolution40I . Since only very short regions of amino acid sequence similarity exist, it is possible that the interaction surface between the two proteins is probably not a linear sequence but a three dimensional one. However, althaugh the rad6 phenotype and these last findings still favour a ftiuction of RAD6 nearby the DNA, the only three "reai" RAD6 targets identified thus far (all recognised with the help of aspecific E3_protein 405 ) are not strictly DNA-assaciated: I) RAD6 is the E2 protein involved in degradation of N-rule targets 322 ,323 (see abave). It interacts specifically via its very conserved N~terminus (see Chapter 4) with the yeast VBR I-encoded E3 protein 322 .406. In reticulocyte Iysates the reaction is independent of the presence of its acidic tail 323 . However, in vivo, in yeast, the acidic tail is important for N~rule degradation407 .408 Yeast RAD6 has apparently three ways of recognising targets: alone, tail dependent, unassisted~C-terminus independent, and E3-a'ssisted C~terminus independent408 [. The tirst mode of action is apparently lost in higher eukaryotes, as the tail is absent from these homologues (see Chapters IT, III and N)]. Thus far the only N-mle target protein for which it has been proven that it involves RAD6, is GPA I, the ex subunit of a yeast G protein implicated in pheromone~dependent signal transduction286 . 2) Gcn4 is a yeast transcriptiollal activator of the bZIP family involved in regulation of the biosynthesis of amino acids and purines. lts normal rapid degradation, dependent upon CDC34 and RAD6, is inhibited under starvation conditions 284 • 3) The p27 mammalian cell cyc1e protein is an inhibitor of cyc1in-dependent kinases. Bath iJl vivo and iJl vitro, the protein is degraded by the ubiquitin-proteasome pathway. The human ubiquitin~col1jugatil1g enzymes RAD6 and UBC3 were specifically iuvolved in its ubiquitination 168. It is for the moment unknown whether Gcn4 or p27 are N-rule targets. 32
Note that measurement of reaction kinetics on total retieuloeyte proteins favour the
implication of RAD6/E2-14K in E3-assisted poly-ubiquitination, although monoubiquitination was also observed but relatively non-specific 375 . This poly-ubiquitination, however, does not always seem to occur via the normal1ysine-48 of ubiquitin (which is used
by RAD6 and UBRI in the N-rule degradation pathway)'95. Recently, it was shown that RAD6 is also capable of forming K-6 (to histones H2B in the absence of an E3 protein) and K-63 (made by RAD6 in an E3-dependent manner) linkages, bath apparently not involved in degradation l96 . The K-63 poly-ubiquitination of RAD6 is the most interesting for DNA repair. Haas and co-workers eonstrueted a yeast in which they replaeed the normal four ubiquitin genes by a ubiquitin mutant encoding an arginine instead of lysine-63 (strain
UbK63R)173, whieh prevents the addition of other ubiquitin moieties at that position of the molecule. The strain grows at wild type levels (see Table III) and degrades a set of short-Iived N-rule proteins. However, the strain which is shown to be epistatie with a rad6L1 mutant is highly sensitive to DNA damaging agents and has a deficiency in DNA damage-induced mutagenesis. Also the most abundant, but rather restricted family of multiubiquitin-protein
conjugates found in wild type S.cerevisiae cells is completely absent. Sa, apparently, this mutant ubiquitin is involved in the DNA mutagenesis pathway. The rad6 phenotype is much more pleiotropic, which suggests that for sporulatioll, resistance to y-rays and N-nlle
degradation, the RAD6 protein uses another type of lysine linkage or mono-ubiquitinates its targets. In the RAD6MUbK63R double mutant a relative high UV resistance is observed when compared to the single md6L1 mutant which indieates that UbK63R is a partial suppressor of RAD6. This suggests that the K-63 ubiquitination persists in the absence of RAD6, and that other ubiquitin conjugating proteins (making K-63 linkages) partieipate in other RAD6-independent repair pathways which work more effieiently in the absence of K-63. So, in conclusion, as already indicated above, all the data suggest that ubiquitin is a very versatiIe signal, as different ubiquitin ehain configurations can be used to perform different funetions. A single ubiquitin conjugating enzyme, like for instanee RAD6, is able to
perform different linkages (Le. K-6, K-48 and K-63) dependent on the type of target/process and the E3 protein involved. The type of linkage as weil as tlle E2 and E3 protein involved, determilles whether a protein can be degraded, deactivated, correctly folded, proteeted against degradation, stabilised, ... leading to a complex pleiotropie phenotype and implicatiolls in many different functions .. .
Ubiquitin: small, but very powerful! In the next chapters the isolation and characterisation of RAD6-homologous genes and proteins is described from the distantly related yeast Schizosaccharom)'ces pombe98 , the fly Drosophila melanogastel'99 and a duplicated locus from man 1OO.409,41O. (Note that tlle HHR6B gene was isolated three times independentl y lOO.117.191.411.) In the course of thls work, also RAD6 proteins from Arabidopsis thaliaIla and wheat 374 .400, Caenorhabditis elegalls 399 , rat 373
33
and rabbit 404 were isolated; the most important data of these articles have been included in this introduction.
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49
Chapter IJ The rhp6+ geile of Schizosaccharomyces pombe: a structural alld functiollal homologue of the RAD6 gene from the distalltly related yeast Saccharomyces cerevisiae.
Ths EMBO Journal vol.9 nO.5 pp.1423-1430, 1990
The rhp6+ gene of Schizosaccharomyces pombe: a structural and functional homolog of the RAD6 gene from the distantly related yeast Saccharomyces cerevisiae P.Reynolds, M.H.M.Koken', J.H.J.Hoeijmakers 1 , S.Prakash2 and L.Prakash Th;:partment of Biophysics, University of Rochester School of MeJicine, Rochester, NY 14&-12, USA, lTh;:partment ofCell Diology and Gene/ks, ErasffiUS University, PO Box 1738, 3000 Dr Rotterdam, The Nelherlands and 2Th;:partment of Biology, Uniwrsity of Rochestcr, Rh'er Campus Station, Rochester, NY 14627. USA Communicated by D.BootslTL1
n.e RAD6 gene of Saccharom)'ces cere~';siae encodes a ubiquitin conjugating enyzme and is re(lulred for DNA repair, DNA-damage-illduced mutagenesis and sporuJation. Here, we show that RAD6 and the rhp~ gene from the distalltly related yeast Schizosaccharolll)'ces pOli/be share a high degree of slrueluraJ and fllllctiollal homoJogy. The predomillantly aeidic carboxyl-terminal 21 amÎllo acids present in the RAD6 protein are absent in the rhp6+ -ellcoded proteln; otherwise, the two proteins are ycry simiJar, nith 77% ldentleal residlles. Ukc rad6, null llIutations of the rhp6+ gene confer a defect in DNA repair, UV mutagellesis and sporulatlon, and the RAD6 and rhp6+ gelles can functionally subslÎlute for one another. These obseryatiollS suggest that functional illteractiollS between RAD6 (rhp6+) protein and othel' components of the DNA rep.'lir complex ha\'c been COJlserYCd amollg eukaryotes.
words: DNA repair/E2 enzyme/RAD6 gene/r1lp~ gene/Schizosacclwromyces pOli/be
Ke)'
Introduction The RAD6 gene of Sacclmromyces cerevisiae is involved in a variety of cellular processes. rad6 mutants are highly scnsiti\'e 10 numerous DNA danlaging agents, including UV, -y-rays and alkylating agents (Cox and Parry, 1968; Game and Mortimer, 1974; Prakash, 1974) and are defectÎ\'e in mutation induction by these agents (Prnkash, 1974; Lawrence and Christensen, 1976; McKee and Lawrence, 1979). rad6 nlUtants are defeclive in post-repIîcalion rcpair of UV damage: DNA strand discontinuities left during DNA repIîcation in the newly synthesized DNA strand across from the non-coding UV lesion remain unrepaircd in rad6 mutants (Prakash, 1981). rad6 mutanls are also defeclh'c in sporulation (Game et al., 1980; Montelone et al., 1981), and they grow poorly and have pOOl' plating eftlciency. The RAD6-encoded protein (M r 19.7 kd) possesses a highly acidic carboxyl lerminus in which 20 of the 23 residues are acid ie (Reynolds et al" 1985). The polyacidic sequence of RAD6 protein forms a disordered Iinear structure that is appended to the globular domain constiluted by the first 149 residues (Morrisoll et al" 1988). RAD6 protein is a ubiquitin..çonjugating enzyme (E2) (Jentsch el al., 1987) that mediates the attachment of multiple
molecules of ubiquitin to hislones H2A and H2B ill l'itro (Sung el al" 1988), Multiple ubiquitination ofhistones may eftèct all open chromatin configurntioll, or it may mark histones for degradation by the ATP-dependent proteolytic system (Hershko el al., 1984a,b; Hershko and Ciechanover, 1986). The acidic domain of RAD6 is required for the multiple ubiquitination ofhislones (Sung el al., 1988). rad6 mutants hearing a deletion of the acidic sequence fail to sporulate, but the DNA repair and UV mutagenesis functiolls are not aftèctcd (MorrÎson el al" 1988). Mutation ofthe sole cysteine residue (eys-88) in RAD6 to alanine or valine abrogates its E2 activity, and these mutants resembie rad6 null mutants in being defective in DNA repair, UV mutagenesis and sponJlation (Sung et al., 1990), suggesting that RAD6 mediates all of its cellular functions via its role as an E2 enzyme. Because of the central role of RAD6 in DNA repair and in DNA-damage-induced mutagenesis, we have become interestcd in determining whether RAD6 is conserved among eukaryotes, A high degree of conservation of RAD6 would also suggest a parallel evolutionary conservation of proteins with which RAD6 might interact in its various cellular roles, In tbis paper, we report our studies with the RAD6 homolog from the evolutionarily divergent fission yeast Schizosaccharom)'ces pombe. Phylogenetic studies with SS ribosomal RNAs indicate that S.poll/be is evolutionarily closer 10 Homo sapiens than to S.cere~'isiae (Huysmans el al., 1983). S.pombe also resembles the higher eukaryotes in the control of the milotic cell cycle (RusselI and Nurse, 1986; RusselI et al., 1989), in the presence of introns in many of its gelles and in the sequence requircments for the splicing of introns (Käufer el al., 1985; RusselI and Nurse, 1986). Therefore, a comparisoll of the structure and function of RAD6 from these Iwo divergent ycast speACGTG
-180
TIG(;GiGCGCCCCATTMïM1GAN>.TACI'GCATATûAGiGAAiGGîCCiACTArrGITCCCIGCCCITCiCCAACMCGCMAiCGGAT
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AatII ATGICMCGACCGCAAG.>,AGAffiTCiCATGeGAGAITITAAG!.il:ACGGCAGTiGiGGAKCGAGMGACilllIATTA....CCl>ATTCI1l-L:.A ï A R R R L M R D F K R
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SU
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TGGACATICTGCAAAACAGGrGGTCTCCAACTIACGATGiGGCAGCJ..AHCITACCTCIATTCAMGGIllGCTITiCAGAAATATTAC,
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ft
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rvs T M i
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R W S P T Y D V A AlL
T S
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L L ft
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ft
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ft
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on
ACAAGAAGGAGTATGIGCGKGAGIACGl\J\AAACiGTAGAAGACTCCIGGG.-\AAGITGATITCMGACATAGCIGiCilMiAATCCCTA
",
TITCAITAGAACITACTCITCITITTCMCCGCC1TiCACATT~.A'ICTAnTAAGiGCITCAAAGilGCCMAAITG'MC'ATAGI"-'''.
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CA'IATACAGACA'lGTACATGCN>.AAïTlAiAHGAKïrAITAAA.CAIMCCiGCCClAAA.CCACACMMAiACCAAA.CCMGCGÇJ;M
1171
AACTCCATIT'ITITAAATACATATGGTCMITIT'iTAAGTAAACCICAACGGCTGITCCCAAMTMiGAITMCGACGITTCAMITC
1261
TICTTICITITACGATA'IGACTGGCCMI"i-IGAGCAGGTGMGC,AA'IGAGCCATCGïrMCGICTICAAMITAGCACGiCrCTTAc-cI
1351
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111
D'l~J uescriocd previously (She-rrnan el 111., 1986), and media for S.pomhe lI"ere prepaR"'d as descnD.-tl by OulZ et 111. (1974) and Nurse (1975). S.pombe Slr,lln CBS356 (Yea.\t Slock Center, o.:lft, The Nelherlands) wa, uscd for preparing the genornic DNA library. GenetIc analyses Standartl genetk lechniques for S.pomb,' (Gutz ct uI., (974) :md for S.arelüiae (Sh.:rman et al., 1986) were used. Ttansformation and o/he, proeedutes Yeast transfomutions wefe p..:rfomJoed aeçording to tho! IJ1Çthod of Ito et lil. treatll~nt wen': çarried oul by prel'ioosly publich,E.F. and Sambrook,J. (198"2) Mo!endar Cloning: A lnbomtor)' MaJl!Iil!. Cold Spring Harbor Laboratm)', Cold Spring Hafbor, NY. McKee,R.H. and Lawrence,C.W. (1919) Gem'rics, 93, 361-313. Mertins,P. and Gallwitz,D. (1987) EMBOJ., 6,1751-1763. Manlelone,B.A., Prakash,S. and Prakash,L. (\981) Mol. Geil. Genet., 184, 410-415. Morrison,A., MilIeT,E.I. and Prakash,L. (1988) Mol. Cello Biol., 8, 1179-1185. Morrison,A., Chris/ensen,R.B., Alley,J., Bxk,A.K., Bemstine,E.G., Lemontt,J.F. and Lawrence,C.W. (1989) J. Bactfriol., 171,5659-5667. Nasim,A. and Smith,B.P. (1975) Genetics, 79, 573-582. Nurse,P. (1975) Nature, 256, 547-551. Phipps,J., Nasim,A. and MilIer,R.D. (1985) Ad~'. Gelift., 23, 1-72. Prakash,L. (1974) Genetics, 78,1101-1118. Prakash,L. (1981) Mol. Gen. Genet., 1s.t, 471-478. Reynolds,P., Weber,S. and Prakash,L. (1985) Proc. Nat!. Acad. Sd. USA, 82, 168-172. Rotenberg,M.D, Chow,L.T. and Broker,T.R. (1989) Virofog)', 172, 489-491. RusseU,P. and Nurse,P. (1986) Cel!, 45, 781-782. RusseH,P., Moreno,S. and Reed,S.1. (1989) Cel!, 57, 295-303. Saiki,R.K., Scharf,S., Faloona,F., MuHis,K.B., Hom,G.T., Erlkh,H.A. and Amheim,N. (1985) Sdence, 230, 1350-1354. Sanger,F., Nkklen,S. and Coulson,A.R. (1977) Proc. NaJ!. Acod. &1. USA, 74,5-.\63-5467. SchupbJ.GTFRLLLEFDEEYPNKPPHVKFLSEHFHPNVYAlIGEICLDILQNRh"TPTYDVASII,
106 106
(S.cerevisiae)
RA"
B
,
D. melanogasler OHm
152 151
S. pombe fhp6t
• ••••• L •••• N ••••• A ••• Q. HreN. KE •• r •• rK ••• D ••• S' 151 TSIQSLfNDP1IPASPJlJIVEAATLFKDHKSQYVKRVKETVEKSWEDDMDDMDDDDDDDDDDDDDE"'O. 172
Human HuE2-17
71% • ••••• LOe ••• N •••• SQ •• Q. YQeN. RE. E ••• SAI. • Q •• H .S~ • ••••• LS •••• ll. ••• ST •• Q. Y. el/rRE. E •••• AC •• Q. FI. ~
~S5% 71%
6
%$7if.,(,
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77%
FIG. 2. (A) Comparison of amino acid sequcnces of RAD6 homologs from Sa. cert'risiae, Sc. pOli/be, D. me!alloxaster. and human. The ln-amino acid-long RAD6 protein is aligned with the 151 amine acids ofDhr6 and rhp6 + proteins and the 152 amine aeids ofthe human homolog. The position ofCys-88, involved in thioesterformation with ubiquitin, is indieated by an open triangle. Sequences are completely coJinear. except for the acidic tail in Sa. cerel';siae RAD6. Dols indicate identity, whereas small letters indieate conservati\'e çhanges in Sc. pombe, D. me/aflogaster, and human proteins çompared with the Sa. cern'isiae protein. Similaraminoadds: R = K, E = D, I = V = L, S = T. (D) Percent identical amino acid residues shared among RAD6 homologs from Sa. arel'isiae, Sc. pombe, D. me!allogasler, and Homo sapiells. Only the residues present in both homologs were çonsidered; thus, camparisan afDhr6 and rhp6+ proteins with eachotherand with RAD6and HuE2-17 prateins included 151 residues, and the comparison of HuE2-17 with RAD6 protein included 152 residues.
DISCUSSION The protein encoded by the Dlu6 gene of Drosophila shares a high degree of homology with the RAD6. rhp6+ , and E217k proteins of Sa. cerel'isiae, Sc. pOli/be, and humao, respectively. However, the acidic carboxyl-terminal domain present in the Sa. cerel'isiae RAD6 protein is absent in the Sc. pOli/be, Drosophila, and human homologs. Two regions, one flanking the Cys-SS residue, and the other consisting of the amioo-terminal 15 residues, have been in particular highly conserved among these homologs. Because the Cys-SS residue is involved in thioester formation wÎth ubiquitin (6), the region nanking this cysteinc residue is likely involved in interactions with the ubiquitin-aclivating enzyme (El). The high!y conserved amino terminus in these homo!ogs is very basic and shows similarity to nucJear-localization signa! sequences. However, mutationaI studies with RAD6 suggest that this is oot the role ofthis sequence (J. Walkins, S.P., and L.P., unpublished observations). Becallse the high degree of
conservation ofthe amino terminus among the various RAD6 homologs does not extend to olher ubiquitin-coojugating enzymes (23, 24). this sequence may be involved io specific interactions with protein components ofthe DNA-repair and mutagenesis machinery, mther than in interactions with the El enzymc. Genetic studies in Sa. cerel'isiae with Ihe Dhr6 gene reported here cJearly demonstrate conservation of RAD6 function in higher eukaryotes. The Dhr6 gene complemented the UV and y-ray sensitivity and defective UV mutagene sis of rad6à mutant strains. However, whereas UV mutagenesis was restored to wild-type levels, UV survival was complemented to alesser degree. As expected, Dhm did not complement the sporulation defect ofthe rad6à/rad6à strain because the RAD6 acidic-!ail sequence required for sporulation in Sa. cerel'isiae is absent in the Dhr6 protein. In Drosophila and other eukaryotes (7, S), a different protein may perform the role of the RAD6 acidic domain. '000 , - - - - - - - - - - - ,
~
8
~
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1QOO-fold by llllR6A and HHR6B, respectively (Fig. SA). The IJIJR6A and IJIJR6B genes also restore UV mutagenesis in the rad6A strain to wild-type levels (Fig. SB). In contrast, the two human homologs confer only a Jow level of sporu· lation abilily (=5%) to the rad6ilJrad6il strain. DISCUSSION In this paper, we have identified two c1ose!y related ho· mologs oflhe S. cerevisiae RAD6 gene in hu man, one ofthem being identicallo the E2 (Mr 17,000) protein recenlJy de· scribed by Schneider et al. (17), who isolated an incomplete cDNA on the basis of a partial amino acid sequence. Dur extensive analysis ofa large number of independent genomic DNA clones points to the existence of only a single RAD6 gene in S. cerel'isiae, Sc. pOli/be, and D. melal/ogas/er. The very high degree of amino acid sequence conservation throughout eukaryotic evolulion points to extremely strong sequence constraints imposed on the RAD6 protein. As shown in Fig. 4, the human and yeast RAD6 homologs share =70% sequence identity and the Drosophila homolog is the one most c10sely relatcd to the human HHR6 proteins
(85-87% identity). The Dhr6 and HHR6 proteins share almost the same degree of sequence homology (68-69% identity) to RAD6, whereas the rhp6+ gene product is only somewhat more homologous to the S. cerel'isiae protcin (77% idcntity). Based on the degree of divergence between the various RAD6 homologs, we calculale that the duplication found in hUll1ans (and also in mouse and kangaroo; unpub· lished results) must have occurred =200 x lW years ago, in the Jurassic era. Fig. 3 (top five Iines) shows that among the RAD6 ho· mologs, the N-terminal part and the central region, in par· ticular, have been highly conscrved. The middle portion contains the invariant Cys-88 residue that is involved in thiol ester linkage with ubiquitin and that is crucial for all RAD6 functions, as its substitution by valine or alanine produces a rad6 null phenotype (24). The C terminus, on the olher hand, has diverged much more. Thc S. cerel'isiae RAD6 protein is unique in harboring an acidic tai! sequence. Mutational analysis has shown the acidic domain 10 be essential for sporulation in S. cere~'isiae(4). A possible cxplanation forthe absence of an acidic C·tenninal extension in other RAD6
68
.. (1) HHR6A
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FIG. 3. Comparison of amine add sequences of HHR6A and HHR6B proteins with various RAD6 homologs and with other ubiquitinconjugating enzymes. (Upper) Comparison ofvurious RAD6 homologs: S. cerel'isiae RAD6 (3), Sc. pombe rhp6+ (10), D. melarlogasler Dhr6 (11), and human HHR6A and HHR6B (human E2; AIr 17,000) (this paper; ref. 17). (LQwer) Comparison of the other published ubiquitinconjugating (132) proteins; S, cerel'isiae UBC1 (20); UnC4 and UBC5 (21), involved in protein degradationi S. cere~'iJiae CDC34, involved in eeU eycle regulation (22); and wheat E2(Mr 23,000) (23). Dols indicate identity, whereas lowerease letters indicate strongly eonserved rcsidues eompared with the yeast RAD6 protein. Conserved amino acids: Rand K; E and D; I, V, and L; Tand S, (1), Horizontal bars, aminoacid residues exclusively conserved in all members oflhe RAD6 family; (2), consensus sequence present in all 10 E2 enzymes. Doldface lelters, amine acid residues occurring at Ihis position in all 10 ubiquitin·conjugating enzymes; lightface letters, the most likely possibility at thls position (occurring in 80% or more of the cases); circles, hydrophilic residue at lhis posÎlion in alilhe proteins; crosses, hydrophobic residues in all 10 positions. Cys-88 residue, used for ubiquitin attachment, is boxed in all E2 family members. homologs is that in the olher species this domain may have evolved into a protein of its own or it may have become incorporated into a different prolein. The comparison of RAD6 with the other ubiquitinconjugating enzymes presented in Fig. 3 (bottom six lines)
8 ~
, • ~ •m •~ ," ,," ,," 0
6S 6. 69 S. pombe rhp6+ 70 71 71 .4 O. melanoga$tElr OHR6 74 77 .7 85 % Identity 95 Man HHR6A 74 7. 90 Man HHAaB 74 7. '9 9. S. eerevls!aEl RAD6
77
%Sîmilarity
Fm. 4. Identical and similar amino acid residues shared among RAD6 homologs. Percentage identity is given above Ihe diagonal, and percentage similarity is given below the diagonal. See Fig. 3 legend for classification of conserved residues,
69
reveals marked similarity, especially in Ihe central part around the Cys-SS residue (sec overall consensus sequence 2 in Fig. 3). This segment is Iikely involved in binding of ubiquitîn andjor interaction with the ubiquitin-activating enzyme El that donates a ubiquitin moiety from an internal cysteine residue 10 the cysleine in E2 enzymes. The amino acid sequence around eys-SS in E2 enzymes bears resemblance 10 the sequence context of Cys-908 and ·866 of the recently cloned ubiquitin·activating enzymes (El) of wheat and human, respectively (25, 26), and may define a ubiquitin binding domain in El enzymes as weil. The strict conservation of the N terminus among RAD6 homologs does nol extend to the olher E2 enzymes. This part may therefore be implicated in important RAD6-specific functions such as interaction with protein components of Ihe DNA repair and mUlagenesis machinery. Finally, it is remarkable that all E2 proteins begin with the sequence MS(Sj Proteins starting with serine are frequently subject 10 N-terminal acetylation (27)_ It is not known whelher RAD6 or any olher E2 enzyme is acelylated at the N terminus. The high degree of amino acid sequence conservation of RAD6 is also reflected at the functionallevel. Both human
n.
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homologs restore normalieveis of UV mutagenesis and effect a substantial increase in UV resistance in S. cerc\'isiac rad6 mutants. On the other hand, human homologs confer only a very low level of sporulation ability 10 rad6/rad6 mulants. This result is expected in view ofthe absenceoftheacidic tail sequence in the human proteins and prevÎous observations that this domain is essential for sporulation but not for DNA repair or UV mutagenesis (4). The availability of 1I11R6 genes should make it possible 10 examine their role in various cellular processes in mammals such as mutagenesis, postreplication repair, and recombination. Because of Ihe involvement of RAD6 in sporulation, it will be of special interest to examine whether the HHR6 genes are implicated in meiosis and gametogenesis. At the flnal stages of spermalogenesis, histones are replaced by prolamines. One can envisage that Ihe capability ofRAD6 10 polyubiquitinate histones is ulilized al this stage to mark hislones for degradation by Ihe ATP-dependent ubiquitinspecific protease complex. For these studies, it will be necessary 10 obtain HHR6 mutanls. One way toward identifying such mutants wil! be 10 screen mutant celllines from human DNA repair disorders or from the existing collection of in vitro generated repair-deficient rode nt ceillines. Alternalively, HHR6 mutanls could be generated by gene disruption utilizing recently developed melhods of gene replacement (28). It is passible 10 perform Ihis in totipotent mouse embryonic stem eells and in thaI way la ereale an HHR6 defeetive mouse model. An obvious eamplication, however, is Ihe presence of Iwo genes, whose function is likely 10 overlap cansiderably, necessitating Ihe simultaneous inaclivation of both genes. We thank Mirko Kuit nnd Tom de Vries Lentsch for photography and Sjozef van Daal for computer assistance. This work was sup· ported by the Dutch Cancer Society (Project IKR 88-2 and 90-20), the European CommunityContract Bl6-141-NL, and U.S. Public Health Service Gronts GM19261 and CA41261 from the National Institutes ofHealth and DE-FG01·88ER60621 from the Department ofEnergy. 1. Prokash, S., Sung, P. & Prakash, L. (1990) The EllkaryalÏC' Nucleus, eds. Straus, P. R. & Wilson, S. H. (felford Press, CaIdweIl, NI), Vol. I, pp. 275-292.
4
6 8 UV, Jfm'L
10
12
FIG. 5. Complementation of UV sensitivity and UV Îmmutability of the S. cerel';siae rad6Jl mutation by human HlIR6A and HHR6B genes. Survival afIer UV irradiation (A) and UV-induced reversionofmet14(B) in theS. cen'\'isiae. rad6tJ. stroin EMY8 carrying the HHRM or HlIR6B gene on the ADel plasmid, eells were grown in synthetic complete medium lacking tryptophan for selection of the plasmid and were harvested in midexponential phase. After plating on appropriate medium, eells were irradialed v.ith UV light at a dose rale of 0.1 J'm- 2'sec- I and incubated in the dark 10 avoid photoreactivation. 0, EMY8 + pR67 (CEN RAD6); e, EMY8 + pR611 (radM); "', EMY8 + pRR510 (ADC HHR6A); t;. EMY8 + pRR518 (ADC HHR6B).
2. Picologlou, S., Brown, N. & Lieberman, S. (1990) Mol. Celt. Biol. 10, 1017-1022. 3. Reynolds,P., Weber,S. & Prakash, L. (1985) Proc. Na/I. Acad. Sci. USA 82, 168-172. 4. Morrison, A., Miller, E. J. & Prakash, L. (1988) Mol. Cell. Biol. 8, 1179-1185. 5. Jentsch, S., McGrath, J. P. & Varshavsky, A. (1987) Na/ure (LondOI/) 329, 131-134. 6. Sung, P., Prnkash, S. & Prnkash, L. (1988) Genes De\'. 2, 14761485. 7. Hmhko, A. (1988) I. Bial. Chem. 263, 15237-15240. 8. Rechsteiner, M. (1988) UbiqllÎtin (Plenum, New York). 9. Jentseh, S., Seufert, W., Sommer, T. & Reins, H.-A. (1990) Trends Biochem. Sei. 15, 195-198. 10. Reynolds,P.,Koken,M. H. M.,Hoeijmakers,J. H. J.,Prakash,S. & Prakash, L. (1990) EMBO 1.9,1423-1430. H. Koken, M. H. M., Reynolds, P., Bootsma, 0., Hoeijmakers, J. H. J., Prakash, S. & Prakash, L. (1991) Proc. Na/I. Acad. Sci. USA 88, 3832-3836. 12. Feinberg, A. P. & Vogelstein, B. (1983) Allal. Biochem. 132,6-13. 13. Sambrook, J., Fritsch, E. F. & Maniatis. T. (1989) Mole('lIlar Cloning: A Labara/OI)' Marmot (Cold Spring Harbor Lab., Cold Spring Harbor, NY). 14, SangeT, F., Nicklen, S. & CouJson, A. R. (1977) Proc. No/I. Acad. Sci. USA 82, 168-172. 15. Sherman, F., Fink, G. R. & Hicks. J. B. (1986) Me/hads in Yeas/ Gene/iC!: Lahora/of)' Course Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY), 16. Sung, P., Prakash, L., Weber, S. & Prakash, S. (1987) Proc. Nall. Acad. Sci. USA 84, 6G4S-fiO.I9. 17. Schneider, R., Eckerskom, C., Lottspeich, F. & Schweiger, M. (1990) EMBOJ. 9,1431-1435. 18. Bimstiel,M. L., Busslinger,M. & Strub, K. (1985)CeIl41,349-359. 19. Wickens, M. (1990) Trends Biochem. SC!. 15,277-281. 20. Scufert, W., McGrath, J. P. & Jentseh, S, (1990) EMBO J. 9, 4535-4541. 21. Seufert, W. & Jentseh, S. (1990) EMBO J. 9, 543-550. 22. Goebl, M. G., Yochem, J., Jentseh, S., McGroth, J. P., Varshavsky, A. & B)'ers, B. (1988) Science 241, 1331-1335. 23. Sullivan, M. L. & Vierstrn, R. D. (1989) Proc. Na/I, Acad. Sci. USA 86, 9861-9865. 24. Sung, P., Prakash, S, & Prakash, L. (1990) Proc. Nall. Acod, Sci. USA 87, 2695-2699. 25. Hatfield, P. M., Callis, J. & Vierstrn, R. D. (1990)/. Biol. Chem. 265,15813-15817. 26. Handley, P.M., Mueckter, 1.1., Siegel, N. R., Ciechanover, A, & Schwartz, A. L. (1991) Proc. Nall. Acad. Sci. USA 88, 258-262, 27. Persson, B., Flinta, C., von Heijne, G. & Jömvall, H. (1985) Ellr. I. Biochem. 152, 523-527. 28. Capecchi, M. R. (1989) Sc!enee 244, 1288-1292.
70
Chapter V Localization of two hllmall IlOmologlles, HHR6A and HHR6B, of the yeast DNA I'epair geile RAD6 to chromosomes Xq24-25 alld 5q23-31
I I
GRNOMICS 12,447--453 (1992)
Localization of Two Human Homologs, HHR6A and HHR6B, of the Yeast DNA Repair Gene RAD6 to Chromosomes Xq24-q2SandSq23-q31 M.
H.
M. KOKEN,' E. M. E. SMIT, I. JASPERS·DEKKER, B. A. OOSTRA, A. HAGEMEIJER, D. BOOTSMA, AND J. H. J. HOEIJMAKERS
Department of Ceff Bio/ogy and Genetics, Medical Genetics Center, Erasmus University, P.O. Box 1738, 3000DR Rotterdam, The Netherlands Received luly 19, 1991; revised October 10, 1991
'rhe chromosomalloealizalions of two closely relaled human DNA rcpair genes, llllR6A and llllR6B, were delermined by in situ hybridization with hiotinylated probes. HHR6A and HHR6B (human homolog of yeast RAn6) eneode ubiquitineonjugating enzymes (E2 enzymes), likely 10 be involved in postreplicalion repair and induced mulagenesis. TheHHR6B gene was assigncd to human chromosomc 5q23-q31, whcreas the HHR6A gene was localized on the human X chromosorne (Xq24-q25). This latter assignment was confirmed wUh an X-specific human-mouscjhamsler somatic eeU hybrid panel. Southern blot analysis points 10 an X and an autosomallocalization of HHR6A and HHR6B, respectivcly, in Ihc mouse. Thc potcntlal involvemenl of these genes in human genetic disorders is discussed. © 1992 MaMml~P...s, In~.
INTRODucnON
Recently, we reported the cloning oftwo human genes, designated HHR6A and HHR6B, homologous to the Saccharomyces cerevisiae RAD6 gene (Koken et al., 1991b). As deduced from the very pleiotropie phenotype of yeast rad6il mutants, t,he RAD6 protein plays an important roie in various cellular processes, including postreplication repair (a poorly defined, error-prone repair pathway), damage-induced mutagenesis, sporulation, and recombination (for a review, see Prakash et al., 1990). The RAD6 functions are accomplished by a 172amino-aeid protein with an N-ferminal globular structure and an extended C-terminal aeidie tail (Reynolds et al., 1985). 'fhe acidie domain is specifically required for sporulation but is not essential for the other RAD6 functiOllS (Morrison et al., 1988). An important finding concerning the biochemical activity of the RAD6 protein was the discovery t,hat. the gene eneodes a ubiquitin-conjugating enzyme (Jentsch et al" 1987). Ubiquitin, a widespread, highly conserved 76-amino-acid polypeptide, is llHR6A nnd HHR6B are not HGMW approved gene sYlllbols. I To whom correspondence should be addressed.
covalently attached to specifie cellular proteins that in this way are targeted for selective degradation, (re}folding, or stabilization (for recent reviews, see Hershko, 1988; Reehsteiner, 1988; Jentsch eta!., 1990). Ubiquitination of proteins occurs in a multistep reaction. First, a ubiquitin-activating enzyme (or El enzyme) binds and activates a ubiquitin molecule. This is subsequently transferred to one of a set of ubiquitin-conjugatillg enzymes (or E2 enzymes). The E2 enzyme ligates t.he ubiquitin moiet.y to a target protein with or without the help of an E3 ubiquit.in protein ligase molecule. The RADG protein was found to attaeh one (Jentsch et al., 1987) or multiple (Sung et al., 1988) ubiquitin moieties to histones H2A and H2B in uitro. If histones are also the main targets of RAD6 in uiuo, it is likely that RAD6 mediates chromatin remodeling required for t.he processes impaired in a rad6û mutant. RAD6 is very st.rongly conserved in eukaryotie evoluHon, and this property permitted us to clone by evolutionary walking two human homologs (Koken et al., 1991b) using the Schizosaccharomyces pombe (Reynolds et al., 1990) and Drosophila melarwgaster (Koken et al., 1991a) homologs as "intermediates." The human HHR6A and HHR6B proteins (HHR for human homolog of RAD6) share ~95% amino acid sequence identity with eaeh oUler and ~70% amino acid identity wit.h their yeast counterparts, but notably lack the acidie Cterminal domain, the occurrence of which seems to be limited to S. cereuisiae RAD6. r...loreover, the human polypeptides were found to substitute funct.ionally for the repair and mutagenesis functions of RAD6 in a S. cereuisiae rad6il mutant but not for its role in sporulation. This indicates that the proteins of the repair and mutagenesis maehinery with which RAD6 interacts are also conserved to a significant extent. belween man and yeast. Furthermore, it is likely that. the HHR6 proteins in man have a funetion similar to that of RAD6 in yeast, i.e., catalyzing ubiquitin conjugation as an essential step in t.he repair and mutagenesis pathways. This conclusion makes the gene a eandidate for human inherited
72
repair disorders, in particular the variant cOIllplementatiOll group of t,he cancer-prone repair syndrome xeroderma pigmentosum (XP) in which the postreplication repair pathway is considered to be impaired (Lehmann et al., 1975). Here we present the chromosomallocalization of these two human genes by in situ hybridization using biotinylated pro bes and by Southern blot hybridizations to DNA of rodentjhuman ceU hybrids. 1tATERIALS AND METHODS Cell lineslDNitS. The somatic eell hybrids conlalning vaTÏous parts of the human X chlOmosome used in this sludy have been describcd elsewhere. The hamsterfhuman hybrids were X3000, Xq24qtcr (Nussbaum et al., 19S6); 90SKlBlS, Xq24--q26 (Schonk et 0/., 19S9); 8121, Xpler--q27.1; and 238-1, Xpter--q27.2 (Patterson et 0/., 1987). The mousefhuman hybrids were RJK734, Xq26--qter (Scott et al., 1979); and CY34A, Xq24--q27 (Suthers et al., 19S9) See Fig. 2B for schematic diagram of the human X-chromosome segments in these hybrids. Restrictioll enzyme digests and Southem blot hybridizations. Enzyme digestions and Southern blotting procedures were essentially tho same as described previously (Koken et al., 1991b; Sambrook et af., 1989). In brief, 20 pg of reslriction enzyme·dig(>sted genomic DNA was size-fraclionated on O.S~ agarose gels and transferred onto nylon (ZetaplOhe) membranes. Hybridization occurred overnight at 65°C in a hybridization buffer containing lOX Denhardt's solution, 10% dOll:tran sulfate, 0.1% SDS, 3X SSC, and 50 pg/ml sonicated salmon sperm DNA. Washings were performed extensi\'ely up to 0.3X SSC containingO.l% SOS at 65°C. The L7-kb HHR6A cDNA probe, H28, containsa full-length HHR6A cDNA on a Sall fragment (Koken et al., 1991b). The HHR6B eDNA probe, H13"-8' harbors the complete HHR6B open reading fwme on an O.S-kb fragment starting with an rutificial BcoRI site at the position ofthe ATG and ending at a nalural BroRI site in the cDNA (Koken et al., 1991b). In situ hybridization_ III situ hybridization was performed essenfiany as described (Lanclegent et al., 1985; Pinkel el al., 1986). Human lymphocyte metaphase spreads were trcated with 100 pg RNase A/mi in 2X SSC for 1 h at 37°C, rinsed in 2X SSC, and dehydrated in alcohol. After a pepsin (0.1 pg/ml 0.01 N HCI) treatment at 37°C for 10 min, the slides were washed in PBS, postfixed with 1% formaldehyde in PBS containing 50 mM MgCI 2 , washed for 5 min in PBS, dehydrated in ethanol, and air-dried. The hybridization mÎltture (10 pI per slide) eonsisted of 50% formamide, 2x SSC, 40 mM sodium phosphate (pH 7.0), 10% doxtwn sulfate 50 ng labeled probe, 1 pg sonicaled salmon sperm DNA, and 1 pg Bscherichia cali tUNA. The genomic probes, B3.0, B2.3, 112.7, HO.75, and HS2.7 (HHR6A) and E2.3, E6.0, E4.5, and E1.3 (IlIlR6B), representing most of the genomic region of bath genl'S (Koken et ol., manuscript in prcparation), were biotin-Iabeled. A cocktail of the genomic prohes for each gene was used for ill situ hybridizalion. Probes were denatured al 70°C for 5 min in hybridization mixture (specified aboye). Competition for repeat sequences present in the genomic subclones was achieved by incubation for 6 h (HHR6A probes) or 2days (IlIlR6B) witha 100 times eltcess of thymus DNA (HHR6A) or a 1000 times excess ofhuman con DNA (IIHR6B) at 37°C in hybridi?ation buffer. This was Ilecessary because ofthe elttremely high content ofrepeats in the genomic dones used as probes. Thechromosomespreads weredenatured in 70% formamide for 2.5 min at ?OoC. After competition, the probes were incubated oyernight with tho slides and then washed once with 50% formamide in 2x SSC at 39°C followed by three times for 5 min in 2X SSC, three times for 5 min in O.lX SSC at GOoC, and ollce for 5 min in 4x
SSC, 0.05% Tween20 at room temperature. Finally, the slides were blocked in 4X SSC, 5% nonfat dry milk for 20 min al 37°C. Slides were incubated with 5 pg avidin D-FITC (Vector, U.S.A.), and the fluorescent signaI was amplified with biolinylatcd goat antÎ-avidin D, washed, dehydrated with ethanol, and air-dried_ The slides were embedded and stained in 9 pruts glycerol containing 2.3% (w/v) l,4-diazobicyclo-(2,2,2)-octane (DABCQ) and 1 part 0.2 MTris-HCl, 0.02% NaNJ , pH 8.0, containing 4',6'-diamino-2-phenylindole (DAPI) to a final cOllcentration of 0.5 pglpl.
RESULTS
In Situ Hybridization to Metaphase Chromosomes For mapping the HHR6A and HHR6B loci, in situ hybridization experiments on metaphase spreads were performed lIsing biotinylated genomic probes. A representative in situ hybridization for each ofthe two genes ofthe more t.han 50 metaphases analyzed is depicted in Fig. 1. As shown in Fig. IA (HHR6A), a specific signal (arrow) is found on the long arm of only one chromosome in every metaphase analyzed. Because cells in this experiment were derived from a male donor, this finding strougly suggests that t.he gene is located on the X chromosome. This interpretation was conftrmed by simultaneous hybridization with an X-specific centromere prohe, pBamX5 (Willard et al., 1983), clearly identifying the hybridizing chromosome as the X chromosome. [The weak hybridization with the centromeric regions of four otller chromosomes (9 and 17) is due to cross-hybridization of the X-centromere probe to the centromeres of chromosomes 9 and 17 (Willard aud Waye, 1987).) From t.hese results we deduce that t.he HHR6A gene resides on the lower part of the q arm of the X chromosome. Figure IB shows the hybridization with biotinylated HHR6B gene probes (arrows). Using the DAPI staining procedure, the hybridizing chromosome was identified as chromosome 5 (Fig_ 1B). Therefore, the gene was unambiguously assigned to 6q23-q3L
Southem Hybridization of HHR6A Probes 10 DNA of a Panel of HumanjRodent Somatic Cel! Hybrids To confinu the assignment of HHR6A aud to obtain a more precise subchromosomal localization, Southern blot analysis was carried out using genomic DNA from a panel of human-mousejhamster hybrids containing specific parts of the human X chromosome (Fig. 2B). As shown in Fig. 2A the HHR6A cDNA probe recognizes the human fragments (3.0, 2.6, and 0.75 kb, indicated by arrowheads) in hybrid celllines X3000, 8121, and 2384. This indicates that the HHR6A gene maps on Xq24-q25 ceutromeric of the breakpoint in the X chromosome found in the RJK734 hybrid and distal ofthe breakpoint
FIG. 1. In situ hybridization of metaphase chromosomes to biotinylatcd genomÎC HHR6 probes. (A) Hybridization with a cocktail ofall genomic 1I1IR6A probes specified uncler Material and Methods. The arrow Îndicates Ihe hybridization signaion chromosome Xq. This chromosome shows also the X-specific hybridization ofthe pBamX5 probe. The prohe weakIy closs-hybridizes to chromosomes 9 and 17 as indicated. (B) Hybridization with a cocktail ofaIl genomic HHR6B probes (indicated under Materials and Methods). The arrowspoint 10 the regions with aspecific signal on chromosome 5q23--q31. In panels I the in situ hybridization is shown. In panels II the DAPI banding ofthe same melaphases is shown.
73
A
B
74
B A
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.
0 0 0
~
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"
~
N
....... -
~
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'"
MW ~b}
., .... '" 4111
5.0
.W
3.0 2.6
....... "" ...
1.1 0.75
" " " "
I
I I
11
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+
Hyb/Jd
""..'il "'",I' +\9"V 1,-1;>~"b"'1;l',s>0,'4
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FIG. 2. (A) Southern blot analysis ofX-specific hybrid panel with the HIlR6/\ cDNA probe. Thc source ofthe gcnomic DNA is inclicated. DNA is digcsted with HilldIli nnd size-fmctionated on an 0.8% agarose gel. Tbc molecular weight (MW) indicated on the right refers to tha hyhridizing frllgments at the corresponding posiHons in the autoradiogram. Tbc fragments of 3.0, 2.6, nnd 0.75 kb are of the human IIIIR6A gene. (H) Reprcsentation ofthe human X-chlOmosome fragments (indicated by tinas) retained in the rodent/human hybrids used in tbis shldy. Thc + or - sÎgn above the hybrid-narnes indicates whether or not DNA fIOm tbis specific celiline hybridizes with the human probe.
in the X3000 hybrid cellIine, confirming the data found via in situ hybridization. Hybridizations to t.he somatic ceU hybrids 90BK1B18 and CY34A were also positive with HHR6A probes (data not shown). Chromosomal LocalizatioTi of the Mouse Homologs of HHR6A alid HHR6B
To assess whether also in the mouse one gene is 10cated on the X chromosome and the ot.her on an autosame, a Southern blot with equal amounts of genomic DNA from a male and femaie mouse was hybridized consecutively with both human cDNA probes. As shown in Fig. 3, the hybridization with the HHR6A gene c1eady shows an approximately twafold difference in hybridizaHon intensity between the DNA of the male and the female mouse, whereas with the HHR6B probe and the same blot, na difference between male- and female-derived DNA is detectabie. This strongly suggests that also in the mause the HHR6A gene is X-linked, whereas the HHR6B gene is on an autosame. DISCUSSION
Localizatio!l of Genes lnvolved in DNA Repair or in Ubiquitin 8ystems
This paper describes the localization of two human homologs, HHR6A and HHR6B, of the yeast DNA repair gene RAD6 to human chromosomes Xq24-q25 and 5q23-q31, respectively. The HHR6A gene is the first human DNA repair gene lacated on X. Among the DNA repair genes isolated thus far, no clustering is apparent,
75
with the possible exception of the q13.2 area of chromosame 19 onto which at least three repair genes have been lacalized (Mohrenweiser et al., 1989; \Veeda et al., 1991; Smeets et al., 1990; Thompson, 1989). This, however, could be due at least in part to the presenee of large regions of hemizygosity in the Chinese hamster eells used to generate the repair mutant eeU lines with which these three genes were cloned. The hemizygosity favors the isolation of mutants in genes located in those areas (Siciliano et al., 1983). In contrast to a dispersed localization of DNA repair genes over the genome, it is of interest to note that a clustering of genes far different components of the ubiquitin system may exist on the X chromosome. \Vith the exception ofubiquitin itself, encoded by several polyubiquitin and ubiquitin fusion genes on a number of different autosomes (\Vebb et al., 1990), the other two ubiquitin-system genes cloned thus far are both located on X. The GdX gene (HGMW symbol DXS254), with exten~ sive hamology to ubiquitin, has been localized onto Xq28 (Tonialo et al., 1988). Moreover, the gene for one of the human ubiquitin-activating enzymes (El, HGMW gene symbol UBE1) has been assigned to the X chromosome (Ohtsuba and Nishimoto, 1988; Kudo et al., 1991), more precisely to Xpll.2-p11.4 (Zackenhaus and Sheinin, 1990; Handley et al., 1991; McGrath et al., 1991).
Duplication of HHR6 In the lower eukaryotes (8. cerevisiae, S. pombe, and D. melarzogaster), we eould identify only a single RAD6 locus situated on an autosame (Reynolds et al., 1990; Koken et al., 1991a). As ealculated from divergenee data,
HHR6A
HHR6B
Probe
MWlld"-l!o'So'ONA
kb
4.5
2.'
1.1
0.' 0.5
0.34
.---
FIG. 3. Southern blot analysis ofgenomic liver DNA from a male and a femele mouse. MW: Molecuier weight marker, i.e., phega" DNA digested with PstI. Left: The autorüdiogram of a blot with HindfII + EroRI + BamHI triply digested mouse DNA hybridized with e human HHR6A cDNA probe. i\IiddJe: An autoradiogram of the same blot hybridized with a human HHR6B cDNA probe. Hight: A photograph ofthe ethidium-stained genomic gel. The probes used and the ó (male) or 2 (femala) sex ofmouse from which the DNA was isolated are indicated above the autoradiogram.
the two HHR6 genes in human and mouse (unpublished data) may have arisen from a gene duplication event in the Jurassic era about 200 million years ago, early in the history of mammais, i.e., weIl before the separation of evolutionary lines Jeading to rodents and primates. Duplieation has several advantages and is more often found for essential genes. One advantage could be that it permits differential gene regulation and/or functional divergence of the proteins. Finally, the synteny conservation for the X chromosome between mouse and man as found for HHR6A supports Ohno's law that there is astrong selection against chromosomal rearrangements involving the sex chromosomes and autosomes (Ohno, 1969; Nadeau, 1989).
The Chromosomal Context of HHR6A and HHR6B; Possible Involvement of HHR6 in Human Disorders Yeast rad6D. mutant cells show a very pleiotropie phenotype, with sensitivity to many DNA damaging agents, a defect in postreplication repair, no induced mutagenesis, and a complete lack of sporulation. In human, only ceUs of a single syndrome are known to be aft'ected in postreplication repair: the variant complementation
group of the rare DNA repair disorder, xeroderma pigmentosum (XP) (Lehmann et al., 1975). In this complementation group, constituting about 30% of all XP patients, no indications favoring an X-Iinkage have been found. This renders it unlikely t.hat. HHR6A is the gene responsible for this disorder. However, the HHR6B gene remains a possible candidate, although ceUs from XP variant patients have an elevated frequency of uv-in~ duced mutations, and in t.hat respect differ from the yeastphenotype (Maheretal., 1976; Myhr et al., 1979). A systematic search for abnormalities in DNA, mRNA, or protein structure or expression in (families 00 XP variant patients should resolve this issue. In addition, two mammalian post.replication repair-deficient ceU mu~ tants that are potent.ial HHR6A mutants have been characterized, UV1 of Chinese hamster origin (Hentosh et al., 1990) and .SVM (derived from Indian Muntjac) (PiIlidge et al., 1986) . Two human disorders have been assigned to the q24~ q25 region of the X cluomosome where HHR6A is 10cated (Human Gene Mapping 10 and 10.5): first, the X-linked lymphoproliferat.ive syndrome, which results in fatal infeetious mononucleosis, hypogammaglobulinemia, and malignant Iymphoma-celis from these patients seem to be dist.urbed in t.he appropriate immune response to Epstein-Barr virus (Skare et al., 1989); and second, the oculocerebrorenal syndrome of Lowe, char~ acteTÎzed by congenital cataract, mental retardation, and a defective renaI tubular function (Reilly et al., 198B). Although these diseases apparently map to the same region of the X cluomosome as HHR6A, to our knowledge there is no evidence for a DNA repair defect associated with any of them. A final X-linked disorder not assigned ta a certain subchromosomal region with a possible defect. in DNA repair is the N syndrome. Patients sutfering from this disease display mental retar~ dation, malformat.ions, development ofT-cellleukemia, and chromosome breakage (Floy et al., 1990). The last two phenotypic traits resembie those of the DNA repair disorder Fanconi anemia. Although it has been proposed that malfunction of DNA polymerase a (X-linked) could be the cause for N syndrome, the evidence is based on aphidicolin inhibitioJl studies which provide only indirect indications. HHR6B resides in a region of cluomosome 5 containing a large cluster of growth factor genes, i.e., the genes for IL3, IL4, IL5, and CSF2 (Human Gene Mapping 10 and 10.5). These genes have recently been assigned to chromosome 11 in mouse (ATCC/NIH,1990). 'I'hepossibility exists that-due to synteny conservation-the murine HHR6B gene is also located on this chromosome. In situ hybridization should be performed to verify this proposition. Thus far, t.he human 5q23-q31 region has not been associated with any hereditary disease (Humun Gene Mapping 10 and 10.5). '1'0 our knowiedge, the only syndrome to be linked to chromosome 5 with a possible defect in DNA repair is Gardner syndrome (HGMW gene symbol APC), a dominant disorder with a predispo· sition to cancer, especially of the large intest.Îlle. It has been found that ceUs from same of these patients are
76
hypersensitive to uv light, X rays, and mitomyein C (Little et 01., 1980); however, thus far no specific repair defect has been reported in eells of these patients (Henson et al., 1983). Because post.replication repair was not investigated, a possible involvement of HHR6B in this disorder is not ruled out on the basis of these findings. However, the recent cloning ofthe APC gene, responsibie for familial adenomatous polyposis (FAP) and Gardner sYlldrome (I{inzler et al., 1991), excludes any link with HHR6B. It is reasonable to assume that HHR6A and HHR6B have largely overlapping functions in view of their high sequence homology and their ability to complement yeast rad6 repair functions. This functional redundancy would require the unlikely event of simultaneous inactivation ofboth HHR6 genes for clinical symptoms to become manifest. Alternatively, eonsidering the pleiotropic and severe yeust rad6 phenotype, it is possible that inaetivation of one or both HHR6 genes is let.hal in mammais. These propositions couldprovide an explanaHon for the possible absence of known disorders associated wit.h HHR6. The recently developed Illethodology of targeted gene replacement in mouse embryonal stem eeUs (Capeechi, 1989) opens the possibilit.y ofgenerating HHR6-defective celllines or mice in the laboratory. In that way the role of these genes at the level of the ceU and organism eau be established. ACKNOWLEDGMENTS We are indebted to Des. D. Nelson (Houston) and B. Wiednga (Nijmegen) for providing us with thc DNAs from the rodent/human Xch:romosome-specific hybrids; Dr. H. Willa:rd (Stanford) for providing us with the pBamX5 probe; Mirko Kuit and Tom de Vlies Lentsch for photographYi and DIS. A. Gemts van Kessel (Nijmegen) and J. den Dunnen (Leiden) for initial efforts to map these two genes. This work was supported by tho Dutch Cancer Society (P:rojed IKR 88·02 and 90-20) and European Community Contract B16-141-NL.
REFERENCES ATCCjNIH (1990). Repository Catalogue ofHuman and Mouse DNA probes and Libralies, September 1990, p. 23. Capecchi, 1\1. R. (1989). Alteringthc genome by homologous :recombination. Science 244: 12B8-1292. Floy, KM., Hess, R 0., !lnd Meisner, L. F. (1990). DNA polymew.se alpha defect in the N syndrome. Am. J. Med. Genet. 35: 301-305. Handley, P. M., Mueckler, M., Siegel, N. R, Ciechano\'el, A., and Schwartz, A. L. (1991). Molocular dOlling, sequence, alld tissue dist:ribution ofthc human ubiquitin~aclivating enzyme El. Proc. Nat/. Acad. Sci. US,t 88: 258-262 and 7456. Henson, P., Fornace, A. J., and Little, J. B. (1983). Normal :repair of ultraviolet-induced DNA damage in a hype:rsensitivc strain offibroblasts fIOm a patient ...oith Garrlner's syndrome. Mutat. Res. 112: 383-395. Hentosh, P., Collins, A. R S., Correll, L., Fomace, A. J., Giaccia, A., and Waldren, C. A. (1990). Genetie and biodHlmical cha:racterization ofthe CHO-UV-l mutant rlefective in postreplication :recov(1)' of DNA. Cancer Res. 50: 2356-2362. Hershko, A. (1988). Ubiquitin-mediated protcin degradation. J. Rial. Chem. 263: 15237-15240. Human Gene Mapping 10 and 10.5 (1990). Cytogenet. ('eli Genet. 55. Jentsch, S., McGrath, J. P., and Varshavsky, A. (1987). The yeast
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DNA :repair gene Rt1fJ6 encodes a ubiquitin-conjugating enzymc. Nature 329: 131-134. Jentseh, S., Scufert, W., Sommer, '1'., and Reins H-A. (1990). Uhiquitin-conjugating enzyIDes: No\'el regulators of euka:ryotic cells. Trends Biochem. Sci. 15: 195-198. Kinzler, KW., Nilbert, i\I. C., Su, L-K., Vogelstein, B., Bryan, T. M., Levy, D. R, Smith, KJ., Preisinger, A. C., Hedge, P., McKechnie, D., Finniear, R, Markham, A., G:roffen, J., Boguski, M. S., Altschul, S. F., HOlii, A., Ando, H., l\fiyoshi, Y., 1\1iki, V., Nishisho, I., and Nakamura, Y. (1991). Identification of FAP genes f:rom chromosome 5q21. Seience 253: 661-669. Koken, M. H. 1\1., Reynolds, P., Bootsma, D., Hoeijmakers, J. H. J., Prakash, S., and P:rakash, L. (1991a). Dhr6, a Drosophila homolog ofthe yeast DNA-repair geneRAD6. Proc. Nat{. Acad. Sei. USA 88: 3832-3836. Koken, M. H. M., Reynolds, P., Jaspels-Dekker, 1., Pcakash, L, Prakash, S., Bootsma, D., and Hoeijmakers,J. H.J. (1991b). Structural and fllnctiollal conservation of two human homologs of the yeast DNA repair gene RADS. Proc. Not{. Acad. Bei. USA 88: 8865-8869. Kudo, M., Sugasawa, K, Hod, T-A., Ellomoto, T., Hanaoka, F., and Ui, 1\1. (1991). Human \lbiquitin-activating enzyme (El): Compensation for heat-labile mOllse El and its gene loealization on the X ch:romosome. Exp. Cell Res. 192: 110-117. Landegent, J. E., Jansen in de \Val, N., Van Ommen, G-J. B., Baas, 1- - -:::,
encoded human proteins are bath structurally and functlonally hlghly conserved: they share approximalely 70% sequence Idenlity with S.c8r8vlslae RAD6, are able 10 ublquitinate hlstones In vi/ro, and both human gene produets can suhslitule for Ihe mulagenesls and UV reslslance functIon of Ihe yeast proteln, but nol for ils mIe in sporulalion. This lalter funcUon rsqulres In S,cerevlslae an acldle C·termlnal extension, In S.pombe however, liks In Ihs Drosophl1a and mammalian homo!ogs, Ihe aeldle laU Is absent and not needed lor sporulation (Koken et ar., 1991a and 1991b; Reynolds et alo, 1990; Schneider et al" 1990). The subcellular localizaUon of HR6 In the euchromatic regIons of the nucleus (Koken et al., 1996) suggests th at lts funclion Is related to active chromatfn conformation. Bath mammalian genes are expressed In alt organs and tissues and are nol subjecl la milotic cell cycle regulaHon. Furthermore, expression of bath genes Is elevaled in mouse spermatids (post-meioHc spermalogenic cells), colnelding with Ihe developmenlal sleps al whlch a complex series of chromalÎn modlflcalfon events takes place (Koken et at, 1996). These events Involve replacemenl of somatic and leslfs-specJflc hlstones by transition proteins TPl and TP2, and subsequently by prolamines Pl and P2 (Barhom, 1989; KIstIer, 1989; Meistrieh, 1989). In rat spermatlds, occurrence of highly acetylated hislone H4 Is 'ound la be associaled wilh hislone displacement (Melslrich el aL, 1992). Ublqullfnalfon of hislones and olher nuclear proteins mlght also be Involved in Ihis process, because ublqulUnatlon of hlslones has been observed during chicken and Irout spermalogenesls (AgelI et aL, 1983; Agell and Mezquila, 1988; Nickel el ar., 1987; Oliva and Dlxon,
1991), Studies on Ihe blological and molecular funcUon of HR6 and other enzymes implicaled in the ubiquilin palhway in higher organlsms Is hampered by lack of mulanls. The central role of RAD6 In multiple
96
Figure 1. Targeted disruplion ol Ihe mHR68 gene by homologous recombination. (A) Genomlc organlsallon and disruption slrategy lor mHR68 showing Ihe gene, Ihe largeling construcl and Ihe largeted mHR6B aIleIe. The neo cassette Is lnserted in Ihe Sen site ol exon 1, Introduc!ng a diagnosllc EcoA! site. Note thai insertion ol Iha domlnanl marker disrupls Ihe gene Immedialety behlnd Iha AlG translallon iniliaHon codon. Shown ara tha relevant restriction sites (E, EcoAl; S, SaA; N, NsA; K, Kpr/.; V, EcoR.V; P, SphI). Iha posiHon ol the 3' probe and Ihe neo proba lor Soulhern blol analysls are indicated abova tha mulated locus. Unes on top and bottom Indlcata tha estlmaled Jength of the Iragmants dataclad In Southern blol analysls ol EcoR/ digested DNA. Roman numerals mark tha axons. (B) Soulhern analysls of EcoR/ digested DNA from nlne littermates alter hybridization with Iha 3' probe. Ihe position ol tha wild·type allele (6.8 kb) and Ihe targeted allele (5.4 kb) are Indicaled. (C) Western blot analysls ol testes ax/ract of wild·type (+I+), haterozygous (+/-) and homozygous mulant (./-) animais. In the left panel mHR68 protein was detected uslng tha anUserum ralsed agalnst Ihe C·termlnus ol mHR6BIIlHR6B (u-A81). In Ihe right panel Iha raactlon with Ihe u-RAD6 antiserum Is prasented. On Iha lalt of both panels the position ol mHR6B Is Indicated (aHow). On Ihe right slda the positIons of the relevant molecular weight markers ara shown.
processes makes iI an inleresting large 1 for generaUng a knockoul mouse mulant. Here we demonstrale Ih al mlce deflclent for the murine verslon of HR68 {mHR68} are viabIe and phenotyplcally normal, presumably due 10 functional redundancy wllh mHR6A. The mHR68-delicienl male mlce, however, are InfertiIe, whereas mHR68-deficienl females show normal ferillily. The defect in spermatogenesis is consistent wilh impalrment of Ihe complex post-melotic chromalin remodelling process, and provIdes evldence for Involvemenl of the ubiquitin palhway In chromatin dynamics. Moreover, our flndlngs may have clinical Impllcatlons for underslanding male inlertility in man. Results Maln features of the mouse HR6B gene and
cONA Ta permit Ihe design of largeting conslructs, mouse cDNAs and the correspondlng gene were Isolated using cross-hybridizatlon to a human HR6B (hHR68) probe. Ta lacHJtate homologous recombination wilh high efflclency, the mouse homolog of RAD6 (mHR68) was cloned from a À phage fibrary of genom!o mouse siraln 129/Sv DNA, Isogenlo 10 Ihe embryonal slem cell IIne used lor gene targetlng. The hIgh conservation of the gene Is apparent Irom the finding thai its predieled amino acid sequence is completely conserved belween mouse and man. A nOlable feature Is the 100% conservation ol a sequence ol al least 309 base pairs in Ihe 3'UTR of the mHR68 mRNA belween all mammals investigaled (man, mouse, rat and rabbit). Thls slrelch corresponds with nucleoli des 575 10 884 of the publlshed human cDNA sequence {Koken el al., 1991b}. Ta our knowledge th Is represenls the longesl nucleotide stretch slrictly preserved over such an evolulionary dislance. The function ol thls excepUonally stabIe, non-coding
nucleot!de sequence elemenl is unknown. Figure lA presenls Ihe architeclure of Ihe murine m H R 6 B gene. The gene spans a region of approximalely 15 kb and Is comprised of six exons. Interestlngly, Ihe locatIon of two inlrons Îs exacUy preserved In Drosophifa and even S. pombe (Koken et al., 1991a; Aeynolds el al., 1990), presumably reflecting a high imporlance for the gene. The gene was mapped on mouse chromosome 13 In a region syntenic wilh human chromosome 5, and evidence was oblained for a pseudogene on mouse chromosome 11 (Aoller et al., 1995). Inactlvatlon of the mHR6B gene and generatIon of mouse mutants In deslgnlng a knockoul targeling construct, we envlsloned the possibility thai any Iruncaled mHR6B prolein may exert unpredlclable elfecls. Particularly Ihe highly conserved N-termlnus, encoding a site lor proteln-proteln Interaction, could interlere wilh other processes resulting in semi-dominant consequences. Therelore, we chose to inactivale Ihe mHR68 gene Immediately alter Ihe translational slart codon by insertion ol the dominanlse!eclable neomycln or hygromycln marker, ruling out Ihe synlhesls ol any part of Ihe prolein. The largeUng construct depicled In Figure 1A conlains 3.5 kb and 2.8 kb ol homology al Ihe 5' and 3' slde flanking !he dominant-seleclable marker, respectively. Two verslons, each with a dHferent selectable marker, were constructed 10 permit Inactivation ol both autosomally localed alleles In ES cells. Translecllon of the neo cassette-containing largeling construct (Figure 1A) by electroporation and selection lor sta bIe uplake of Ihe dominant selectable marker gene yielded a frequency ol 16% largeted Iranslormants (27 homologous recombinanls/166 tolal transformanis; no selecHon was applled againsl random integration). Homologous recombinanls were checked tor accurate InlegraUon ol Ihe construct by Soulhern blol aflalysls using exlerna! and inlernal pro bes and were lound to be correct (dala nol shown). The multiple engagemenls ol RAD6 on the one hand and the presence ol a 95% Identical mHA6A proteln on the olher, make 11 dilficuH to predlct a phenolype lor a mHR6Bdeliclent mouse. To find out whelher a homozygous mHR68 inactivaUon Is viabIe, at least at the cellular level, Ihe second aUele was targeted using Ihe hygro casselle-contalnlng construct. The Irequency of largeUng directed 10 Ihe wild-type aUele was 8% (11/143), indlcaling Ihat there was no selection againsl mHR68 InaclivaUon and Ihal inacUvation of both mHR68 alleles Is not lelha!. Therelore, we perlormed injectiofl of ES cells ol Iwo independeni, neomycln-resistanl clones (80 and 134) Inlo biastocysis of C57BU6 mice, resulting In the generation of chlmaeras. Male chimaeras Irom both Independent clones were bred and bolh gave germline Iransmission. Soulhern brot analysis on DNA isolated Irom tal I biopsies was used 10 delermine Ihe genotype of Ihe offsprJng. HybridizatIon with Ihe 3' external probe vlsualized a 6.8 kb EcoAI fragment In Ihe case of a normal allele and a 5.4 kb fragment lor a targeted allele (Figure 1B). Heterozygotes were interbred and yielded homozygous mHR68 mulants wilh Ihe expecled Mendelian Irequency. The results of a represenlative Huer are shown In Figure 18. We verWed that Ihe largeling ol mHR68 Indeed resulted In a null-mutatlon al Ihe ANA and proleln
levels. Northern blol analysis confirmed absence of significant amounts ol mHR68 Iranscripis (dala not shown), indicaUng Ihal Ihe presence ol the domInant marker Inlerfered wlth proper transcription and/or processing of Ihe altered mRNA. The mHA6A and mHR68 proteins, like hHR6A and hHR6B, are 95% Identical and migrate al Ihe same molecular welght In SOS-PAGE. To dlsllngulsh batwaen these highly homologous polypepHdes, we look advanlaga of tha fact Ihat wllhln the 14 C-terminal amino acids, Ihe A and 8 produets djffer al 2 posJl/ons. A peptide Idenlical 10 Ihe 14 C·termlnal amlno acids ol HA6B was synlhesized and utlllzed 10 raise a polyclonal anllserum Ihal specifically recognlzes Ihls proleln. Sloce In testis both proleins are expressed In hIgh quanlities, lolal testis exlracls ware analysed. Figure tC (Ieft panel) shows thai no mHA68 prolein is detecled in mHR68-/- mlce, whereas Ihe proleln Is present In mHR68+/- aod mHR68+/+ lillermales. The decrease in Intensity in Ihe lestis exlract of Ihe heterozygous anlmal suggests that Ihese animals conlain roughly approximately half Ihe amount of mHA6B proleln as compared to Ihe normal animaIs. Thls argues against upregulatlon ol the unlargeled allale to compensate for the loss of expression ol the largeled copy. An antiserum againsl yeasl RAD6, recognlzlng both HABA and HR6B (Koken el al., 1996), shows a positive reaction In the mHA6B'/- sample, indicating thaI the mHR6A gene is expressed (Figure 1C, right panel). These results verily the nuH stalus ol Ihe mHR68 mulation and also show Ihal mHA6A prolein is still present. Phenotyple eharaeterlstles of mHR6B-/- mlee and ce lis The mHR68-1- mlce proved normally viabIe with a lifespan exceeding 14 monlhs. Excepl for Ihe feature dlscussed below, no apparent phenolypical or pathological abnormalilies were lound. Furlhermore, no differences were noled belween Ihe main phenolypic characterist!cs ol Ihe mHR68-1- mice derived Irom the independenUy largeted ES recomblnanls and belween mlce from crossings between different slrains (129xFV8/J, 129x C57BU6). This rules out Ihe possibilily thai by acctdent other genetic alteraUons had occurred Ihal mighl influence Ihe phenotype or Ihal Ihe genetic background Is of major importance. Since AAD6 in yeast accounts for much of Ihe cellular resislance agalnsl a wide speclrum of genoloxlc agenls, we lnvesligated UV aod y-ray sensitivily in mouse cells. To lest UV sensitivity, mouse embryonic fibroblast cell !ines were established Irom mHR68+1+, +1-, and -I- mice and tesled for Iheir cellular survival, as measured by [3HI-thymidine incorporallon, after irradiation w!th different doses ol UV. For y-ray sensillvlty Ihe doublelargeted ES cell line was irradialed and cloning efficIency was compared wilh irradlated, nontargeled ES cells. No differences belween mHR6B·deflclent and -proficlenl cells were observed for Ihese DNA damaging agenls (dala nol shown). Thus no overl defect in DNA repair was delected. Thls Is possibly caused by a redundant effect ol a lunclional mHR6A gene. Spermatogenesls In mHR6B-deficlent mlce In breedlng experimenls, lt soon became apparent that Ihe mHR68·1- male mice were conslslently intertife. Copulatory behaviour was judged 10 be normal,
97
Figure 2. Testicular hlstology of normat and mHR6B knockout mlce. The hlstologlcal secUolls were prepared as descrlbed In Experimental Procedures, and stalned with periodic acid Schiff (PAS). The p:lI1els to the lelt (A,e,E) show the lesticular hlslology of norma! m!CXl, Ihe panels 10 the right (B,O,F) thai of knockoul animaIs. A.B: a·day-old mlCXl (x400): C,D: 4o-day·old mIca (x200); E,F: g-month-old mIca (x200).
and copulation plugs were lound, but none of Ihe tested males induced pregnancy in lerWe lemales (out ol at least 27 malings with 11 knockoul ma1es no pregnancJes were recorded). Histologlca1 evaluation of the testes and epldidymldes ol adult mHR6B·j- males showed astrong derallmenl of spermatogenesis (> 10 males Investigated). However, conslderable varlalion in the severily ol Ihe deficiencies in different adult mlce was observed, Involving early as weil as laler steps of spermalogenesis, precluding Identification of the exact slep at which spermatogenesls Is affected. Therelore, the onsel ol spermalogenesls was closely 10110wed in these mlce. In Immature mHR6B-j- mlce, an intact lubularslruc-
98
lure with normal development ol Sefloli cells was observed (Figure 2A and B). Subsequently, in!!iatlon of spermalogenesis showed no over! abnormal!tles, with proper development of sparmalogonia, and timely onsel and progression ol Ihe meiolIe prophase and divisions. It is unlikely that mHR6B is indispensable lor meiosis, a[so because Ihe mHR6B-j- lemales showed normal lerlillty (not shown). Clear signs of spermatogenlc tallure were observed when Ihe flrst waves ol spermatogenic ce lis reached the more advanced sleps of spermiogenesis, In 4 10 5-week-old mHR6B·j· mice (mlce analysed al 8 days, 2.5, 3.5, 4.5, and 5.5 weeks). In general, Ihe spermatogenlc epilhellum slarted to show a number of trregufarities,
Tabls 1. Body anti organ weighls, and epJdidymal sparm counlln norrnaJ and mHR6B knockout mica. Intact' Knockout .. (Mean±SD) (mJce no. 1;2;3) Body welght{g) 44 ±6 40; 49; 63 Testis (mg) 99 ±17 55; 26; 48 Eplcfldym!s (mg) 42 i5 38; 29; 39 Semlnal veslclss(mg) 109 ±16 86; 79; 109 Sperm count (xt0 6 ) 15.5±2.7 0.9; -:0.1; 0.9 'Contro! group conslsted of flve 8·month·old mlce (r.vo +1+ and three +/') "Individual data of three 8-month·o!d .,. mice (no.l;2;3)
Includlng Ihe formatIon of vacuoles wilhln Ihe epithelium and shedding of Immature germ cells, In parlJcular round and more advanced spermaUds. Figures 2C and 20 show histo!ogical sacllons of testes from contral and knockout mica isolaled at Ihe age of 40 days. From Ihls point on, heterogenelty in tesUcular hlstology and variation in regres sion of spermalogenesis was observed between Individual mlce. OccasionaHy (in 10 - 20% of mHR6B-/- males) nearly lolal absence of all germ cell types was found (Figure 2F), bul in most knockout malss we regislered ongolng sparmatogenesis with only low numbers of predomlnanUy abnormal spermatozoa (see below). A marked, but variabIe reduction In testis weight (Tabla 1) Illustrated Ihe pronounced overall regression of spermalogenesis, allhough Inler·lndividual helerogeneity was apparent. In mHR68-/' mice #1 and #3 (Tabie 1), the epldldymls welghls were not signrflcantly decreased, despite Ihe fact thai Ihe
A
B
c
epldidymal sperm counts were less Ihan 10% of the numbers found in mHR68+1+ and +/- mlce. Thls Is probably explained by Ihe abundant presence of Immature germ ceUs in the epididymal lumen (Figure 5; compare C and 0). Epldidymis welght of mutant mouse #2 was lower, due 10 the complete absence of germ ceUs. Mutant mlce #1 and #3 still contalned many immature germ ceUs In the epldldymal lumen_ Seminal vesicle welght is an excellent marker of long-term testosterane aCllon, and Ihe data In Table 1 therefore indlcate Ih al the plasma testosterone concenlration In Ihe mHR68 knockout mice was malntained within Ihe normal range. Furthermore, the plasma fOllicle-sllmulallng hormone (FSH) concenlraUon was not different between mHR6B· deficient mice (37, 38, and 51 nglml in three mice) and inlact mice (40 ± 6 nglml in five mice). To study Ihe remalnlng spermatozoa of mHR68-/mice In more detail, morphology and molillty were examlned uslng Nomarski optfcs of unfixed material and phase contrast mlcroscopy, respeclively. In knockout mice more Ihan 90% of Ihe spermatozoa were c!early morphologicaUy abnorma!. At leasl 70% of these spermatozoa had an aberrant head morphology, In most cases comblnad wllh middle piece deformalJon (see Figure 3). Moreover, the residual spermatozoa appeared almosl Immotile: a few spermatozoa {about 5%} displayed a slugglsh progressfve or nonprogresslve molJlity. These flndings confirmed Ihal Ihe mHR68 gene knockoul does not cause a complete and uniform block of spermalogenesls al a givenpoint in adult animaIs.
o
E
F
Flgure3. Norrna! (A) and abnormal (B-F) ffiOrphology ol spermatozoa from mHR6B knockout micEl. Ths spermatozoa warEl col1ecled from ths cauda epldidymls, and photographed without lixalion uslng Normarski opties (x400)_
99
Figure 4. SchematIc presentation of Iha hls·lone-Ic-
protamine replacemant in mousa spermallds. Ths flgure 10 Iha lelt Is a schamaUc representation of a part of a cross-section of a tubule at Stage VI cf Ihe spermaloganic cyele (Russeli et aL, 1990), showing Ihs lntetrelalionshlp balween a Sertel! cell (S), spermatogonia type 8 (B), pachytene sparmatocytes Hislones (IH2B)c-.""!,,,,,!,_~ lP), (ound spermatlds Siep 6 (6), and condenslng • spermatld$ Siep 15 (15). The righl part of lhe figure shows selecled sleps of spermaUd davelopmenl Transition proleins 1 end 2. . .~_~ (Steps 8,10,13, and 15 of spermlogenssls). Tha bars ProlamInes 1 end 2 rapresanl (Irom [elt 10 right) Ihs following: testlsspeeme hlstone 28 (tH2B) Is present In tound spermatlds (and In spermaIOl:ytes), but Iha rmmunoa)lprasslon ol thls prolain Is incraased ~n elongaling spermatids Sleps 9·11 (Unnr at al., 1995); nuclaar depositIon ol translUon proteins 1 and 2 (TP1 and TP2) ocrors In condenslng spermatids Sieps 12·14 (Allonso and KlstIer, 1993), lollowed by rep!acamant ol Ihe TPs by Iha pro!amlnas (P1 and P2). Round
Elonga\Ïng
Condenslng
Steps 9-11
St-ep512·14
The low number of ceUs al Ihe critlcal step where the fjrst abnormaHties we re seen precluded blochemical analysls of these ceUs in the mHR68'/mlce. However, Ihs Impairment of spermatogenesis In these mlce was deflned more precisely, using Immunohistoehemlslry. As an intro duel Ion to these studies, a brief descriptIon ol chromalin rearrangemenl durlng spermatogenesis, in part!cular during the post-meiolie development of spermallds {spermiogenesis}, Is presented (see also Figure 4). Spermalogonla, prolileratlng through mltolic divlsions, contaln somatIc histones. Wilh Ihe progression ol spermalogenesls, a number ol lestlsspecIfIc histones (IH2B) are synthesized, mainly In prlmary spermatocyles, during the prophase of Ihe melollc divisions. The round and elongaUng spermatids conlaln a mixture of somalic and leslis·specHic histones (Brock el al., 1980; Meislrich el al., 1985). Following the elongation ph ase (Sleps 9-11 ol spermiogenesls In Ihe mouse), Ihe elongated spermatids slarl wlth Ihe process ol nuctear condensation (Steps 12-14), involvlng Ihe synlhesis of IransUton proteins 1 and 2 (TPt and TP2) and protamines 1 and 2 (PI and P2). The transition proleIns appear in Ihs nucleus al Step 12 and are lost at Slep 14 when lurther condensation ol Ihe nucleus takes place, concurrent with Ihe nuclear deposItion ol Ihe protamines (Allonso and KisUer, 1993; Kisller, 1989; Melslrich, 1989). Teslis·speclfic histona H2B (IH2B) is synthesized and deposlted onlo Ihe chromatin, beginning in early primary spermalocytes (Brock et al., 1980; Me!slrich el aL, 1985). n represents a good marker lor Ihe elongatIon phase, showing Intense immunostaining, due 10 increased accessibillty of Ihe epitope In spermatids (Unnl et al., 1995). Figures 5A and SB show that Ihe IH2B·lmmunoposilive spermaUds Ihat remaln presenl In Ihe lestis ol mHR68-/mlee, display an Irregular orienlatlon and distribution, In contrast to Ihe well-organized slruclure of the spermalogenic epithelium In control mice (Figure 5A and B). Inleresllngly, tH2B-lmmunopositive cells were also delecled in the lumen of the epididymls ol mHR68-1 mice (Figure 50). These ceUs were virtually absent In Ihe epidldymis Irom Intacl adult mice, wh1ch was lilled wllh mature spermatozoa (Figure SC). Many ol Ihe epldldymal tH2Blmmunopos!live ce lis are round and elongating spermatids thai have been prematurely released Irom Ihe spermatogenle epithelium and have nol undergone lurlher elongatlon and nuclear condensation. Immunoslalnlng wilh an anl1body against TP2 showed pronounced slalning of elongatedJcondenslng
100
Condensed St"9.1~16
spermatids, at Steps 12-14 of spermiogenesis (Allonso and Kistter, 1993). In control mice, these spermatids are arranged in groups ol eeUs and in a regular paltem, al Stages XII and of Ihe spermalogenlc cycle (Figure 5E). In mHR68-1- mlce, a relatively smal! nu mb er of elongated spermatids showed TP2 Immunostalnlng, and a proportion ol these ce lis showed abnormal morphology and were not weil posilloned wlthln Ihe spermatogenic epllhelium (FJgure SF). Our lindings indlcale that mHR68-1- mice syntheslze TPs, bul Ihat Ihese proteins are not unlformly localed in the nucleus as observed during normal spermalogenesls. Since Ihe general picture Is an overall impairment of spermatogenesis as a consequence of a primary delect in the elongation stage of spermlogenesis, we Investigaled whether apoptosls is elevaled In mHR6B-I- mlce. Figure 6A shows sections through seminlferous tubules of testis ol 6-waek-old mHR6B+I+ and -I· mlce stained using Ihe TUNEL assay. A 4·lold increase In the number ol apoptotic cells was calculaled and represented as Ihe number of poslt1vely-stalned ceUs per 100 tubull (Figure 6B). Moreover, Ihe apoptotic cells were cluslered and predomlnantly locallzed in the germ cell layers Ihal contain primary spermatocyles. These data Indicale an elevaled level ol apoptosls as a consequenee of mHR68-deficlency.
'-lil
DIscussIon In spile ol the pleiotropIe lunctions and lundamenlal Importance of the ublquitin system, no mammalian mulants aflected In this palhway are avalIabIe that reveal Ihe biologleal ramificalions and impact ol Ihis process al Ihe level ol the organism. In Ihe present reporl, we describe Ihe phenolype of mice deflclenl In Ihe ublqu1t!n-conjugating enzyma mHR6B. In bolh ES cells and in mlce, Ihe 10ss ol function of mHR6B is compalible wilh viabllily. Although yeast rad6 deletion mutants are viabIe, Ihey display a severe phenolype. The finding thaI this is not the case In Ihe mHR68 knockout mouse can ba explained by funclivnal redundancy ol Ihe HR6A and HR68 gene produels. The hHR6A and hHR6B proteins are expressed to approximalely Ihe same extent in most somaUe ceUs and tissues (Koken el al., 1996). The two gene produets show 95% amlno acid sequence identity, and thus probably calalyze very simIlar reaclions. Furthermore, both proteIns are lunclional and complement the same delecls of a rad6 null allele (Koken et al., 1991b). Apparently, Ihe approxlmal 50% of remaining aetivily derived Irom Ihe mHR6A gene is sufflclenl 10 permil relatively normal developmenL
Flgure 5. Immunohlstochemlcal locallzaUon of tesUs.speciflo hlstone H2B (IH2B) and transitIon proteln 2 (TP2) In lestis and epldidymls ol Intact end mHR68 knockoul mIca. Tha Immunohls\ochemlslry was parformad as describad In Exparimenlal Procedures. The panels 10 Ihe lelt (A,e,E) show tissues Irom a g·month-old Intact mousa, and Ihe panels 10 Iha r1ght (B,O,F) represent a g·month-old mHR68 knockout mOUS9. A,B: aH2B ImmunO$lalnlng or tastis; e,o: IH2B Immunoslalnlllg of epldldymls; E,F: TP2 Immunostalnlng or les\ls (x400).
We lalled 10 observe any defecl In DNA repak. However, Ihls does nol exclude a subIIe effecl of parllal loss of mHR6 actlvlly on mulagenesls and carcinogenesis, whlch remalns 10 be studled. Experlmenls almed al generatlng mHR6Adeflcient mlce, In order 10 assess Ihe phenolype ollhese and full mHR6AlmHR6B double knockout mlce, are In progress. The mosl promlnenl phenolyplc express!on of Ihe mHR6B gene knockoul detecled 10 date is Impairment of spermatogenesis, resulUng In greatly reduced numbers of malnly abnormal spermatlds and spermalozoa. However, In Ihe adult leslis, Ihe
causatlve slep Is difflcult 10 pinpoInt, because of Ihe considerable Inlerlndlvldual variallon In Ihe manifestations, and Ihe facl Ihal early as weil as late steps of spermalogenesls seem 10 be Impalred. Oetal!ed analysls of Ihe first wave of spermatogenesls, however, allowed IdenUflcatlon of the prlmary delective slage: progression Ihrough Ihe elongating and condenslng steps of spermat1d developmenl Is Impalred. Probably as a secondary consequence, earlier steps ol spermatogenesls also become deregulated (see below). Prevlously, we found e!evaled levels of mHR6A and mHR6B mRNAs In spermatids during narmal rat
lOl
A
B
+/+
+/Genotype
Flgufe 6. Analysls of apopiosis In semlniferous tubule cross secUons of slx-week-old mloo. . (A) Nuclear DNA fragmentatlon visuallzed uslng Ihe TUNEl assay. TM upper panel shows a section through a lestis of a homo~gous mutant, end the lower panel Is a teslis section of a wild-type anima!. A clusler of apoplotic eells Is present In the
upper lelt corner of Ihe upper panel.
102
spermalogenesis (Koken el ar., 1996). In fact, HR6A Is the fifS! X-Ilnked gene lor whlch post-malotic expression, In mouse spermatlds, has been documenled (Hendriksen at al.. 1995). In additIon, imrnunohlslochemlcal experiments show Ihe presence of HR6 proteins in Ihe nuclei ol round and elongating rat spermalids. However, n Is Important 10 note thai In two-dimensional Immunoblot analysis of all cells tested, elongating spermatids and spermatozoa were Ihe only cell types In which the mHR6A level appeared slgn!f!cantly ]ower relative to Ihat of mHR6B (Koken el at, 1996). Thus, il Is conceivabJe thai in Ihe absence of mHR6B, the relatively low levels of mHR6A are Insufflclenl for performing Ihe HR6 function requlred In these ceUs. Unfortunalely, the [ow number of elongaling spermaUds in Immature mHR6B'/- mice precludes biochemicai analysis of HR6 activity In this way. AlIhough different hypotheses can be put forward 10 explaln our findings, such as delecls in Sertotl ce1ls, which IIke germ cells, express high levels ol both mHR6A and mHR6B (Koken et aL, 1996), we consider Ihe following scenario most consislent with all observatIons. The nuclei ol early round spermalids conlain a mixture of somatic hislones and testisspecific hlstones. Following elongation of spermaUds. chromatin Is reorganlzed, and the hlstones are rep[aced by transitlon proteins (TPs) and then by protamines (Balhorn, 1989; Klsller, 1989; Meistrich, 1989). Two types ol histone modifIcation have been documenled during spermalogenesls_ In rat spermatids, occurrence of hlghly acetylaled H4 Is associaled with h!slone displacemenl (Melstrlch et al., 1992), and during chicken and troul spermatogenesls poly·ublquJtlnation of hlstone H2A has been observed (AgelI et aL, 1983; Agell and Mezquita, 1988; Nicke! et al.. 1987; Ollva and Dixon, 1991). In preliminary experimenls we have detected mono- and polyublqultinated forms ol hlstones In nuclear exlracls of mouse spermatocytes and spermatlds (our unpubtlshed observallons). ConsiderIng Ihe abllily ol RAD6 10 polyublqultlnate hislones In vi/ro, and ils in vivo role in yeast sporulalion, Ihe most plauslble hypothesis Is thai. in mammalian spermalids, Ihe lunclional homologs of RAD6 poly-ublquilinate h!stones. This aHows for thelr degradation and replacement by transmon proteins and, subsequently, by protamlnes. A shortage of Ihe enzyme at thls critica I stage could Interlere wlth thls process. However, It remains 10 be shown that the ubiqultinconjugatlng enzyme actlvlty targeUng speclflc histones In spermallds Is, befow a critical threshold level In spermatids from mHR6B knockoul mice. Spermatogenesls Involves elimination and modiricatlon of many proleIns. A novel ubiqulUn-conjugating enzyme E2 (E2 17kB) was recenlly also found to be highly expressed In testis (Wlng and Jain, 1995). In additIon. the Y-chromosomal gene Sbyor Ube1y, encodlng ubiquilin-acllvaling enzyme Ei, shows testis-specUlc expression In Ihe mouse, and Is consldered a candidate spermatogenesls gene (Kay et (B) Quantilicallon of apopiosis In wild·type (+1+), helerozygous (+I-) and homozygous mutanl (-1-). Slides ware randJmly manoeuvred under a Ilght microscops and all apoplotic ceHs presenl In at laast 100 lubuIa cross sections were counlad and divldad by Ihs numbar of lubulas< These dala ware reca!culated 10 glve Iha number of apop!otlc caHs per 100 lubuias cross secUons.
aL, 1991: Mitchall al aL, 1991). TasUcular axprasslon Is dapendanl on Iha prasance of garm caUs (Mitchall at al., 1991) and Ube1y mRNA has been detected in round spermatlds (Hendriksen et at., 1995). The homologous gene on Ihe X chromosoma (Sbx or Vbe 1x) Is expressed In all male and female tissues (Milchell at aL, 1991), and also In spermatogenlc eeUs (Handriksen et alo, 1995). Assuming thai disturbance of chromatin remooeting in spermatids of mHR68daftclent mlce is the primary cause of lhe InfertJltty, how can thls defect laad 10 formatIon of vaeuo[es In Sertoll ceUs and the release of Immature garm ceUs? A clue Is provided by a recent finding on the effect ol ectoplc expression of avlan protamine (galline) in spermatids of transgenlc mlce. This expression induces dlsruptJon ol the norma! dense chromatJn slruclure of spermatozoa, and resulls In Inlertllity (Rhlm et at., 1995). As lor mHR68'/' mlce, the spermatogenlc epithelium of these transgenlc males showed many vacuoles and loss of Immalure germ ce lis. Thus, disruption of chromatIn conformatfon by ectopic prolamIne expression leads to very similar types of spermatogenlc abnormalities as observed In mHR68'/' mlce, In agreement wilh Ihe Idea that HR68 deficiency affects chromatIn conformatIon. Posslbly, Sertoli eeUs are adversely affecled by degenerallng late spermatlds. These spermalids mighl release protamines, whlch are known to exert toxlc effects on epithelial eelis (Peterson and Gruenhaupt, 1992). In eoncordance wJlh Ihis, Ihe clustered apoptosls of primary spermatocytes In lestIs of mHR68-j - mlce (Flgure 6) pOints 10 local Sertoll eeU damage, It Is not elear why most ol the male mHR68 knockout mlce show productIon of spermatozoa wlth a wlde range ol morphological abnormallUes, Other defects In spermalogenesis affecllng spermatocytes rather Ihan spermallds, can also give rise 10 abnormal spermatozoa, Such a defect In spermalogenes!s was observed In mice Ihal we re mulaled in Ihe DNA mismatch repak gene pMS2 (Baker et al., 1995). Thls defecl resulls In abnormal ehromosomal synapsis in meiosIs and male lnfertilily, wilh production of a smalt number of spermalozoa with abnormal morphology. In a conslderable number of male inferlilily patlenls, the cause of Ihe InferUlily mighl be relaled 10 dlslurbance ol the hlstone-to-protamlne replacemenl during spermalogenesis, Several reports deseribe Ihal sperm Irom InfertIIe men can show abnormal protein compiemenIs, wllh persistent elevaled levels of hlstones and/or an allered protamine P1/P2 rallo (Chevailtler et al., 1987; De Yebra et ar., 1993; Foresta et al" 1992). NOlwilhstandlng the relative genetIc uniformlty of HR68- j - mlce, a marked variatIon ol testis hlstology and sperm morphology was observed. The pronounced vartabllity in features is reminiscent of the test/cu lar manjleslat/ons assoclaled wllh Infertility in man. The lacl, Ihal an HR68 defecl In mice ean be transmilled not only by helerozygous carriers bul even by homozygous knockout temales enhances Ihe posslblllty, that Ihe ldentlcal human enzyme may be Impllcated In male inlerliilly eondillons, Probably as many as one In three ol alt cases ol human male inlerllllty are ol unknown testlcular orlgin, These cases cannot be explalned by chromosome abnormaliUes, endocrine dysfunclion, elc. (Wong et al., 1973). In unexplalned male inferllllty, there is often Ihe productIon ol a low number ol spermatozoa (o!lgozoospermla) and/or abnormal sperm
morphology (teratozoospermia) (Aitken el at., 1995), Severa! haltmarks ol thls variabIe condilJon are shared with mHR68-j - mlce. Apotentlal Involvement of a defect In the ublquilin-palhway in cases ol human male Infertllily Is presenUy under lnvesligalion. A linal implication from Ihe flndings reported here Is Ihe parallel emerglllg belween spermalogenesls In mamma!s and sporulalion in yeasl (Game el ar., 1974, Montelone et at., 1981), The laller process also appeared to be accompanled by gross changes In chromaUn conlormatlon In whlch RAD6 may play a simIlar role as HR6B in higher organisms. Interestingly, the yeast UBC1 enzyme Is found 10 be requlred for recovery of growth alter germInation of ascospores (Jenlsch, 1992), Thls enzyme may thus accomptish Ihe reverse ol Ihe reacllon eatalysed by RAD6, namely Ihe decondensatlon of chromatin, Experlmentel
Procedures
Isolellon end sequence of murine mHR68 cON A clones A 784 bp H/nalll·BamHi cONA fragment conla!nlng Ihe complele open reading frame (ORF) of Ihe human hHR68 gene, !ncluding 5' and 3' lIanking sequences (176 and 149 nucleoUdes, respectively) (Koken el ar., 1996), was used 10 screen an 129/01a mouse testis library (A ZAP) for hHR68 homologous mouse cONAs. Seven positive plaques were Iso!aled of whlch 2 contalned the complete ORF, The nucleotide sequenea of the ORF ol mHR6B and (part ol) Ihe 3' unlranslaled region ware delermlned uslng T7-po!ymerase (Pharmacla Blolech, Uppsala, Sweden) and deposlled In Ihe GenbankJEMBl nucleotide sequence dalabase under accession number X968S9. ConstructIon of the mouse mHR6S-targetlng vector and Iransfeelion An EMBl-3 À phage genomlc library construcled Irom tha CCE ES caU Hne derived from mouse siraln f29/Sv (a kind gift of dr. G. Grosveld) was screened with Ihe 784 bp human HR68 cDNA fragmen\. Posiliva genomlc clones wera rescreened with a y,32P-ATP labeled primer, complementary 10 nucleolides 28·69 of Iha mouse HR6B coding region, A genomlc clone was lsolated, deslgnated G28, encompasslng Ihe exons encoding Ihe 5' end of lhe mHR6B COding region. Thls genomlc clone was digested with SaA and subcloned In pTZ19R (Pharmacla Blotech), The two SaA subclonGs flanklng Ihe Salt·site at the 5' end of Ihe ORF, were cloned Inlo Ihe vector pGEM-7Zf(+) (promega Corp., Madlson WI), In Ihls way. a unlque Kpnl·site was crealed allhls position. A cassetIe with Ihe neomycln reslslance gene driven by Ihe TI(promoter (Thomas and Capecchl, 1987) was Inserted allhls Kpnl site, resulting in a targeting vector wilh 3.2 and 3.5 kb of homologqus sequences flankln9 the mulatlon al Ihe 3' and 5' poslUon, respectively. Thls nao cassetIe was Insarled In Ihe anllsense orlenlation wilh raspect 10 Ihe IranscripHonal orienlatlon of Ihe mHR68 gene. The resu!ting plasmld was tinearized wilh Ns~, reducing'lhe homologous region 3' of Ihe neo cassette 10 2.8 kb, and electroporated Inlo 129fOla-de
