Mutation Research, 250 (1991) 205-210 © 1991 Elsevier Science Publishers B.V. All rights reserved 0027-5107/91/$03.50 ADONIS 002751079100179K

205

MUT 02526

D N A r e p a i r in the fission yeast,

Schizosaccharomycespombe

A.R. Lehmann a, A.M. Carr a, F.Z. Watts b and J.M. Murray b MRC Cell Mutation Unit and b Microbial Genetics Laboratory, School of Biology, Sussex University, Falmer, Brighton BN1 9RR (Great Britain) (Accepted 16 April 1991)

Keywords: DNA repair in S. pombe; Fission yeast, DNA repair in

Summary Mutants of the fission yeast Schizosaccharomyces pombe which are sensitive to UV a n d / o r -y-irradiation have been assigned to 23 complementation groups, which can be assigned to three phenotypic groups. We have cloned genes which correct the deficiency in mutants corresponding to 12 of the complementation groups. Three genes in the excision-repair pathway have a high degree of sequence conservation with excision-repair genes from the evolutionarily distant budding yeast Saccharomyces cerevisiae. In contrast, those genes in the recombination repair pathway which have been characterised so far, show little homology with any previously characterised genes.

The budding yeast, Saccharomyces cerevisiae, is, apart from Escherichia coli, the organism in which the mechanisms of DNA repair have been characterised in most detail. Despite the identification of over 50 genes involved in DNA repair processes in S. cerevisiae, our understanding of the role that the gene products play in the repair processes is still rudimentary. DNA-repair genes in S. cerevisiae have been assigned to three different epistasis groups. The RAD3 group is involved in excision-repair of ultraviolet-damage, the RAD6 group in mutagenic repair, and the RAD52 group in recombination repair, particularly of ionizing radiation induced damage (Friedberg, 1988). Many of these genes have been cloned

Correspondence: Dr. A.R. Lehmann, MRC Cell Mutation Unit, School of Biology, Sussex University, Falmer, Brighton, BN1 9RR, Sussex (Great Britain).

and characterised, but their precise function is, with few exceptions, still unknown.

Repair pathways in S. pombe A study of DNA repair in S. pombe has a number of attractions. (1) Several features of this organism have more in common with higher eukaryotes than do those of S. cerevisiae. The majority of S. pombe genes, like those of higher eukaryotes, contain introns. The cell cycle and its control have been shown to be very similar in S. pombe and mammalian cells (Lee and Nurse, 1987; Nurse, 1990), mammalian promoters are efficiently expressed and mammalian introns are excised in S. pombe (Kaufer et al., 1985; Jones et al., 1988; Toyama and Okayama, 1990), and conserved sequences have been identified between the mating-type locus of S. pombe and the sex-determining sequences in man (Sinclair et al., 1990).

2{~

(2) Like S. cerecisiae, S. pombe is very amenable to genetic manipulation, but thc two yeasts arc, in evolutionary terms, as diverged from each other as cach ycast is from man (Sipiczki, 1989). Thus identification of sequences conserved between homologous genes in the two yeasts will serve to highlight important parts of the structure of the corresponding gcne products (e.g. active sites. p r o t e i n - p r o t e i n interactions), and the conscrvcd sequences may also provide new routes for isolating corrcsponding genes from higher organisms. (3) Many mutants scnsitivc to UV- and y-radiation have bccn identified in S. pomhe and thcsc putative D N A - r e p a i r mutants have bccn assigned to 23 complemcntation groups (Phipps et al.. 1985; Licbcrman et al., 1989). It is in gcncral a relatively straightforward procedure to clone the genes corresponding to these complemcntation groups by correction of the radiation sensitivity of an appropriate mutant, following transformation with a gene bank constructed from wild-type S. pombe DNA. In this short review wc bricfly describe the characteristics of the S. pomhe mutants and wc summarize our recent work, in which wc have cloned many of the genes defcctivc in these mutants. Early work on most of the S. pombe radiationsensitivc mutants was reviewed in 1985 by Phipps ct al. The mutants have not bccn studied in nearly as much detail as the S. cererisiae mutants, and there arc insufficient data on double mutants to enable them to be assigned, as in S. cerel'isiae, to different epistasis groups. Nevertheless, based on their published properties, it is possible to make tentative assignments of the mutants to three phenotypic groups (Table 1). Early work showed that wild-type S. pombe was sensitized to thc lethal effects of UV-irradiation by post-irradiation treatment with caffeine, and that this caffcine sensitization was retained in some mutants but not in others. Evidence was presented to suggest that the caffeine-sensitivc process represented recombinational repair ( G e n t n c r ct al., 1978), and that mutants which retained thc caffeine sensitization might be deficient in excisionrepair. On this basis we can tentatively assign to a putativc excision-repair group, those mutants which retained caffeinc sensitization to UV (Group 1). As expected from mutants deficient in

TABLE 1 T H R E E I)HEN(.)'I'YI)I(. ' G R ( ) U P S (.)F I)NA R E P A I R M t ' T A N T S IN S. P O M B E ('ompiled and modified from Phipps et al. (1985~ Group I Excision-repair

Group 2 Recombination repair

(Jroup 3 y-Ray repair

nzd2 rad5 rad 7 radio rad I ] rad I3 rad 15 rad 10 radl 7

rad 1 rad3 rad4 rad6 rad,%' rad9 rad 12 rad 1h' radl9 tad20 tad23 rhp6 "

rad21 rad22

E R ( "( "-3sP

excision-repair, many of these mutants have elevated levels of UV-mutability (Gentncr ct al., 1978), but, unlike most excision-repair-deficient mutants in E. coli, S. cerecisiae and mammals, several of the S. pombe mutants in this group appear to be sensitive to ionizing radiation as wcll as to UV light (Phipps et al., 1985). A further puzzling observation is that, despite their marked UV-sensitivity, nonc of the mutants showed a greater than 50% reduction in excision of pyrimidinc dimcrs (Birnboim and Nasim, 1975). In contrast, the ut'rABC mutants of E. coli, several mutants o[" the rad3 group of S. ceret'isiae, and mutants in scvcral of the rodent complcmenration groups are totally defective in excision-repair. This could imply that either more than one excision-repair pathway operates in S. pombe, or that the mutants are leaky. Construction of null mutants by gene disruption following cloning of the genes should resolve this issue. The mutants that we have assigned to Group 2 are those that have been reported to havc lost caffeine-sensitivity after UV damage. It would thus appear that these mutants have lost the caffeinc-sensitive pathway, and they are thought to be defective in recombinational repair. Growth, protein synthesis and a duplicated genome arc required for this pathway to operate (reviewed in Phipps et al., 1985). All mutants in this group are sensitive to both UV- and y-radiation. Most of

207 them have a reduced UV-induced mutant frequency (Gentner, 1977), suggesting that this repair pathway may have a mutagenic component. One of the mutants, in this group, rad4, is temperature-sensitive for growth as well as being sensitive to UV- and y-radiation (Duck et al., 1976). It has been suggested that there might be more than one recombination-repair pathway (Gentner, 1977). Group 3 contains two mutants, rad21 and rad22, reported to be sensitive to y-radiation but not to UV. In our hands, however, rad21 is UV-sensitive. This may require that it is reassigned to a different phenotypic group. Cloning of the genes

We have cloned many of the genes defective in the UV-sensitive mutants, in most cases by complementation of the UV-sensitivity of the mutants with an S. pombe gene bank, but in a few cases by other means.

Group I (putative excision-repair mutants) The RAD3 gene of S. cerevisiae codes for a DNA helicase, capable of unwinding DNA in the 5'-3' direction (Sung et al., 1987). In the RAD3 protein, 7 domains conserved in DNA helicases have been identified (Gorbalenya et al., 1987). The human ERCC-2 gene, recently cloned and characterised by Weber et al. (1988, 1990) is homologous to the RAD3 gene with 55% sequence identity at the amino acid level. Using a truncated RAD3 gene as hybridization probe, we isolated the homologous gene from an S. pombe genebank (Murray et al., manuscript in preparation). Subsequently we showed that the S. pombe homologue had the same sequence as a gene which we had cloned by its ability to correct the UV-sensitivity of the radl5 mutant of S. pombe. Thus the S. pombe radl5 gene is the homologue of the S. cerevisiae RAD3 and human ERCC-2 genes. The sequence conservation is comparable (50-65% identity) between the three organisms, confirming the equivalent evolutionary divergence. As expected, the helicase domains are very highly conserved between the three species. The sequences between these helicase domains are

also conserved, but to a lesser extent (Murray et al., manuscript in preparation). McCready et al. (1989) showed that an S. pombe radl3 mutant could be partially complemented by the S. cerevisiae RAD2 gene. We have cloned the S. pombe radl3 gene and our preliminary sequence data show that the functional homology is matched by sequence conservation. Two tracts of 60-100 amino acids show 60-70% identity between RAD2 and radl3, the rest of the sequence having little conservation (Carr et al., unpublished observations). We have also cloned the S. pombe radl6 gene and preliminary sequence data indicate homology to S. cerevisiae RADI. In this case the degree of identity is lower (30-35%) but extends over a longer region (Carr et al., unpublished observations). The RADI, RAD2 and RAD3 genes of S. cerevisiae are all involved in an early (incision or pre-incision) step of excision-repair of UV damage (Friedberg, 1988). Our results demonstrate convincingly that this pathway is conserved in the two evolutionarily distant yeasts and it is likely to be conserved also in higher eukaryotes. In this regard it is already known that the human ERCC-1 and ERCC-2 genes are respectively homologous to the S. cerevisiae excision-repair genes radiO (van Duin et al., 1986) and tad3 (Weber et al., 1990). Furthermore, the ERCC-3 gene has homologues in both S. cerevisiae and S. pombe, which had not been previously identified (Koken et al., personal communication). These homologies are summarized in Table 2.

Group 2 (mutants in caffeine-sensitive pathway) Cloning and characterisation of the S. pombe radl gene was reported by Sunnerhagen et al. (1990). The predicted gene product had no special features and did not show sequence homology to other genes "in the data banks. We have cloned several genes in this phenotypic group, and their characterization is underway. The rad4 gene has been fully sequenced. It contains a zinc finger and nuclear location signal (Fenech et al., manuscript submitted). A small region of the tad4 gene is homologous to a region of the human XRCCI gene (Thompson et al., 1990), which is involved in the repair of strand breaks. The conservation is not as extensive as for the exci-

2(18 TABLE 2 H O M O L O G I E S B E T W E E N D N A R E P A I R G E N E S IN S. ('EREVISIAI£ S, POMBE A N D M A N S. c'eret'isiae RADI

S. pombe radl6

RAD2

radl3

RAD3

radl5

RADIO

Man

ERC('-2

ERCC-I

ER('('-3 s(

ER('('-3 sv

I'R('('-3

RAI)b

rhp6 ~

E2

('D('9

c&'17

I)NA ligasc

Reference C a r r c t al.. unpublished Cart ctal., unpublished Murray el al.. unpublished; W e b e r ct al.. 1990 Van l)uin et al.. 1986 Kokcn ctal.. personal communication Reynolds et al., 199(); Schneider ctal.. 1990 Barker ct al., 1987; Barnes el al.. 19q(I

sion-repair genes described above. It may reflect a conserved domain or active sitc. The rad9 genc has also been sequenced and does not show any homology to other DNA-repair genes or to other sequences in the data bank (Murray ct al., manuscript submitted). The radl, rad4 and rad9 gene are all expressed at very low levels in S. pombe cells. The lack of extensive homology between the charactcrised genes in this phcnotypic group and other sequences in the data banks raises two intriguing possibilities. Firstly, the S. pombe genes in this pathway could be homologous to as yet uncharacterised genes acting in an equivalent pathway in S. ceret,'isiae. Our future experiments will be aimed at isolating corresponding S. eerecisiae genes by their ability to complement the S. pombe mutants. Alternatively these genes may be involved in a pathway which does not exist in S. ceret'isiae, This in turn raises the question as to whether this pathway exists in S. pombe and man but not in S. cerecisiae, or whether it might bc peculiar to S. pombe. In either of these cases, further study of these genes should provide new information on the mechanisms of recombination repair.

Other DiVA-repair genes The S. cerecisiae RAI)6 gene is the prolt)typc m e m b e r of the RAI)6 cpistasis group, being involved in mutagcnic repair, meiotic recombination and sporulation. The g e n t codes for an ubiquitin-conjugating cnzymc E2, which in vitro is able to ubiquitinate histone H2 (Jentsch el al., 1987). The S. pombe homologuc of this gent. designated rhp6' was isolated by hybridization techniques using a truncated RAI)6 gcnc as probe (Reynolds et al.. 1990). Subsequently, homologous genes have bccn isolated l'rom 1), melatlogaster (Koken ct al., 1~)91) and man (Schneider ct al., 1990; Koken c t a l . , personal communication). The degree of sequence conservation is consistently very high, between all these species, e.g. 69% identity, 84~:'; homology between the S. cerecisiae and human genes. This gene, and by implication the pathway in which it is inw)lved, is, therefore, also conserved through the cukaryotic evolutionary ladder. The r h p O ~ gone does not correspond to any of the S. p o t n b e genes that we have cloned by complementation. Its relationship to other S. pombe genes is not yet known, but this will no doubt become apparent in future studies. S. pombe strains mutant at the cdcl7 locus arc temperature-sensitive for growth and have deficient DNA ligasc activity (Nasmyth, 1977). The cdcl7 gene has been cloned and charactcrised (Barker ct al., 1987) and is 53% homologous to the S. cerecisiae cde9 D N A ligasc gene, with much greater sequence conservation in the ('terminal half of the gcnc. The recently cloned human D N A ligase e D N A also shows 511-61)r?~• homology to both yeast I)NA ligases (Barnes et al., 1990). Ot'erlap o( repair genes Mutants in three of the genes involved in the mating-type switch (swi mutants) in S. pombe arc sensitive to UV-irradiation (Schmidt ct al., 1989) Swi9 mutants have been shown to bc allelic to rad10, radl6 and rad20, suggesting that these four genes may bc one and the same (Schmidt et al., 1989). Mutants in these three rad genes arc however reported to havc quite different properties (Phipps et al., 1985). Sequcncing of the genc complex and identification of the mutations in

209

the various rad genes should clarify the relationship between them.

Such experiments cannot yet be readily carried out in mammalian systems.

Physical mapping of rad genes Notl digestion of S. pombe DNA produces 16 fragments which can be separated using pulse field gel electrophoresis. We have been able to assign the cloned rad genes to these fragments by hybridization. Our results show that there is no significant clustering of genes, but most of them appear to be located on chromosome 1 (Broughton et al., 1991).

Acknowledgements

Conclusions and future prospects Our work on the cloning and analysis of the rad13, radl5 and rad16 genes has demonstrated convincingly that the excision repair pathway is conserved between S. cereuisiae and S. pombe. We have also cloned the rad2, radll and radl7 genes from the putative excision-repair pathway (Group 1). The sequencing of these genes will reveal whether further homologies exist. In the recombination repair pathway, the cloning and sequencing of the rad4 and rad9 genes in our laboratory, and of the radl and rad3 genes in the laboratory of Subramani (Sunnerhagen et al., 1990 and personal communication) have not revealed any homologies with previously cloned genes, with the exception of a short region of homology between the rad4 and XRCC-1 genes. We are currently sequencing the rad8 and radl8 genes in this pathway. Studies of DNA-repair genes in S. pombe are providing information on important domains in the gene products, and on the evolutionary conservation of DNA-repair pathways. In addition, they should open up new ways of cloning human DNA-repair genes, by using sequences conserved between the two evolutionarily distant yeasts, S. pombe and S. cereuisiae, to synthesize degenerate oligonucleotides which can be used either as hybridization probes or as primers for PCR using human cDNA. Once a yeast homologue to a human gene has been identified, gene function can be readily investigated in the yeast ceils by genetic manipulation techniques, such as site-directed mutagenesis and creation of null alleles.

Work from the authors' laboratories was supported in part by EC contract B16-E-142-UK (A.R. Lehmann and A.M. Carr) and by MRC Project grant G88/03213CB (F.Z. Watts and J.M. Murray).

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