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Yeast as an honorary mammal
Mutation Research 451 Ž2000. 1–11 www.elsevier.comrlocatermolmut Community address: www.elsevier.comrlocatermutres
Editorial to Special Issue on Yeast DNA Repair and Human Implications: Past, Present and Future Perspectives on DNA Repair in Yeast
Yeast as an honorary mammal
To those who have had the opportunity to devote much of their research careers to the study of what used to be referred to as a simple or lower eukaryote, there has been the pleasure of watching often esoteric research turn into highly relevant investigations of human biology and disease. Aging, cancer, neurological disorders, and immunological defects are just few of the frailties that can be traced to DNA repair. Our little friends, the budding yeast Saccharomyces cereÕisiae and the fission yeast Schizosaccharomyces pombe have been the molecular and genetic workhorses for much of our understanding of DNA repair in eukaryotes. They have provided insight into the integrated circuits that connect initial DNA injury, lesion processing, cellular checkpoint response, and eventual genetic consequences, resulting in an increasingly detailed view of the maintenance of DNA integrity and control of genetic variation. In essence, they have provided a holistic portrait of cellular damage response. Human genetic defects associated with repair can often be addressed directly in yeast because of evolutionary conservation of genes and systems. In this Special Edition, we have tried to cover all the different systems which the two yeasts use to repair or limit the effects of damage to DNA. We saw these at first as distinct systems: nucleotide excision repair, double-strand break repair, DNA synthesis in repair and mutagenesis, mismatch repair, base excision repair, photorepair, and checkpoint control. Many of these processes are, in fact, integrated and extend to normal cell activities. Therefore, we include damage-independent DNA transactions that are a normal part of the yeast lifecycle, 0027-5107r00r$ - see front matter. Published by Elsevier Science B.V. PII: S 0 0 2 7 - 5 1 0 7 Ž 0 0 . 0 0 0 3 6 - 1
namely meiotic recombination and telomere maintenance.
Linking the past with the present Much of the early DNA metabolism and repair work in yeast was guided by the even more accessible bacterial and phage systems that provided rapid experimental turnover and relative ease of genetic manipulation, in particular the ability to expose genomic DNA separately from cells. These systems led to the identification of several important repair phenomena: host cell reactivation Žhcr.; multiplicity reactivation; photoreactivation as a process that removes DNA damage; damage-induced reactivation ŽWeigle reactivation.; and the isolation of genes controlling transformation efficiency in Diplococcus pneumoniae. These experiments foreshadowed much of the current array of repair systems identified in prokaryotes and eukaryotes. The link between recombination and repair was prompted in part by studies in another eukaryote, Ustilago maydis. Robin Holliday had proposed a recombination model that implicated excision repair-type enzymes, such as those that had recently been found in Escherichia coli w1x. This model was followed by his isolation of UV-sensitive, recombination deficient mutants. In E. coli, furthermore, it had been found that Recy mutants were sensitive to both UV and ionizing radiation w2x. The Holliday model for recombination along with the bacterial studies on repair mutants inspired Nakai and Maysumoto w3x, Snow w4x, Moustacchi w5x, Resnick w6x,
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Averbeck et al. w7x and Cox and Parry w8x in the first mutant hunts for genes affecting UV and ionizing radiation resistance in Sac. cereÕisiae. Naively, perhaps, we assumed that if enough mutants with a radiation-sensitive phenotype were found, some would be involved in recombination. It was indeed gratifying to find that some of the mutants had the expected secondary phenotypes. For example, two of the first characterized mutants were meiotic lethals. In uÕs6ruÕs6 Žs rad6 . diploids, meiosis aborted before spore formation and in uÕs5ruÕs5 Žs rad50 ., spores formed but were inviable. These were considered significant phenotypes in view of the important role chiasma formation plays in meiosis. The uÕs6 mutants also turned out to be totally refractory to induction of mutations. The set of X-ray sensitive mutants isolated by Resnick w6x and by Game and Mortimer w9x, now called rad52 to rad57, turned out to be especially good predictors of defects in various aspects of mitotic and meiotic recombination. Subsequently, studies with rad52 mutants provided direct evidence for the importance of damage-induced double-strand breaks and led to the first model that linked DSB repair to recombination w10x. In the beginning, there appeared to be only two repair systems. Nakai and Matsumoto w3x, who isolated one UV-sensitive mutant, UVs-1 Žs rad1. and Xs-1 Žs rad51. an X-ray sensitive mutant, showed that the genes were operating in different repair systems by demonstrating the super-sensitive phenotype of the double mutant to UV. Complexity was advanced by one notch when Game and Cox w11,12x added a third presumptive pathway of repair by showing that rad6 and rad18 were epistatic with one another but synergistic in UV sensitivity with either rad1 or rad51. These groups corresponded neatly with their respective phenotypes with members of the RAD6 epistasis group all being sensitive to both UV and ionizing radiation. It also seemed that they were sufficient to account for the recovery of cells from all measurable UV-induced damage w13x. The passage from three repair systems, which we were happy to characterize as excision repair Žthe RAD1 group., recombinational repair or doublestrand break repair Žthe RAD52 group. and post-replication or error-prone repair Žthe RAD6 group. — all concepts borrowed without much evidence from
the growing understanding of DNA repair in bacteria and bacteriophages — to the much more complex picture now being painted has been full of surprises and accompanied by a growing revelation of the conservation of DNA repair in yeasts and mammals. Firstly, there was a growing list of mutants isolated with different phenotypes relevant to repair, recombination, and mutagenesis. Jeff Lemontt isolated mutants defective in spontaneous mutation Ž reÕ1 to reÕ3 . w14x. Louise Prakash isolated a large number of mutants sensitive to methyl methane sulphonate Žmmsy. , identifying 20 or so loci differing from those already involved in repair w15x. Ethel Moustacchi isolated mutants sensitive to psoralen, implying a repair system able to deal with DNA cross-links w16x and Mike and Shelley Esposito initiated a genetic analysis of meiosis by isolating mutants defective in sporulation, fittingly designated spoy w17x, some of which are involved in meiotic recombination itself. An important gene XRS2 that has subsequently been shown to be essential in meiotic recombination and is part of the MRE11 associated complex, was also isolated in a broad screen of radiation sensitive mutants w18x. Many other genes have arrived in the DNA repair category by other routes, among them MRE11, SSL2 Žs RAD25 ., DMC1, SPC1rMEC2rBAD1 Žs RAD53 ., SRS2 and SGS1, PMS1, PMS2, and PMS3. Secondly, biochemical approaches have identified enzymes with activities similar to those found both in mammals and bacteria which either reverse alkylation damage in DNA or repair it by excising the relevant base and replacing it with a normal one-base excision. These included transferases, DNA glycosylases, abasic endonucleases and apurinic lyases Žreviewed in MEMISOGLU and SAMSON 1 .. Finally and appropriately, an important set of yeast repair genes has been identified not by mutagenesis or biochemistry, but by their sequences. These include many of the genes involved in mismatch repair, which have been cloned by low-stringency PCR based on either mammalian or bacterial homologues which had already been cloned. Genes identi-
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Reference to reviews in this Special Edition are listed in bold as authors only.
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fied this way include the set of mutator homologues MLH1 to -3 and MSH1 to -6. Three genes in this category had already been identified by mutation: PMS1 Žin the MutL family., PMS2s MLH1, and PMS3s MSH6 ŽBORTS, CHAMBERS and ABDULLAH; HARFE and JINKS-ROBERTSON.. Likewise, RAD26 and RAD30 were identified by a homology search in the yeast genome database using the corresponding sequences of the human genes ŽGAME.. This growth in the numbers of genes identified more modes or systems of repair or damage-tolerance. Base-excision ŽMEMISOGLU and SAMSON., translesion DNA synthesis ŽKUNZ, STRAFFON and VORNAX; BUDD and CAMPBELL., nonhom ologous end-joining Ž LEW IS and RESNICK ., mismatch repair ŽHARFE and JINKS-ROBERTSON; BORTS, CHAMBERS and ABDULLAH. and cross-link repair have all been added to the list of systems established in yeasts in the last 15 years. However, complexity of another kind has become apparent in that many systems can no longer be considered discrete. For example, the endonuclease encoded by the RAD1–RAD10 genes, at first categorized as ‘‘excision-repair’’, also functions in double-strand break repair ŽHABER.. Similarly, the Rad50rMre11rXrs2 complex functions in nonhomologous end-joining, homologous recombination, and telomere maintenance ŽHABER; LUNDBLAD; LEWIS and RESNICK.. Genes for DNA metabolic proteins such as Rpa, Pola , PCNA, and DNA ligase appear to have a role in almost all repair systems. Double-strand break repair is achieved by nonhomologous end-joining and two methods of homologous recombination ŽHABER; SUNG, TRUJILLO and VAN KOMEN; LEWIS and RESNICK.. The two families of MUT gene homologues common to mammals and yeast namely MLH1 to -3, PMS1 and MSH1 to -6, apart from being involved in mismatch repair, play roles in many other DNA maintenance pathways. These processes include replication, excision repair, and homologous recombinational repair. In meiotic recombination, they have the effects both of enhancing homologous recombination and in the presence of excessive mismatches, inhibiting homeologous recombination. ŽHARFE and JINKS-ROBERTSON; BORTS, CHAMBERS and ABDULLAH.. This functional
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diversity highlights them as playing a central role in all aspects of control of genetic variation. Genomic complexity and divergence of function is also apparent in the distribution of members within certain gene families that are involved in different systems of repair and maintenance of DNA integrity. The most remarkable, perhaps, is the proliferation of E. coli RecA homologues. Also interesting is the divergence of function among the nucleases such as S cR A D 27r S pR A D 2r H sF E N 1, S cE X O 1r SpEXO1rHsHEX1, and ScRAD2rSpRAD13r HsXPG. The different complexes participate in Okazaki fragment processing, maintenance of small repeats including triplet repeats, recombination, mismatch repair and in nucleotide excision repair. Similarly the SWI2rSNF2 family, which modulates protein-DNA interactions in an ATP-dependent manner, contains putative helicase members which appear to be involved several repair pathways Žsee Table 1.. Nucleotide excision repair has been found to have two modes, fast repair associated with DNA undergoing transcription and the slower nucleotide excision in non-active DNA. Some of the nucleotide excision-repair genes are common to both modes, some exclusive to the latter. The article by PRAKASH and PRAKASH neatly organizes the complexities by distinguishing between repair genes and repair factors. The latter are the complexes of gene products, which form and then interact to achieve nucleotide excision repair. In addition to these mechanisms, an alternative pathway of excision repair, UVER, operates in Sch. pombe. This itself is apparently resolved by either of the two processes, one similar to long-patch repair in E. coli and the other by recombination. In Sch. pombe, both the NER and the UVER pathways repair 6-4 photoproducts faster than pyrimidine dimers ŽMcCREADY, OSMAN and YASUI.. In the last 10 years, research in DNA damage tolerance and repair has transited from biochemistry of specific proteinŽs. to organizational biology. The most dramatic example comes from the demonstration by Weinert and Hartwell w19x that cell-cycle progression is interrupted by the presence of DNA damage and that this is important for cell survival. The first gene identified as being required for checkpoint control, RAD9 of Sac. cereÕisiae, has been joined by others in Sac. cereÕisiae ŽFOIANI, PEL-
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LICIOLI, LOPES, LUCCA, FERRARI, LIBERI, MASERATI, FALCONI and PLEVANI. and in Sch. pombe ŽHUMPHREY.. Checkpoint genes may act to interrupt progression at G1rS, within S or at G2rM as a result of either damage in the DNA or a replication blockage. The manner in which the systems in the two yeasts map on to one another is quite remarkable. In the matter of damage-recognition and transduction of signal to the Cdc25 and Cdc2rcyclin B proteins that determine transition through the G1rS and G2rM checkpoints, yeast regulatory mechanisms are strikingly similar to human checkpoint controls. The only significant divergence in checkpoint mechanisms is the absence from yeast of p53, a focal point of cell-cycle regulation and apoptosis in mammals. ŽEven so, yeast has proven highly useful for understanding the functions of this human gene since expression of normal and mutant p53 expression can be easily monitored in yeast w20x.. HUMPHREY lists 19 Sch. pombe checkpoint genes and 15 homologues of these from Sac. cereÕisiae. Nine of the former and four of the latter were first isolated by their radiation-sensitive phenotypes. McCready, Osman and Yasui make the point that Sch. pombe depends upon the checkpoint control system for the purpose of damage tolerance much more than does Sac. cereÕisiae, possibly because Sch. pombe cells spend most of their cell cycle in G2. This raises a question about the balance of checkpoint control relative to damage repair in the damage tolerance of humans. The significance of spatial organization has become clear from studies of nucleotide excision repair, mutagenesis, and recombination. Nucleotide excision repair is conducted by the core transcription factor complex, TFIIH, thus coupling excision repair to transcription ŽPRAKASH and PRAKASH. and giving high priority to repair of DNA being transcribed. The transcription factor becomes a repair complex by the interaction with NER proteins. Other proteins direct the same complex to damage in nontranscribed DNA. The discovery of transcriptioncoupled repair in yeast and humans is a classic story of how information obtained in one organism stimulates progress in the other and is then reciprocated. It started with the discovery that one of the genes in Sac. cereÕisiae implicated in excision repair, RAD3, was an essential gene w21,22x and then that the
protein was an ATPase and DNA helicase w23x. From 1986 to 1989, Hanawalt et al. demonstrated preferential excision repair of transcribed strands of DNA, thus coupling excision repair and transcription in both humans and E. coli ŽPRAKASH and PRAKASH and Refs. w17,18,19x therein.. In 1993 and 1994, the link between transcription and excision in both yeast and human cells was established in a series of papers that followed one another so quickly that clearly this idea’s time had come in several labs. In these years, it was revealed that the human homologue of RAD3 ŽXPD. also coded for a helicase, as did RAD25 ŽXPB.; that mutants of both genes were defective in transcription; that both these proteins were constituents of TFIIH in yeast and human cells and that TFIIH was involved in excision repair ŽPRAKASH and PRAKASH, and Refs. w47, 49, 50, 51, 52, 72, 73,74x therein.. It remained only to have the transcriptionrrepair complex re-constituted in vitro the following year to round off a triumphant passage of research based on the application of information obtained in one organism to experiments in another. Spatial organization is also becoming apparent in recombinational repair involving the RAD51 gene product. This RecA homologue is central in the formation of paired homologous strands of DNA and has been found to form double-strand break-dependent foci in mitotic cells and also RAD54-dependent foci in prophase of meiosis. The process of meiosis, which involved many repair genes, is largely a study of spatial organization and the formation and dissolution of complex organelles like the synaptonemal complex and chiasmata ŽDRESSER.. In his article, HABER raises the question of whether the location of chromosomes within the nucleus is altered by damage such that a search for other broken ends or homologous sequences may be carried out more efficiently.
Humanizing repair in yeast The relevance of yeast repair studies to mammalian systems has been clear for some time and as with checkpoint controls, the extent to which they resemble each other is encouraging and has already
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proved very useful to research in both yeast and mammals. Presented in Table 1 is a list of many of the yeast and repair genes and their corresponding human homologues that deal with UV and gamma ray damage. It is not meant to be exhaustive, but instead to demonstrate the commonality of repair and consequences of mutations. There are likely to be hundreds of genes associated either directly or indirectly with the many types of DNA repair. The similarities in sequence homologies allows genes and functions found in one organism to be identified in others. There is a commonality of organization of processes between different organisms, with the same proteins appearing in the same protein and nucleoprotein complexes. Our Table 1, which focuses on the radiation repair pathways, complements the article by GAME, which presents an overview of the background and the current state of DNA repair studies. He approaches the topic with a geneticist’s point of view, which allows one to appreciate repair better as a holistic interaction of many systems. This is a nice contrast with the more reductionist and analytical approach of our other contributors. Table 1 largely excludes genes involved in translesion DNA synthesis, checkpoint controls and mutator genes, since the Special Edition articles on these subjects present much better tables than we could aspire to. The use of knock-out mice makes it clear that many sequence homologues play similar roles in mammals and yeast. For example, mouse rad6 knockouts abort spermatogenesis in early prophase I, the identical point at which sporulation aborts in yeast. However, knockouts can sometimes obscure the likely function of a gene. For example mouse RAD51yry knockouts abort as embryos, limiting analysis of their functions by this means. Nevertheless, mammalian homologues of the RAD51 series of genes involved in both double-strand break repair and meiotic recombination in yeast are expressed in testis, as would be expected of genes involved in meiotic recombination. Tissue-specific expression like this is common among mammalian homologues of yeast repair genes and obviously provides an indication of their function and significance. Sometimes, the homologies are not exact. The Xrs2 protein in the yeast Rad50rMre11rXrs2 complex required for nonhomologous endjoining is replaced in humans by a protein only remotely related to it,
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Nbs1p. Mutations in Nbs1 result in Nijmegen breakage syndrome. As expected, there is a greater genetic complexity apparent in mammals compared with yeast. Some yeast genes have more than one mammalian homologue; RAD51 has several, and clearly they are not merely redundant. Surprisingly, the RAD52 gene, which is essential to recombination and meiosis in yeast, has much less importance in mammals cells possibly because of redundancy. While many tricks are available, understanding the functions of human genes within human cell systems is usually limited because of difficulties with genetic manipulation. The yeasts have often provided a bridge because of the evolutionary conservation of genetic function between these organisms, particularly in the case of DNA repair. Two approaches have been used in which yeast provide surrogate assays for human genes, complementation, and interference. Many human or mammalian genes have been identified that can complement yeast repair defects as well as other deficiencies and these are presented in Table 2 Žalso see the XREF database http: r r www.cbi. nlm.nih.gov r xrefdb r pubtools. html.. Included are homologues of S.c. RAD6 ŽHHR6A and HHR6B., S.p. RAD9 ŽhRAD9., and S.c. APN1 ŽhAPE.. Ligase I is essential to many DNA metabolic and repair activities, and the human ligase I can complement a S.c.lig1 mutant. Surprisingly, human DNA polymerase b can complement the MMS sensitivity of a yeast strain with a DNA polymerase d defect. The human flap endonuclease Fen1p has been proposed to function in both the maturation of Okazaki fragments during DNA replication and in base excision repair. The hFEN1 gene can fully complement a yeast rad27 mutant and a nuclease-defective Fen1p has similar effects in yeast to the corresponding yeast mutant w24x. Similarly, it is possible to complement mutants that are deficient in double-strand break recombinational repair, particularly rad50, rad54, and the endjoining mutant Ku70. Thus, the consequences of defects in human genes can be studied in yeast. The complementation approach can be reversed. For example, expression of the Sac. cereÕisiae genes RAD10 w25x can alleviate corresponding defects in mammalian cells. Human genes involved in DNA repair responses can also be examined in yeast through their ability to interfere with yeast functions Žsee Table 2.. This is
6 Table 1 UV and ionizing radiation repair genes of Sac. cereÕisiae, their activities and their homologues in humans and in Sch. pombe, with occasional reference to E. coli, U. maydis and mice Roman superscripts refer to the chapters in this volume in which the genes are discussed and in which appropriate references may be found. Abbreviations and symbols: ) s complex in humans; ) sassociated; CSs Error-free translesion synthesis; EP-TLSs Error-prone trans-lesion synthesis; ER s nucleotide excision repair; FENs flap endonuclease; H.s.s human; mut-sdeficient in induced mutation; NHEJs non-homologous end joining; PRR s post replication repair; rec-srecombination deficient; Rep.s replication; SMC sstructural maintenance of chromosomes; spo-ssporulation deficient; S.p.s S. pombe; TLSs translesion synthesis; UVER salternative excision repair; UBC s ubiquitin conjugation; uvs sUV sensitive; XPs xeroderma pigmentosum; xs ssensitive to ionizing radiation. S.c. Yeast gene
RAD1
II,V ,V I, X
Homologues
Family
H.s.XPF S.p.Rad16
Repair system
X
5 ss endonuclease
ER
3 endonuclease ATP-dependent X X 5 -3 helicase
ER
DNA helicase component of TFIIH putativeATPaser DNA helicase conjugates ubiquitin to histone
H.s.XPG S.p.Rad13 H.s.XPD H.s.TTD S.p.Rad15 ERCC2
RAD4 II
XPC
RAD5 X I REV2
S.p.Rad8
Swi2rSNF2
EF-TLS
RAD6 X I, X V I
HHR6A HHR6B S.p.Rhp6
UBC
TLS
RAD7 II
S.p.rhp7
REV3 X I, X V I RAD8
H.s. REV3
Polj
ER nontranscribed strand EP-TLS
RAD9 X II
S.p.crb2 XIV
DNA synthesis and damage checkpoint
X
Complex
RAD10rTFIIH RAD10rMSH2r MSH3rSRS2r RAD52 TFIIH TFIIH
Ži. TFIIH Žii. RAD23) proteasome26
Mutant phenotype Yeast
Human
uvs
XP
uvs missense mutant is uvs, essential for transcription uvs
XP XP, TDD and mouse TDD model
XP
uvs, mut proteasome26, sub-unit 5r RAD18p
uvs, xs, spo mut
TFIIH
uvs decreased induced mutagenesis uvs, xs
mouse rad6 K.O. is malesterile at meiotic prophase I chromosome condensation stage
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ER DSBHR
RAD2 II RAD3 II
FEN1
Activity
RAD10 II,V
ERCC1 S.p. Swi10
RAD14 II RAD16 II
XPA S.p.rhp16
RAD17 X II
X
5 endonuclease
lesion recognition ATPase
RAD18 X I, X V I
H.s.RAD1 S.p.Rad1XIV U.m. rec1 HR18A HR18B
ER ER Žnontranscribed DNA. check-point for Ži.DNA damage; Žii. meiotic reccombination TLS
RAD23 I!
HR23A HR23B
ER
targets RAD6p to lesions 23A stimulates XPC-activity 23B)apoptosis
RAD24 X II r r1s RAD25rSSL2 II
H.s.RAD17 S.p.Rad17 XIV XPBrCSC
RAD26 II
CSBrERCC6
DNA damage checkpoint Ži. ER nontranscribed, Žii. DNA cross-links ER
RAD27 X I, X V I
H.s.FEN1
Swi2rSNF2
FEN1
Okazaki fragment processing. Limits triplet array expansion
S.p.Rad2 XIV
UVER
RAD28
CSA
RAD30 X I, X V I
H.s.RAD30A ŽXP-V. H.s. RAD30B
required for strand specific repair EF-TLS
DNA polymerase h
X
RAD1rTFIIH RAD1rMSH2r MSH3rSRS2r RAD52 TFIIH DNA helicase
X
putative 3 - 5 exonuclease
uvs
uvs uvs
XP
uvs
RAD6prssDNA
uvs, xs
26S-proteasomer RAD4p
uvs
DNA helicase
TFIIH
null is lethal
XPrCS
inhibits transcription in presence of ER; DNA dependent ATPse X X Flap-5 -3 endonuclease
TFIIH
hypermutable
CS
chromosome breakage; mutator; high mitotic recombination; duplications; defective lagging strand synthesis hypermutable
proposed to affect triplet repeat expansion
uvs
XP
By-pass DNA synthesis
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Ži. ER Žii. DSBHR
CS
(continued on next page)
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Table 1 Ž continued . S.c. Yeast gene
RAD50
V ,V I, X V ,V II
RAD51V ,V I, X V ,V II
RAD52 V ,V I, X V ,V II
Family
H.s.RAD50
SMC
S.c.DMC1 H.s.RAD51A ŽXRCC2. H.s.RAD51B ŽXRCC3. ŽREC2. H.s.RAD51C,D S.p.rhp51 etc. S.p. Rad52 H.s. RAD52
RecA
S.p. cds1XIV H.s. chk2 S.p. rhp54 H.s.RAD54
Repair system
NHEJ; DSBHR telomere maintenance DSBHR; homologous pairing and recombination
DSBHR; homologous recombination
Swi2rSnf2
Repair and damage checkpoint DSBHR
RAD55 V ,V I, X V ,V II
RecA
DSBHR
RAD57 V ,V I, X V ,V II
RecA
DSBHR
Activity
Complex
MRE11rXRS2
ds and ss DNA X X 5 -3 strand transfer
Mutant phenotype Yeast
Human
uvs, xs, meiotic rec-; spores inviable; mitotic recy
radiation sensitive in rodent cells
ds and ss DNA binding; ssDNA annealing with RPArssDNA stimulates RAD51 homologous pairing kinase
RAD52rRAD54r RAD55rRAD57r RP-Arss and dsDNA ) H.s.RAD51Ar BRCA1rBRCA2r BARD1 ) p53 i.RAD51rRAD54r RAD55rRAD57 Žstrand-invasion. ii.RAD1rRAD10r MSH2rMSH3 ŽssDNA annealing. Mec1
dsDNA-dependent ATPase
RAD52,-55,-57r RP-Arss and dsDNA
xs; recy
RAD52,-54r -57r RP-Arss and dsDNA RAD52,-54,-55r RP-Arss and dsDNA
xs at 238C not at 368C xs at 238C not at 368C xs; recy xs, rec-in meiosis
RAD59 X V II XRS2 V ,V I, X V , X V II
S.c. RAD52 NBS1 Žvery limited.
RAD1 DSBHR NHEJ
ss annealing
MRE11V ,V I, X V XRS4 RAD58
H.s.MRE11A H.s.MRE11B S.p.Rad32 E.c. SbcCD
i.NHEJ ii. Homologous recombination iii. telomere maintenance
Mgqq-dependent ss DNA endo X and 5 exonuclease
RAD50rMRE11r DNAligase IVr KU70r80 RAD50rXRS2r DNAligase IVr KU70r80 ) XRCC4rNbs1 ) KU70rKU80
DSBHR-in rodent cells
xs; recy; meiotic lethal
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RAD53 X II SPC1r MEC2rSAD1 RAD54 V ,V I, X V ,V II
Homologues
xs some primary cancers; xs and rec-in mouse K.O.
Nijmegen break syndrome
Table 2 Complementation and interference of yeast repair related functions by human Žor rodent. genes Yeast host
Complementation
HHR6A and 6B hMMS and CROC-1 hAPE hOGG1 hMGMT hAAG hPOL b rodent
rad6 Sac. cereÕisiae mms2 Sac. cereÕisiae apn1 Sac. cereÕisiae ogg1 Sac. cereÕisiae mgt1 Sac. cereÕisiae Sac. cereÕisiae Sac. cereÕisiae
Repair MMS sensitivity MMS sensitivity Reversal of mutator phenotype Reversal of alkylation sensitivity
hPOLL b rodent
Sac. cereÕisiae
hFEN1
rad27 Sac. cereÕisiae
Complements MMS sensitivity of DNA polymerase d mutant Complements repair and genome stability defect
hEXO1 hRAD1
rad27 Sac. cereÕisiae exo1 Sac. cereÕisiae rad1Žq. Sch. pombe
hRACH2
rad1-1 Sch. pombe
hRAD2 hRAD3 hRAD9
rad2 Sch. pombe rad3 Sac. cereÕisiae rad9 Sch. pombe
hKu70 hRAD50 hRAD54 hMSH2 and hMSH6 hTOP2 hLIGI Žligase.
hdf1 Sac. cereÕisiae rad50 Sac. cereÕisiae rad54 Sac. cereÕisiae Sac. cereÕisiae top2 Sac. cereÕisiae cdc9 Žligase defective. Sac. cereÕisiae sgs1 Sac. cereÕisiae
hBLM ŽBloom’s syndrome helicase.
Interference
Mutator phenotype Dominant negative, MMS sensitivity cause by mutant alleles
Reference Koken et al., Proc. Natl. Acad. Sci. 88 Ž1991. 8865 Xiao et al., Nucl. Acid Res. 26 Ž1998. 3908 Wilson et al., Nucl. Acid Res. 23 Ž1995. 5027 Radicella et al., Proc. Natl. Acad. Sci. 94 Ž1997. 8010 Xiao and Fontaine, Mutat. Res. 336 Ž1995. 133 Glassner et al., Proc. Natl. Acad. Sci. 95 Ž1998. 9997 Clairmont and Sweasy, J. Bacteriol. 178 Ž1996. 656
Blank et al., Proc. Natl. Acad. Sci. 91 Ž1994. 9047 dominant inhibition of growth effect and MMS sensitivity caused by mutant allele
Reversal of mutator phenotype MMS sensitivity UV sensitivity and checkpoint control UV sensitivity of the rad1-1 allele and checkpoint control UV sensitivity Rescues viability UV sensitivity and checkpoint control Rescues growth MMS sensitivity MMS sensitivity
Greene et al., Hum. Mol. Genet. 8 Ž1999. 2363
Tishkoff et al., Cancer Res. 58 Ž1998. 5027 Qiu et al., J. Biol. Chem. 274 Ž1999. 17893 Freire et al., Genes Dev. 15 Ž1998. 2560
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Gene
Davey and Beach, Mol. Biol. Cell 6 Ž1995. 1411 Murray et al., Mol. Cell. Biol. 14 Ž1994. 4878 Guzder et al., J. Biol. Chem. 270 Ž1995. 17660 Lieberman et al., Proc. Natl. Acad. Sci. 93 Ž1996. 13890
Growth Growth
Barnes and Rio, Proc. Natl. Acad. Sci. 94 Ž1997. 867 Kim et al., Gene 235 Ž1999. 59 Kanaar et al., Curr. Biol. 6 Ž1996. 828 Clark et al., Nucl. Acid Res. 27 Ž1999. 736 Jensen et al., Mol. Gen. Genet. 252 Ž1996. 79 Barnes et al., Proc. Natl. Acad. Sci. 87 Ž1990. 6679
Reduces hyperrecombination
Neff et al., Mol. Biol. Cell 10 Ž1999. 665
High mutator
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not surprising since related proteins could interact with each other or with DNA products in such a way as to prevent function. The overexpresion of human mismatch repair genes in yeast can increase mutability dramatically, providing opportunities to study these genes. Similarly, mammalian DNA polymerase b mutants can interfere with repair of base damage when overexpressed in Sac. cereÕisiae. The idea of interference with normal function has been applied to screens for human genes that influence repair and DNA metabolic processes in genetically sensitized E. coli w26x and yeast ŽPerkins and Resnick, unpublished.. These examples of complementation and interference suggest that future yeast-based investigations of human DNA repair will include using yeast as in vivo test tubes for examining multicomponent human repair functions in yeast. Since most repair pathways are multicomponent, it may be necessary to tailor the human proteins for examination in yeast or alternatively to introduce the multiple human components into yeast. Parts replacement might also help. For example, the S.c. Rad27p and H.s. Fen1p each bind to PCNA. Replacing the hPCNA-binding domain of H.s. Fen1 with that of S.c. Rad27, creating a FEN1rRad27 chimera results in a protein adopting more of the properties of the yeast holoenzyme w24x. Futhermore, since many wholly or partially heterologous functional systems have been or are being constructed in yeast, it will be possible to examine, among other things the consequences of human DNA repair polymorphisms. The general incidence of polymorphisms is estimated to be around 10–20%. Subtle variations in individual genes or combinations of genes, even as heterozygotes, are likely to influence the impact that the environment can have on genome stability, as shown for DNA replication Ždiscussed in Gary et al. w27x.. Subtle changes may be detected through the use of various functional assays in yeast wherein the affected proteins are expressed singly or in combination with other human proteins. Yeast offers much more sophisticated systems for exploring recombination ŽKUPIEC; HABER. and the roles of mismatch repair genes in all aspects of replication, recombination and repair than are at present available in mammalian cells ŽBORTS, CHAMBERS and ABDULLAH; HARFE and
JINKS-ROBERTSON.. Along with these systems are the panels of yeast mutants that provide opportunities to characterize functional homologues from mammals. Equivalent models in mammalian systems remain to be developed.
Unifying repair in yeast and humans Early studies envisioned only remote relationships between DNA repair in yeast and humans. The new millennium will undoubtedly see the continued exploitation of yeast and other model eukaryotes for understanding mechanisms and consequences of human DNA repair. The many contributions in this Special Edition that review the past, examine the present, and anticipate the future will help point the way. We believe that yeasts as in vivo test tubes may support partial or even entire pathways taken from the human system. Already, the first steps in this endeavor are being taken with the expression of many components of the mammalian apoptosis pathway in yeast. There may, of course, be limitations to the utility of yeasts as human surrogates due to differences in molecular environment and the more complex genetic interactions in humans. The opportunity to characterize complete genomic responses for all genes in an organism using microarray techniques, as was recently done for exposure to the alkylating agent MMS w28x, will provide matrices in which to understand the cellular interpretation of damage. The in vivo test tubes can also be used for rapid screening of environmental agents that affect human gene activities and DNA repair systems within yeast. They may even be used to provide drug screens to deal with repair deficiencies. Although yeasts are not quite human, they will continue to help us to understand ourselves.
Acknowledgements We greatly appreciate the critical review of the manuscript and suggestions by Kevin Lewis, Jake Kirchner, Alberto Inga, and Dmitry Gordenin.
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References w1x R. Holliday, A mechanism of gene conversion in fungi, Genet. Res. 5 Ž1964. 282–304. w2x R. Holliday, Radiation sensitive mutants of Ustilago maydis, Mutat. Res. 2 Ž1965. 557–559. w3x S. Nakai, K. Maysumoto, Two types of radiation sensitive mutants in yeast, Mutat. Res. 4 Ž1967. 129–136. w4x R. Snow, Mutants of yeast sensitive to ultraviolet light, J. Bacteriol. 94 Ž1967. 571–575. w5x E. Moustacchi, Cytoplasmic and nuclear genetic events induced by UV light in strains of Saccharomyces cereÕisiae with different UV sensitivities, Mutat. Res. 7 Ž1969. 171–185. w6x M.A. Resnick, Genetic control of radiation sensitivity in Saccharomyces cereÕisiae, Genetics 62 Ž1969. 519–531. w7x D. Averbeck, W. Laskowski, F. Eckardt, E. Lehman-Brauns, Four radiation sensitive mutants of Saccharomyces, survival after UV and X-ray-irradiation as well as UV-induced reversion rates from iso-leucine-valine dependence to independence, Mol. Gen. Genet. 107 Ž1970. 117–127. w8x B.S. Cox, J.M. Parry, The isolation, genetics and survival characteristics of ultraviolet light-sensitive mutants in yeast, Mutat. Res. 6 Ž1968. 37–55. w9x J.C. Game, R.K. Mortimer, A genetic study of X-ray sensitive mutants in yeast, Mutat. Res. 24 Ž1974. 281–292. w10x M.A. Resnick, The repair of double-strand breaks in DNA: a model involving recombination, J. Theor. Biol. 59 Ž1976. 97–106. w11x J.C. Game, B.S. Cox, Epistatic interactions between four rad loci in yeast, Mutat. Res. 16 Ž1972. 353–362. w12x J.C. Game, B.S. Cox, Synergistic interactions between rad mutants in yeast, Mutat. Res. 20 Ž1973. 35–44. w13x J.C. Game, B.S. Cox, Repair systems in Saccharomyces, Mutat. Res. 26 Ž1974. 35–44. w14x G. Lemontt, Mutants of yeast defective in mutation induced by ultraviolet light, Genetics 68 Ž1971. 21–23. w15x L. Prakash, S. Prakash, Isolation and characterisation of MMS-sensitive mutations of Saccharomyces cerreeÕisiae, Genetics 86 Ž1977. 33–55. w16x J.A. Henriques, E. Moustacchi, Isolation and characterisation of pso mutants sensitive to photo-addition of psoralen derivatives in Saccharomyces cereÕisiae, Genetics 95 Ž1980. 273– 288. w17x M. Esposito, R.E. Esposito, The genetic control of sporulation in Saccharomyces cerreÕisiae: I. The isolation of temperature-sensitive sporulation-deficient mutants, Genetics 61 Ž1969. 79. w18x N.G. Suslova, I.A. Zakharov, Genetic control of radiosensitivity in yeast: 7. Identification of genes conferring sensitivity to X-rays, Genetika 6 Ž1970. 158–163. w19x T.A. Weinert, L.H. Hartwell, The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cereÕisiae, Science 241 Ž1988. 317–322. w20x J.M. Flaman, T. Frebourg, V. Moreau, F. Charbonnier, C. Martin, P. Chappuis, A.P. Sappino, I.M. Limacher, L. Bron, J. Benhattar et al., A simple p53 functional assay for screen-
w21x
w22x
w23x
w24x
w25x
w26x
w27x
w28x
ing cell lines, blood, and tumors, Proc. Natl. Acad. Sci. U. S. A. 92 Ž1995. 3963–3967. D. Higgins, S. Prakash, P. Reynolds, R. Polakowska, S. Weber, L. Prakash, Isolation and characterisation of the RAD3 gene of Saccharomyces cereÕisiae and inviability of rad3 deletion mutants, Proc. Natl. Acad. Sci. U. S. A. 80 Ž1983. 5680–5684. L. Naumovsky, E. Friedberg, A DNA repair gene required for the incision of damaged DNA is essential for viability in Saccharomyces cereÕisiae, Proc. Natl. Acad. Sci. U. S. A. 80 Ž1983. 4818–4821. P. Sung, L. Prakash, S.W. Matson, S. Prakash, RAD3 protein of Saccharomyces cereÕisiae is a DNA helicase, Proc. Natl. Acad. Sci. U. S. A. 84 Ž1987. 8951–8955. A. Green, J. Snipe, D. Gordenin, M.A. Resnick, Functional analysis of human Fen1 in Saccharomyces cereÕisiae and its role in genome stability, Hum. Mol. Genet. 8 Ž1999. 2363– 2373. C. Lambert, L.B. Couto, W.A. Weiss, R.A. Schultz, L.H. Thompson, E.C. Friedberg, A yeast DNA repair gene partially complements defective excision repair in mammalian cells, EMBO J. 7 Ž1988. 3245–3253. E.L. Perkins, J.F. Sterling, V.I. Hashem, M.A. Resnick, Yeast and human genes that affect the E. coli SOS response, Proc. Natl. Acad. Sci. U. S. A. 96 Ž1999. 2204–2209. R. Gary, M. Park, J.P. Nolan, H.L. Cornelius, O.G. Kozyreva, H.T. Tran, K. Lobachev, M.A. Resnick, D.A. Gordenin, A novel role for Fen1rRad27 PCNA-binding in DNA metabolism and implications for genetic risk, Mol. Cell. Biol. 19 Ž1999. 5373–5382. S.A. Jelinsky, L.D. Samson, Global response of Saccharomyces cereÕisiae to an alkylating agent, Proc. Natl. Acad. Sci. U. S. A. 96 Ž1999. 1486–1491.
Michael A. Resnick a, ) Chromosome Stability Group, Laboratory of Molecular Genetics, National Institute of EnÕironmental Health Sciences, NIH, P.O. Box 12233, 111 Alexander DriÕe, Research Triangle Park, NC 27709, USA E-mail address: [email protected] a
Brian S. Cox b School of Biological Sciences, UniÕersity of Kent, Canterbury, CT68EJ, UK E-mail address: [email protected] b
)
Corresponding author.
Editorial to Special Issue on Yeast DNA Repair and Human Implications: Past, Present and Future Perspectives on DNA Repair in Yeast
Yeast as an honorary mammal
To those who have had the opportunity to devote much of their research careers to the study of what used to be referred to as a simple or lower eukaryote, there has been the pleasure of watching often esoteric research turn into highly relevant investigations of human biology and disease. Aging, cancer, neurological disorders, and immunological defects are just few of the frailties that can be traced to DNA repair. Our little friends, the budding yeast Saccharomyces cereÕisiae and the fission yeast Schizosaccharomyces pombe have been the molecular and genetic workhorses for much of our understanding of DNA repair in eukaryotes. They have provided insight into the integrated circuits that connect initial DNA injury, lesion processing, cellular checkpoint response, and eventual genetic consequences, resulting in an increasingly detailed view of the maintenance of DNA integrity and control of genetic variation. In essence, they have provided a holistic portrait of cellular damage response. Human genetic defects associated with repair can often be addressed directly in yeast because of evolutionary conservation of genes and systems. In this Special Edition, we have tried to cover all the different systems which the two yeasts use to repair or limit the effects of damage to DNA. We saw these at first as distinct systems: nucleotide excision repair, double-strand break repair, DNA synthesis in repair and mutagenesis, mismatch repair, base excision repair, photorepair, and checkpoint control. Many of these processes are, in fact, integrated and extend to normal cell activities. Therefore, we include damage-independent DNA transactions that are a normal part of the yeast lifecycle, 0027-5107r00r$ - see front matter. Published by Elsevier Science B.V. PII: S 0 0 2 7 - 5 1 0 7 Ž 0 0 . 0 0 0 3 6 - 1
namely meiotic recombination and telomere maintenance.
Linking the past with the present Much of the early DNA metabolism and repair work in yeast was guided by the even more accessible bacterial and phage systems that provided rapid experimental turnover and relative ease of genetic manipulation, in particular the ability to expose genomic DNA separately from cells. These systems led to the identification of several important repair phenomena: host cell reactivation Žhcr.; multiplicity reactivation; photoreactivation as a process that removes DNA damage; damage-induced reactivation ŽWeigle reactivation.; and the isolation of genes controlling transformation efficiency in Diplococcus pneumoniae. These experiments foreshadowed much of the current array of repair systems identified in prokaryotes and eukaryotes. The link between recombination and repair was prompted in part by studies in another eukaryote, Ustilago maydis. Robin Holliday had proposed a recombination model that implicated excision repair-type enzymes, such as those that had recently been found in Escherichia coli w1x. This model was followed by his isolation of UV-sensitive, recombination deficient mutants. In E. coli, furthermore, it had been found that Recy mutants were sensitive to both UV and ionizing radiation w2x. The Holliday model for recombination along with the bacterial studies on repair mutants inspired Nakai and Maysumoto w3x, Snow w4x, Moustacchi w5x, Resnick w6x,
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Averbeck et al. w7x and Cox and Parry w8x in the first mutant hunts for genes affecting UV and ionizing radiation resistance in Sac. cereÕisiae. Naively, perhaps, we assumed that if enough mutants with a radiation-sensitive phenotype were found, some would be involved in recombination. It was indeed gratifying to find that some of the mutants had the expected secondary phenotypes. For example, two of the first characterized mutants were meiotic lethals. In uÕs6ruÕs6 Žs rad6 . diploids, meiosis aborted before spore formation and in uÕs5ruÕs5 Žs rad50 ., spores formed but were inviable. These were considered significant phenotypes in view of the important role chiasma formation plays in meiosis. The uÕs6 mutants also turned out to be totally refractory to induction of mutations. The set of X-ray sensitive mutants isolated by Resnick w6x and by Game and Mortimer w9x, now called rad52 to rad57, turned out to be especially good predictors of defects in various aspects of mitotic and meiotic recombination. Subsequently, studies with rad52 mutants provided direct evidence for the importance of damage-induced double-strand breaks and led to the first model that linked DSB repair to recombination w10x. In the beginning, there appeared to be only two repair systems. Nakai and Matsumoto w3x, who isolated one UV-sensitive mutant, UVs-1 Žs rad1. and Xs-1 Žs rad51. an X-ray sensitive mutant, showed that the genes were operating in different repair systems by demonstrating the super-sensitive phenotype of the double mutant to UV. Complexity was advanced by one notch when Game and Cox w11,12x added a third presumptive pathway of repair by showing that rad6 and rad18 were epistatic with one another but synergistic in UV sensitivity with either rad1 or rad51. These groups corresponded neatly with their respective phenotypes with members of the RAD6 epistasis group all being sensitive to both UV and ionizing radiation. It also seemed that they were sufficient to account for the recovery of cells from all measurable UV-induced damage w13x. The passage from three repair systems, which we were happy to characterize as excision repair Žthe RAD1 group., recombinational repair or doublestrand break repair Žthe RAD52 group. and post-replication or error-prone repair Žthe RAD6 group. — all concepts borrowed without much evidence from
the growing understanding of DNA repair in bacteria and bacteriophages — to the much more complex picture now being painted has been full of surprises and accompanied by a growing revelation of the conservation of DNA repair in yeasts and mammals. Firstly, there was a growing list of mutants isolated with different phenotypes relevant to repair, recombination, and mutagenesis. Jeff Lemontt isolated mutants defective in spontaneous mutation Ž reÕ1 to reÕ3 . w14x. Louise Prakash isolated a large number of mutants sensitive to methyl methane sulphonate Žmmsy. , identifying 20 or so loci differing from those already involved in repair w15x. Ethel Moustacchi isolated mutants sensitive to psoralen, implying a repair system able to deal with DNA cross-links w16x and Mike and Shelley Esposito initiated a genetic analysis of meiosis by isolating mutants defective in sporulation, fittingly designated spoy w17x, some of which are involved in meiotic recombination itself. An important gene XRS2 that has subsequently been shown to be essential in meiotic recombination and is part of the MRE11 associated complex, was also isolated in a broad screen of radiation sensitive mutants w18x. Many other genes have arrived in the DNA repair category by other routes, among them MRE11, SSL2 Žs RAD25 ., DMC1, SPC1rMEC2rBAD1 Žs RAD53 ., SRS2 and SGS1, PMS1, PMS2, and PMS3. Secondly, biochemical approaches have identified enzymes with activities similar to those found both in mammals and bacteria which either reverse alkylation damage in DNA or repair it by excising the relevant base and replacing it with a normal one-base excision. These included transferases, DNA glycosylases, abasic endonucleases and apurinic lyases Žreviewed in MEMISOGLU and SAMSON 1 .. Finally and appropriately, an important set of yeast repair genes has been identified not by mutagenesis or biochemistry, but by their sequences. These include many of the genes involved in mismatch repair, which have been cloned by low-stringency PCR based on either mammalian or bacterial homologues which had already been cloned. Genes identi-
1
Reference to reviews in this Special Edition are listed in bold as authors only.
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fied this way include the set of mutator homologues MLH1 to -3 and MSH1 to -6. Three genes in this category had already been identified by mutation: PMS1 Žin the MutL family., PMS2s MLH1, and PMS3s MSH6 ŽBORTS, CHAMBERS and ABDULLAH; HARFE and JINKS-ROBERTSON.. Likewise, RAD26 and RAD30 were identified by a homology search in the yeast genome database using the corresponding sequences of the human genes ŽGAME.. This growth in the numbers of genes identified more modes or systems of repair or damage-tolerance. Base-excision ŽMEMISOGLU and SAMSON., translesion DNA synthesis ŽKUNZ, STRAFFON and VORNAX; BUDD and CAMPBELL., nonhom ologous end-joining Ž LEW IS and RESNICK ., mismatch repair ŽHARFE and JINKS-ROBERTSON; BORTS, CHAMBERS and ABDULLAH. and cross-link repair have all been added to the list of systems established in yeasts in the last 15 years. However, complexity of another kind has become apparent in that many systems can no longer be considered discrete. For example, the endonuclease encoded by the RAD1–RAD10 genes, at first categorized as ‘‘excision-repair’’, also functions in double-strand break repair ŽHABER.. Similarly, the Rad50rMre11rXrs2 complex functions in nonhomologous end-joining, homologous recombination, and telomere maintenance ŽHABER; LUNDBLAD; LEWIS and RESNICK.. Genes for DNA metabolic proteins such as Rpa, Pola , PCNA, and DNA ligase appear to have a role in almost all repair systems. Double-strand break repair is achieved by nonhomologous end-joining and two methods of homologous recombination ŽHABER; SUNG, TRUJILLO and VAN KOMEN; LEWIS and RESNICK.. The two families of MUT gene homologues common to mammals and yeast namely MLH1 to -3, PMS1 and MSH1 to -6, apart from being involved in mismatch repair, play roles in many other DNA maintenance pathways. These processes include replication, excision repair, and homologous recombinational repair. In meiotic recombination, they have the effects both of enhancing homologous recombination and in the presence of excessive mismatches, inhibiting homeologous recombination. ŽHARFE and JINKS-ROBERTSON; BORTS, CHAMBERS and ABDULLAH.. This functional
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diversity highlights them as playing a central role in all aspects of control of genetic variation. Genomic complexity and divergence of function is also apparent in the distribution of members within certain gene families that are involved in different systems of repair and maintenance of DNA integrity. The most remarkable, perhaps, is the proliferation of E. coli RecA homologues. Also interesting is the divergence of function among the nucleases such as S cR A D 27r S pR A D 2r H sF E N 1, S cE X O 1r SpEXO1rHsHEX1, and ScRAD2rSpRAD13r HsXPG. The different complexes participate in Okazaki fragment processing, maintenance of small repeats including triplet repeats, recombination, mismatch repair and in nucleotide excision repair. Similarly the SWI2rSNF2 family, which modulates protein-DNA interactions in an ATP-dependent manner, contains putative helicase members which appear to be involved several repair pathways Žsee Table 1.. Nucleotide excision repair has been found to have two modes, fast repair associated with DNA undergoing transcription and the slower nucleotide excision in non-active DNA. Some of the nucleotide excision-repair genes are common to both modes, some exclusive to the latter. The article by PRAKASH and PRAKASH neatly organizes the complexities by distinguishing between repair genes and repair factors. The latter are the complexes of gene products, which form and then interact to achieve nucleotide excision repair. In addition to these mechanisms, an alternative pathway of excision repair, UVER, operates in Sch. pombe. This itself is apparently resolved by either of the two processes, one similar to long-patch repair in E. coli and the other by recombination. In Sch. pombe, both the NER and the UVER pathways repair 6-4 photoproducts faster than pyrimidine dimers ŽMcCREADY, OSMAN and YASUI.. In the last 10 years, research in DNA damage tolerance and repair has transited from biochemistry of specific proteinŽs. to organizational biology. The most dramatic example comes from the demonstration by Weinert and Hartwell w19x that cell-cycle progression is interrupted by the presence of DNA damage and that this is important for cell survival. The first gene identified as being required for checkpoint control, RAD9 of Sac. cereÕisiae, has been joined by others in Sac. cereÕisiae ŽFOIANI, PEL-
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LICIOLI, LOPES, LUCCA, FERRARI, LIBERI, MASERATI, FALCONI and PLEVANI. and in Sch. pombe ŽHUMPHREY.. Checkpoint genes may act to interrupt progression at G1rS, within S or at G2rM as a result of either damage in the DNA or a replication blockage. The manner in which the systems in the two yeasts map on to one another is quite remarkable. In the matter of damage-recognition and transduction of signal to the Cdc25 and Cdc2rcyclin B proteins that determine transition through the G1rS and G2rM checkpoints, yeast regulatory mechanisms are strikingly similar to human checkpoint controls. The only significant divergence in checkpoint mechanisms is the absence from yeast of p53, a focal point of cell-cycle regulation and apoptosis in mammals. ŽEven so, yeast has proven highly useful for understanding the functions of this human gene since expression of normal and mutant p53 expression can be easily monitored in yeast w20x.. HUMPHREY lists 19 Sch. pombe checkpoint genes and 15 homologues of these from Sac. cereÕisiae. Nine of the former and four of the latter were first isolated by their radiation-sensitive phenotypes. McCready, Osman and Yasui make the point that Sch. pombe depends upon the checkpoint control system for the purpose of damage tolerance much more than does Sac. cereÕisiae, possibly because Sch. pombe cells spend most of their cell cycle in G2. This raises a question about the balance of checkpoint control relative to damage repair in the damage tolerance of humans. The significance of spatial organization has become clear from studies of nucleotide excision repair, mutagenesis, and recombination. Nucleotide excision repair is conducted by the core transcription factor complex, TFIIH, thus coupling excision repair to transcription ŽPRAKASH and PRAKASH. and giving high priority to repair of DNA being transcribed. The transcription factor becomes a repair complex by the interaction with NER proteins. Other proteins direct the same complex to damage in nontranscribed DNA. The discovery of transcriptioncoupled repair in yeast and humans is a classic story of how information obtained in one organism stimulates progress in the other and is then reciprocated. It started with the discovery that one of the genes in Sac. cereÕisiae implicated in excision repair, RAD3, was an essential gene w21,22x and then that the
protein was an ATPase and DNA helicase w23x. From 1986 to 1989, Hanawalt et al. demonstrated preferential excision repair of transcribed strands of DNA, thus coupling excision repair and transcription in both humans and E. coli ŽPRAKASH and PRAKASH and Refs. w17,18,19x therein.. In 1993 and 1994, the link between transcription and excision in both yeast and human cells was established in a series of papers that followed one another so quickly that clearly this idea’s time had come in several labs. In these years, it was revealed that the human homologue of RAD3 ŽXPD. also coded for a helicase, as did RAD25 ŽXPB.; that mutants of both genes were defective in transcription; that both these proteins were constituents of TFIIH in yeast and human cells and that TFIIH was involved in excision repair ŽPRAKASH and PRAKASH, and Refs. w47, 49, 50, 51, 52, 72, 73,74x therein.. It remained only to have the transcriptionrrepair complex re-constituted in vitro the following year to round off a triumphant passage of research based on the application of information obtained in one organism to experiments in another. Spatial organization is also becoming apparent in recombinational repair involving the RAD51 gene product. This RecA homologue is central in the formation of paired homologous strands of DNA and has been found to form double-strand break-dependent foci in mitotic cells and also RAD54-dependent foci in prophase of meiosis. The process of meiosis, which involved many repair genes, is largely a study of spatial organization and the formation and dissolution of complex organelles like the synaptonemal complex and chiasmata ŽDRESSER.. In his article, HABER raises the question of whether the location of chromosomes within the nucleus is altered by damage such that a search for other broken ends or homologous sequences may be carried out more efficiently.
Humanizing repair in yeast The relevance of yeast repair studies to mammalian systems has been clear for some time and as with checkpoint controls, the extent to which they resemble each other is encouraging and has already
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proved very useful to research in both yeast and mammals. Presented in Table 1 is a list of many of the yeast and repair genes and their corresponding human homologues that deal with UV and gamma ray damage. It is not meant to be exhaustive, but instead to demonstrate the commonality of repair and consequences of mutations. There are likely to be hundreds of genes associated either directly or indirectly with the many types of DNA repair. The similarities in sequence homologies allows genes and functions found in one organism to be identified in others. There is a commonality of organization of processes between different organisms, with the same proteins appearing in the same protein and nucleoprotein complexes. Our Table 1, which focuses on the radiation repair pathways, complements the article by GAME, which presents an overview of the background and the current state of DNA repair studies. He approaches the topic with a geneticist’s point of view, which allows one to appreciate repair better as a holistic interaction of many systems. This is a nice contrast with the more reductionist and analytical approach of our other contributors. Table 1 largely excludes genes involved in translesion DNA synthesis, checkpoint controls and mutator genes, since the Special Edition articles on these subjects present much better tables than we could aspire to. The use of knock-out mice makes it clear that many sequence homologues play similar roles in mammals and yeast. For example, mouse rad6 knockouts abort spermatogenesis in early prophase I, the identical point at which sporulation aborts in yeast. However, knockouts can sometimes obscure the likely function of a gene. For example mouse RAD51yry knockouts abort as embryos, limiting analysis of their functions by this means. Nevertheless, mammalian homologues of the RAD51 series of genes involved in both double-strand break repair and meiotic recombination in yeast are expressed in testis, as would be expected of genes involved in meiotic recombination. Tissue-specific expression like this is common among mammalian homologues of yeast repair genes and obviously provides an indication of their function and significance. Sometimes, the homologies are not exact. The Xrs2 protein in the yeast Rad50rMre11rXrs2 complex required for nonhomologous endjoining is replaced in humans by a protein only remotely related to it,
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Nbs1p. Mutations in Nbs1 result in Nijmegen breakage syndrome. As expected, there is a greater genetic complexity apparent in mammals compared with yeast. Some yeast genes have more than one mammalian homologue; RAD51 has several, and clearly they are not merely redundant. Surprisingly, the RAD52 gene, which is essential to recombination and meiosis in yeast, has much less importance in mammals cells possibly because of redundancy. While many tricks are available, understanding the functions of human genes within human cell systems is usually limited because of difficulties with genetic manipulation. The yeasts have often provided a bridge because of the evolutionary conservation of genetic function between these organisms, particularly in the case of DNA repair. Two approaches have been used in which yeast provide surrogate assays for human genes, complementation, and interference. Many human or mammalian genes have been identified that can complement yeast repair defects as well as other deficiencies and these are presented in Table 2 Žalso see the XREF database http: r r www.cbi. nlm.nih.gov r xrefdb r pubtools. html.. Included are homologues of S.c. RAD6 ŽHHR6A and HHR6B., S.p. RAD9 ŽhRAD9., and S.c. APN1 ŽhAPE.. Ligase I is essential to many DNA metabolic and repair activities, and the human ligase I can complement a S.c.lig1 mutant. Surprisingly, human DNA polymerase b can complement the MMS sensitivity of a yeast strain with a DNA polymerase d defect. The human flap endonuclease Fen1p has been proposed to function in both the maturation of Okazaki fragments during DNA replication and in base excision repair. The hFEN1 gene can fully complement a yeast rad27 mutant and a nuclease-defective Fen1p has similar effects in yeast to the corresponding yeast mutant w24x. Similarly, it is possible to complement mutants that are deficient in double-strand break recombinational repair, particularly rad50, rad54, and the endjoining mutant Ku70. Thus, the consequences of defects in human genes can be studied in yeast. The complementation approach can be reversed. For example, expression of the Sac. cereÕisiae genes RAD10 w25x can alleviate corresponding defects in mammalian cells. Human genes involved in DNA repair responses can also be examined in yeast through their ability to interfere with yeast functions Žsee Table 2.. This is
6 Table 1 UV and ionizing radiation repair genes of Sac. cereÕisiae, their activities and their homologues in humans and in Sch. pombe, with occasional reference to E. coli, U. maydis and mice Roman superscripts refer to the chapters in this volume in which the genes are discussed and in which appropriate references may be found. Abbreviations and symbols: ) s complex in humans; ) sassociated; CSs Error-free translesion synthesis; EP-TLSs Error-prone trans-lesion synthesis; ER s nucleotide excision repair; FENs flap endonuclease; H.s.s human; mut-sdeficient in induced mutation; NHEJs non-homologous end joining; PRR s post replication repair; rec-srecombination deficient; Rep.s replication; SMC sstructural maintenance of chromosomes; spo-ssporulation deficient; S.p.s S. pombe; TLSs translesion synthesis; UVER salternative excision repair; UBC s ubiquitin conjugation; uvs sUV sensitive; XPs xeroderma pigmentosum; xs ssensitive to ionizing radiation. S.c. Yeast gene
RAD1
II,V ,V I, X
Homologues
Family
H.s.XPF S.p.Rad16
Repair system
X
5 ss endonuclease
ER
3 endonuclease ATP-dependent X X 5 -3 helicase
ER
DNA helicase component of TFIIH putativeATPaser DNA helicase conjugates ubiquitin to histone
H.s.XPG S.p.Rad13 H.s.XPD H.s.TTD S.p.Rad15 ERCC2
RAD4 II
XPC
RAD5 X I REV2
S.p.Rad8
Swi2rSNF2
EF-TLS
RAD6 X I, X V I
HHR6A HHR6B S.p.Rhp6
UBC
TLS
RAD7 II
S.p.rhp7
REV3 X I, X V I RAD8
H.s. REV3
Polj
ER nontranscribed strand EP-TLS
RAD9 X II
S.p.crb2 XIV
DNA synthesis and damage checkpoint
X
Complex
RAD10rTFIIH RAD10rMSH2r MSH3rSRS2r RAD52 TFIIH TFIIH
Ži. TFIIH Žii. RAD23) proteasome26
Mutant phenotype Yeast
Human
uvs
XP
uvs missense mutant is uvs, essential for transcription uvs
XP XP, TDD and mouse TDD model
XP
uvs, mut proteasome26, sub-unit 5r RAD18p
uvs, xs, spo mut
TFIIH
uvs decreased induced mutagenesis uvs, xs
mouse rad6 K.O. is malesterile at meiotic prophase I chromosome condensation stage
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ER DSBHR
RAD2 II RAD3 II
FEN1
Activity
RAD10 II,V
ERCC1 S.p. Swi10
RAD14 II RAD16 II
XPA S.p.rhp16
RAD17 X II
X
5 endonuclease
lesion recognition ATPase
RAD18 X I, X V I
H.s.RAD1 S.p.Rad1XIV U.m. rec1 HR18A HR18B
ER ER Žnontranscribed DNA. check-point for Ži.DNA damage; Žii. meiotic reccombination TLS
RAD23 I!
HR23A HR23B
ER
targets RAD6p to lesions 23A stimulates XPC-activity 23B)apoptosis
RAD24 X II r r1s RAD25rSSL2 II
H.s.RAD17 S.p.Rad17 XIV XPBrCSC
RAD26 II
CSBrERCC6
DNA damage checkpoint Ži. ER nontranscribed, Žii. DNA cross-links ER
RAD27 X I, X V I
H.s.FEN1
Swi2rSNF2
FEN1
Okazaki fragment processing. Limits triplet array expansion
S.p.Rad2 XIV
UVER
RAD28
CSA
RAD30 X I, X V I
H.s.RAD30A ŽXP-V. H.s. RAD30B
required for strand specific repair EF-TLS
DNA polymerase h
X
RAD1rTFIIH RAD1rMSH2r MSH3rSRS2r RAD52 TFIIH DNA helicase
X
putative 3 - 5 exonuclease
uvs
uvs uvs
XP
uvs
RAD6prssDNA
uvs, xs
26S-proteasomer RAD4p
uvs
DNA helicase
TFIIH
null is lethal
XPrCS
inhibits transcription in presence of ER; DNA dependent ATPse X X Flap-5 -3 endonuclease
TFIIH
hypermutable
CS
chromosome breakage; mutator; high mitotic recombination; duplications; defective lagging strand synthesis hypermutable
proposed to affect triplet repeat expansion
uvs
XP
By-pass DNA synthesis
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Ži. ER Žii. DSBHR
CS
(continued on next page)
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Table 1 Ž continued . S.c. Yeast gene
RAD50
V ,V I, X V ,V II
RAD51V ,V I, X V ,V II
RAD52 V ,V I, X V ,V II
Family
H.s.RAD50
SMC
S.c.DMC1 H.s.RAD51A ŽXRCC2. H.s.RAD51B ŽXRCC3. ŽREC2. H.s.RAD51C,D S.p.rhp51 etc. S.p. Rad52 H.s. RAD52
RecA
S.p. cds1XIV H.s. chk2 S.p. rhp54 H.s.RAD54
Repair system
NHEJ; DSBHR telomere maintenance DSBHR; homologous pairing and recombination
DSBHR; homologous recombination
Swi2rSnf2
Repair and damage checkpoint DSBHR
RAD55 V ,V I, X V ,V II
RecA
DSBHR
RAD57 V ,V I, X V ,V II
RecA
DSBHR
Activity
Complex
MRE11rXRS2
ds and ss DNA X X 5 -3 strand transfer
Mutant phenotype Yeast
Human
uvs, xs, meiotic rec-; spores inviable; mitotic recy
radiation sensitive in rodent cells
ds and ss DNA binding; ssDNA annealing with RPArssDNA stimulates RAD51 homologous pairing kinase
RAD52rRAD54r RAD55rRAD57r RP-Arss and dsDNA ) H.s.RAD51Ar BRCA1rBRCA2r BARD1 ) p53 i.RAD51rRAD54r RAD55rRAD57 Žstrand-invasion. ii.RAD1rRAD10r MSH2rMSH3 ŽssDNA annealing. Mec1
dsDNA-dependent ATPase
RAD52,-55,-57r RP-Arss and dsDNA
xs; recy
RAD52,-54r -57r RP-Arss and dsDNA RAD52,-54,-55r RP-Arss and dsDNA
xs at 238C not at 368C xs at 238C not at 368C xs; recy xs, rec-in meiosis
RAD59 X V II XRS2 V ,V I, X V , X V II
S.c. RAD52 NBS1 Žvery limited.
RAD1 DSBHR NHEJ
ss annealing
MRE11V ,V I, X V XRS4 RAD58
H.s.MRE11A H.s.MRE11B S.p.Rad32 E.c. SbcCD
i.NHEJ ii. Homologous recombination iii. telomere maintenance
Mgqq-dependent ss DNA endo X and 5 exonuclease
RAD50rMRE11r DNAligase IVr KU70r80 RAD50rXRS2r DNAligase IVr KU70r80 ) XRCC4rNbs1 ) KU70rKU80
DSBHR-in rodent cells
xs; recy; meiotic lethal
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RAD53 X II SPC1r MEC2rSAD1 RAD54 V ,V I, X V ,V II
Homologues
xs some primary cancers; xs and rec-in mouse K.O.
Nijmegen break syndrome
Table 2 Complementation and interference of yeast repair related functions by human Žor rodent. genes Yeast host
Complementation
HHR6A and 6B hMMS and CROC-1 hAPE hOGG1 hMGMT hAAG hPOL b rodent
rad6 Sac. cereÕisiae mms2 Sac. cereÕisiae apn1 Sac. cereÕisiae ogg1 Sac. cereÕisiae mgt1 Sac. cereÕisiae Sac. cereÕisiae Sac. cereÕisiae
Repair MMS sensitivity MMS sensitivity Reversal of mutator phenotype Reversal of alkylation sensitivity
hPOLL b rodent
Sac. cereÕisiae
hFEN1
rad27 Sac. cereÕisiae
Complements MMS sensitivity of DNA polymerase d mutant Complements repair and genome stability defect
hEXO1 hRAD1
rad27 Sac. cereÕisiae exo1 Sac. cereÕisiae rad1Žq. Sch. pombe
hRACH2
rad1-1 Sch. pombe
hRAD2 hRAD3 hRAD9
rad2 Sch. pombe rad3 Sac. cereÕisiae rad9 Sch. pombe
hKu70 hRAD50 hRAD54 hMSH2 and hMSH6 hTOP2 hLIGI Žligase.
hdf1 Sac. cereÕisiae rad50 Sac. cereÕisiae rad54 Sac. cereÕisiae Sac. cereÕisiae top2 Sac. cereÕisiae cdc9 Žligase defective. Sac. cereÕisiae sgs1 Sac. cereÕisiae
hBLM ŽBloom’s syndrome helicase.
Interference
Mutator phenotype Dominant negative, MMS sensitivity cause by mutant alleles
Reference Koken et al., Proc. Natl. Acad. Sci. 88 Ž1991. 8865 Xiao et al., Nucl. Acid Res. 26 Ž1998. 3908 Wilson et al., Nucl. Acid Res. 23 Ž1995. 5027 Radicella et al., Proc. Natl. Acad. Sci. 94 Ž1997. 8010 Xiao and Fontaine, Mutat. Res. 336 Ž1995. 133 Glassner et al., Proc. Natl. Acad. Sci. 95 Ž1998. 9997 Clairmont and Sweasy, J. Bacteriol. 178 Ž1996. 656
Blank et al., Proc. Natl. Acad. Sci. 91 Ž1994. 9047 dominant inhibition of growth effect and MMS sensitivity caused by mutant allele
Reversal of mutator phenotype MMS sensitivity UV sensitivity and checkpoint control UV sensitivity of the rad1-1 allele and checkpoint control UV sensitivity Rescues viability UV sensitivity and checkpoint control Rescues growth MMS sensitivity MMS sensitivity
Greene et al., Hum. Mol. Genet. 8 Ž1999. 2363
Tishkoff et al., Cancer Res. 58 Ž1998. 5027 Qiu et al., J. Biol. Chem. 274 Ž1999. 17893 Freire et al., Genes Dev. 15 Ž1998. 2560
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Gene
Davey and Beach, Mol. Biol. Cell 6 Ž1995. 1411 Murray et al., Mol. Cell. Biol. 14 Ž1994. 4878 Guzder et al., J. Biol. Chem. 270 Ž1995. 17660 Lieberman et al., Proc. Natl. Acad. Sci. 93 Ž1996. 13890
Growth Growth
Barnes and Rio, Proc. Natl. Acad. Sci. 94 Ž1997. 867 Kim et al., Gene 235 Ž1999. 59 Kanaar et al., Curr. Biol. 6 Ž1996. 828 Clark et al., Nucl. Acid Res. 27 Ž1999. 736 Jensen et al., Mol. Gen. Genet. 252 Ž1996. 79 Barnes et al., Proc. Natl. Acad. Sci. 87 Ž1990. 6679
Reduces hyperrecombination
Neff et al., Mol. Biol. Cell 10 Ž1999. 665
High mutator
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not surprising since related proteins could interact with each other or with DNA products in such a way as to prevent function. The overexpresion of human mismatch repair genes in yeast can increase mutability dramatically, providing opportunities to study these genes. Similarly, mammalian DNA polymerase b mutants can interfere with repair of base damage when overexpressed in Sac. cereÕisiae. The idea of interference with normal function has been applied to screens for human genes that influence repair and DNA metabolic processes in genetically sensitized E. coli w26x and yeast ŽPerkins and Resnick, unpublished.. These examples of complementation and interference suggest that future yeast-based investigations of human DNA repair will include using yeast as in vivo test tubes for examining multicomponent human repair functions in yeast. Since most repair pathways are multicomponent, it may be necessary to tailor the human proteins for examination in yeast or alternatively to introduce the multiple human components into yeast. Parts replacement might also help. For example, the S.c. Rad27p and H.s. Fen1p each bind to PCNA. Replacing the hPCNA-binding domain of H.s. Fen1 with that of S.c. Rad27, creating a FEN1rRad27 chimera results in a protein adopting more of the properties of the yeast holoenzyme w24x. Futhermore, since many wholly or partially heterologous functional systems have been or are being constructed in yeast, it will be possible to examine, among other things the consequences of human DNA repair polymorphisms. The general incidence of polymorphisms is estimated to be around 10–20%. Subtle variations in individual genes or combinations of genes, even as heterozygotes, are likely to influence the impact that the environment can have on genome stability, as shown for DNA replication Ždiscussed in Gary et al. w27x.. Subtle changes may be detected through the use of various functional assays in yeast wherein the affected proteins are expressed singly or in combination with other human proteins. Yeast offers much more sophisticated systems for exploring recombination ŽKUPIEC; HABER. and the roles of mismatch repair genes in all aspects of replication, recombination and repair than are at present available in mammalian cells ŽBORTS, CHAMBERS and ABDULLAH; HARFE and
JINKS-ROBERTSON.. Along with these systems are the panels of yeast mutants that provide opportunities to characterize functional homologues from mammals. Equivalent models in mammalian systems remain to be developed.
Unifying repair in yeast and humans Early studies envisioned only remote relationships between DNA repair in yeast and humans. The new millennium will undoubtedly see the continued exploitation of yeast and other model eukaryotes for understanding mechanisms and consequences of human DNA repair. The many contributions in this Special Edition that review the past, examine the present, and anticipate the future will help point the way. We believe that yeasts as in vivo test tubes may support partial or even entire pathways taken from the human system. Already, the first steps in this endeavor are being taken with the expression of many components of the mammalian apoptosis pathway in yeast. There may, of course, be limitations to the utility of yeasts as human surrogates due to differences in molecular environment and the more complex genetic interactions in humans. The opportunity to characterize complete genomic responses for all genes in an organism using microarray techniques, as was recently done for exposure to the alkylating agent MMS w28x, will provide matrices in which to understand the cellular interpretation of damage. The in vivo test tubes can also be used for rapid screening of environmental agents that affect human gene activities and DNA repair systems within yeast. They may even be used to provide drug screens to deal with repair deficiencies. Although yeasts are not quite human, they will continue to help us to understand ourselves.
Acknowledgements We greatly appreciate the critical review of the manuscript and suggestions by Kevin Lewis, Jake Kirchner, Alberto Inga, and Dmitry Gordenin.
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References w1x R. Holliday, A mechanism of gene conversion in fungi, Genet. Res. 5 Ž1964. 282–304. w2x R. Holliday, Radiation sensitive mutants of Ustilago maydis, Mutat. Res. 2 Ž1965. 557–559. w3x S. Nakai, K. Maysumoto, Two types of radiation sensitive mutants in yeast, Mutat. Res. 4 Ž1967. 129–136. w4x R. Snow, Mutants of yeast sensitive to ultraviolet light, J. Bacteriol. 94 Ž1967. 571–575. w5x E. Moustacchi, Cytoplasmic and nuclear genetic events induced by UV light in strains of Saccharomyces cereÕisiae with different UV sensitivities, Mutat. Res. 7 Ž1969. 171–185. w6x M.A. Resnick, Genetic control of radiation sensitivity in Saccharomyces cereÕisiae, Genetics 62 Ž1969. 519–531. w7x D. Averbeck, W. Laskowski, F. Eckardt, E. Lehman-Brauns, Four radiation sensitive mutants of Saccharomyces, survival after UV and X-ray-irradiation as well as UV-induced reversion rates from iso-leucine-valine dependence to independence, Mol. Gen. Genet. 107 Ž1970. 117–127. w8x B.S. Cox, J.M. Parry, The isolation, genetics and survival characteristics of ultraviolet light-sensitive mutants in yeast, Mutat. Res. 6 Ž1968. 37–55. w9x J.C. Game, R.K. Mortimer, A genetic study of X-ray sensitive mutants in yeast, Mutat. Res. 24 Ž1974. 281–292. w10x M.A. Resnick, The repair of double-strand breaks in DNA: a model involving recombination, J. Theor. Biol. 59 Ž1976. 97–106. w11x J.C. Game, B.S. Cox, Epistatic interactions between four rad loci in yeast, Mutat. Res. 16 Ž1972. 353–362. w12x J.C. Game, B.S. Cox, Synergistic interactions between rad mutants in yeast, Mutat. Res. 20 Ž1973. 35–44. w13x J.C. Game, B.S. Cox, Repair systems in Saccharomyces, Mutat. Res. 26 Ž1974. 35–44. w14x G. Lemontt, Mutants of yeast defective in mutation induced by ultraviolet light, Genetics 68 Ž1971. 21–23. w15x L. Prakash, S. Prakash, Isolation and characterisation of MMS-sensitive mutations of Saccharomyces cerreeÕisiae, Genetics 86 Ž1977. 33–55. w16x J.A. Henriques, E. Moustacchi, Isolation and characterisation of pso mutants sensitive to photo-addition of psoralen derivatives in Saccharomyces cereÕisiae, Genetics 95 Ž1980. 273– 288. w17x M. Esposito, R.E. Esposito, The genetic control of sporulation in Saccharomyces cerreÕisiae: I. The isolation of temperature-sensitive sporulation-deficient mutants, Genetics 61 Ž1969. 79. w18x N.G. Suslova, I.A. Zakharov, Genetic control of radiosensitivity in yeast: 7. Identification of genes conferring sensitivity to X-rays, Genetika 6 Ž1970. 158–163. w19x T.A. Weinert, L.H. Hartwell, The RAD9 gene controls the cell cycle response to DNA damage in Saccharomyces cereÕisiae, Science 241 Ž1988. 317–322. w20x J.M. Flaman, T. Frebourg, V. Moreau, F. Charbonnier, C. Martin, P. Chappuis, A.P. Sappino, I.M. Limacher, L. Bron, J. Benhattar et al., A simple p53 functional assay for screen-
w21x
w22x
w23x
w24x
w25x
w26x
w27x
w28x
ing cell lines, blood, and tumors, Proc. Natl. Acad. Sci. U. S. A. 92 Ž1995. 3963–3967. D. Higgins, S. Prakash, P. Reynolds, R. Polakowska, S. Weber, L. Prakash, Isolation and characterisation of the RAD3 gene of Saccharomyces cereÕisiae and inviability of rad3 deletion mutants, Proc. Natl. Acad. Sci. U. S. A. 80 Ž1983. 5680–5684. L. Naumovsky, E. Friedberg, A DNA repair gene required for the incision of damaged DNA is essential for viability in Saccharomyces cereÕisiae, Proc. Natl. Acad. Sci. U. S. A. 80 Ž1983. 4818–4821. P. Sung, L. Prakash, S.W. Matson, S. Prakash, RAD3 protein of Saccharomyces cereÕisiae is a DNA helicase, Proc. Natl. Acad. Sci. U. S. A. 84 Ž1987. 8951–8955. A. Green, J. Snipe, D. Gordenin, M.A. Resnick, Functional analysis of human Fen1 in Saccharomyces cereÕisiae and its role in genome stability, Hum. Mol. Genet. 8 Ž1999. 2363– 2373. C. Lambert, L.B. Couto, W.A. Weiss, R.A. Schultz, L.H. Thompson, E.C. Friedberg, A yeast DNA repair gene partially complements defective excision repair in mammalian cells, EMBO J. 7 Ž1988. 3245–3253. E.L. Perkins, J.F. Sterling, V.I. Hashem, M.A. Resnick, Yeast and human genes that affect the E. coli SOS response, Proc. Natl. Acad. Sci. U. S. A. 96 Ž1999. 2204–2209. R. Gary, M. Park, J.P. Nolan, H.L. Cornelius, O.G. Kozyreva, H.T. Tran, K. Lobachev, M.A. Resnick, D.A. Gordenin, A novel role for Fen1rRad27 PCNA-binding in DNA metabolism and implications for genetic risk, Mol. Cell. Biol. 19 Ž1999. 5373–5382. S.A. Jelinsky, L.D. Samson, Global response of Saccharomyces cereÕisiae to an alkylating agent, Proc. Natl. Acad. Sci. U. S. A. 96 Ž1999. 1486–1491.
Michael A. Resnick a, ) Chromosome Stability Group, Laboratory of Molecular Genetics, National Institute of EnÕironmental Health Sciences, NIH, P.O. Box 12233, 111 Alexander DriÕe, Research Triangle Park, NC 27709, USA E-mail address: [email protected] a
Brian S. Cox b School of Biological Sciences, UniÕersity of Kent, Canterbury, CT68EJ, UK E-mail address: [email protected] b
)
Corresponding author.