The pso mutants of Saccharomyces cerevisiae comprise two groups: one deficient in DNA repair and another with altered mutagen metabolism

Mutation Research 489 (2001) 79–96

Review

The pso mutants of Saccharomyces cerevisiae comprise two groups: one deficient in DNA repair and another with altered mutagen metabolism Martin Brendel a , João Antonio P. Henriques b,∗ a

b

Institut für Mikrobiologie, J.W. Goethe-Universität, 60596 Frankfurt, Main, Germany Department of Biophysics, Biotechnology Center, UFRGS, Av. Bento Gonçalves 9.500, 91507-970 Porto Alegre, RS, Brazil Received 2 April 2001; received in revised form 24 July 2001; accepted 24 July 2001

Abstract Yeast mutants that are sensitive to photoactivated psoralens, named pso mutants, were isolated and described more than 20 years ago. Nine genes responsible for the pso phenotypes were identified and seven of them cloned and molecularly characterized. Of the nine PSO genes of yeast seven apparently encode proteins involved in the repair of DNA lesions generated by photoinduced psoralens and by other mutagens, while two, PSO6 and PSO7, are responsible for structural elements of the membrane and for a functional respiratory chain, respectively. Of the seven proven or putative DNA repair genes six directly or indirectly control induced mutagenesis. Four of these PSO loci were found allelic to already known repair genes, whereas two, PSO2 and PSO4, represent new genes involved in DNA repair and in repair/pre-mRNA processing in S. cerevisiae. Gene PSO2 encodes a protein indispensable for repair of DNA interstrand cross-links that are produced by a variety of bi- and poly-functional mutagens and that appears to be important for a likewise repair function in humans as well. © 2001 Published by Elsevier Science B.V. Keywords: Yeast; Psoralen sensitivity; DNA repair; Mutagenesis; Cross-link repair; Pre-mRNA splicing; Ergosterol; Cytochrome c oxidase

1. Introduction Phototherapy with UVB alone or with UVA-activated Psoralen (PUVA) has been developed many years ago in order to treat dermatological disorders ranging from atopic dermatitis to psoriasis and vitiligo

[1]. Photoactivated psoralens may react with proteins and nucleic acids and when PUVA phototherapy was shown to induce DNA lesions [2], it became clear that this might result in induced mutation and an elevated risk for skin cancer [3]. Depending on the type of psoralen used (mono- or bi-functional) the UVA-activated

————– Abbreviations: BER, base excision repair; cis-, transDDP, cis- and transPlatin, respectively; DEO, diepoxyoctane; DSB, DNA double-strand breaks; 8HQ, 8-hydroxyquinolineoxide; 8-MOP + UVA, 3-CPs + UVA, pre-treatment with 8-methoxypsoralen, respectively, 3-carbethoxypsoralen and irradiation with 365 nm UV-light; 4NQO, 4-nitroquinoline N-oxide; HN1, nitrogen half mustard; HN2, nitrogen mustard; ICL, interstrand cross-link; MNNG, N-methyl-N -nitro-N-nitrosoguanidine; NDEA, N-nitrosodiethylamine; NER, nucleotide excision repair; TLS, translesion synthesis; UVC, irradiation with 254 nm UV-light; WT, wild type ∗ Corresponding author. Tel.: +55-51-3316-6087; fax: +55-51-3319-1079. E-mail addresses: [email protected] (M. Brendel), [email protected] (J.A.P. Henriques). 1383-5742/01/$ – see front matter © 2001 Published by Elsevier Science B.V. PII: S 1 3 8 3 - 5 7 4 2 ( 0 1 ) 0 0 0 6 6 - 7

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photoaddition of the psoralen molecule to the thymine bases of DNA will generate monoadduct or diadduct lesions, the latter constituting the highly genotoxic interstrand cross-links (ICL) [4,5]. In order to study the photochemical, biological, and genetical consequences of PUVA treatment to living cells, Henriques and Moustacchi [6] chose the simple unicellular eukaryote Saccharomyces cerevisiae as a model. While easy to propagate and with well-understood genetics, this organism has nearly all the features of a higher eukaryote and thus results obtained with yeast may indicate what to expect after likewise treatments in mammalian cells. This is the third review on the physiology, genetics and molecular biology of pso mutants of the yeast S. cerevisiae. Since the last two overviews on this topic in 1990 and 1997 [7,8] there has been considerable progress in elucidating the molecular biology of some of these mutants and also two new PSO loci have been defined. The pso mutants of yeast were first selected for their sensitivity against the action of photoactivated mono- and bi-functional psoralens [6]. Initially, three mutants pso1, pso2 and pso3, each defining a distinct locus, were phenotypically and genetically characterized [6,9,10]. Since then more pso mutant alleles have been selected via their photoactivated psoralen sensitivity phenotype, so that in 1997 we could present data on seven distinct yeast

PSO loci [8]. Presently the number of PSO genes stands at nine. The original screening of a mutagenized yeast culture for psoralen + UVA sensitive isolates seems to have favoured selection of mutants with impaired functions in steps of error-prone DNA repair ([7–11], this review) since six of the seven pso mutants believed to be deficient in DNA repair show reduced mutability and recombination [10,12,13]. Our present review will demonstrate that these genes do not encode a group of proteins with similar functions. While the majority plays a role in the repair of DNA lesions induced by photoactivated psoralens and by other mutagens, and most of them can be integrated into the epistasis groups of yeast repair genes [7,8], at least two of the nine pso mutants are not deficient in DNA repair, but derive their sensitivity phenotypes from physiological deficiencies that influence the psoralens’ (or other mutagens’) genotoxicity rather than the repair capacity of the respective mutant. The majority of these repair-deficient pso mutants have been found allelic to already known DNA repair mutants (cf. Table 1) but because the pso mutants’ group also comprises mutants with other physiological defects either related to a special step in DNA repair or even unrelated to DNA repair they are seldomly included in mainstream reviews on DNA repair in yeast which concentrate on the more popular and intensively researched RAD genes [14], of

Table 1 The nine PSO loci: allelism, function, and phenotypes Gene

Protein (kDa) function

Phenotype of mutant

PSO1/REV3

PSO3

173; catalytic subunit of DNA polymerase zeta 72; late unknown step in ICL removal, repair (␤-metallo lactamase?) Unknown (base excision repair?)

PSO4/PRP19 (essential)

56.7; spliceosome associated protein

PSO5/RAD16 PSO6/ERG3 PSO7/COX11

91.3; DNA helicase NER of silent genome 43; ergosterol desaturase 28; cytochrome c oxidase

PSO8/RAD6

19.6; ubiquitin conjugating DNA repair

PSO9

Unknown DNA repair?

Sensitive to radiation and chemical mutagens; low mutability Sensitive to all ICL-inducing treatments; slightly UVC-sensitive Low induced mutability and recombination; sensitive to H2 O2 , paraquat, 3-CPs + UVA Mutagen-sensitive; no mitotic recombination; low mutability at 30◦ C; no sporulation; pso4-1 thermoconditional mutant allele UVC-, oxidative damage-sensitive Sensitive to oxidative damage Growing cells 4NQO, 8HQ, NDEA sensitive; pso7-1 leaky mutant allele UVC sensitive; variable sporulation; spontaneous mutator; low induced mutability UVC sensitive; normal sporulation; low induced mutability

PSO2/SNM1

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which many encode proteins also involved in repair of PUVA-induced DNA damage. In the present review on the nine PSO loci we will offer a compact but comprehensive summary on their genetic and physiological role in yeast, including their interaction with other repair genes of this organism. In order to facilitate the functional comparison of the PSO genes we will abide to the above-mentioned grouping, i.e. we will first describe the seven pso mutants presumably deficient in DNA repair or RNA processing and then those not impaired in their DNA repair capacity. 2. PSO genes involved in DNA repair or nucleic acid processing 2.1. PSO1 Mutant pso1-1 was amongst the first pso mutants to be isolated [6] and phenotypically studied. In addition to its sensitivity to photoactivated monoand bi-functional psoralens, its low mutability with these and other mutagens was noted [9,10]. Epistasis/synergism analyses [15,16] revealed that PSO1 belongs to the RAD6 group of repair genes that make up two error-free and one error-prone postreplication repair pathway [17]. PSO1 was found to be allelic to the REV3 gene [18], whose mutant allele rev3-1 had been isolated, amongst other non UVC-mutable yeast mutants, by Lemontt [19]. REV3 encodes the catalytic subunit of the rev3p–rev7p dimer that constitutes the translesion polymerase zeta (pol ␨), the protein responsible for error-prone translesion synthesis (TLS) [20]. The activity of pol ␨ is controlled by Rev1p, so that a loss of function in either one of these three REV loci inhibits error-prone TLS and hence UVC mutability in yeast [21], and mutationally induced alterations in REV3 or REV7 gene expression influence the UVC mutability of the yeast cells [22]. The relative contribution of Rev3p to postirradiation repair synthesis is rather small, as the presence of either polymerase delta or epsilon suffices to perform normal repair synthesis [23]. However, the relative high sensitivity to the radiomimeticum HN2 of resting rev3/pso1 mutant cells indicates an important contribution of TLS in processing of intermediates of ICL repair [24]. Pol ␨ is, however, not the only polymerase involved in mu-

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tagenesis as a mutation in Pol32p, a subunit of polymerase delta, renders yeast deficient in UVC-induced mutagenesis and influences the activities of several mutator genes in spontaneous CAN1 forward mutation [25]. The role of error-prone TLS in repair of damaged DNA has been recently summarized [26]. Finally it was found that the rev3 mutants are sensitive to selenite, indicating that selenite-induced DNA lesions can also be processed by the REV3-dependent error-prone TLS [27]. DNA repair is common to all forms of life and while there may be a large diversity of types of DNA repair, the increasing availibility of whole genome sequences has permitted a phylogenetic study that has revealed many evolutionary links between DNA repair pathways of different species [28]. REV3 homologs have been found in Aspergillus nidulans [29], in Drosophila (mus205) [30], in mouse (Rev3L) [31] and two in human cells (hREV3) [32,33] and hREV3L [34]. Disruption of mouse rev3L gene caused embryonic lethality [35–37], whereas the antisense RNA blocking of hREV3 had no influence on the in vitro growth of cells, though slightly enhancing their UVC sensitivity and significantly lowering their UVC-induced mutability [38]. Since human REV7 and REV1 homologs also exist [39,40], it seems that at least the three components of a controllable polymerase ␨ known from yeast are also present in humans and may have the same function there [40]. A recent report shows that human polymerase ␨ plays a major role in Ig and bcl-6 somatic hypermutation [41]. While error-prone TLS may play a role in human carcinogenesis it was noted that the human Rev3p was ubiquitously expressed in normal and malignant tissues [42]. 2.2. PSO2 Mutant pso2-1, also amongst the first psoralen sensitive isolates, was found to be especially sensitive to the bi-functional 8-MOP + UVA treatment and to nitrogen mustard (HN2) [6,10]. When allelism of pso2-1 and the snm1 mutants (sensitive to nitrogen mustard) [43] was established [18], it was clear that a non-functional Pso2p/Snm1p leads to a new phenotype: specific sensitivity to the highly cytotoxic bi- or poly-functional mutagens [44] that, apart from monofunctional lesions, are able to produce bi-functional DNA lesions, amongst them the DNA–ICL. Sensitivity

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Table 2 A unified nomenclature for PSO2 and SNM1 alleles in S. cerevisiaea Allele

New designation

Reference

PSO2/SNM1 pso2-1 snm1-1 snm1-2ts snm1:Tn10LUK (URA3) snm1-cin4:TRP1

PSO2 pso2-1 pso2-11 pso2-12ts pso2:Tn10LUK (URA3) pso2-cin4:TRP1

[18] [6] [161] [55] [161] [57]

a This unified nomenclature for the PSO2 and SNM1 alleles is chosen in order to avoid the confusion with a novel SNM1 locus, defining a S. cerevisiae gene encoding a protein component of the yeast RNase MRP [53].

of the pso2/snm1 mutants is principally independent of the type of bases involved in formation of such ICL, i.e. all tested pso2 mutant alleles (cf. Table 2) were shown to be specifically sensitive to ICL, irrespective of their chemical make-up. These included N7-diguaninyl ICL, induced by mustard gas [45] and by HN2 as well as by its derivatives cyclophosphamide and chlorambucil [46], O6 -diguaninyl ICL, produced by mitomycin C [43], thymine–psoralen–thymine ICL, produced by 8-MOP + UVA [47], cisDDP- and transDDP-induced ICL [48], diepoxyoctane-induced ICL [49], and acetaldehyde induced ICL [50], to name some representative ICL species. Since incision of cross-linked DNA by enzymes of the NER is normal in snm1/pso2 mutants, Snm1p/Pso2p is thought to be specifically involved in a post-incision step [51] of the repair of DNA–ICL lesions, irrespective of their chemical nature [8]. Interestingly, snm1-1 and snm1 mutants exhibit a slight sensitivity towards UVC which may be explained by the fact that UVC produces ICL, albeit as a minor DNA lesion [52]. Following suggestions of the Stanford Yeast Group we have recently agreed to change the names of all SNM1 alleles to the PSO2 nomenclature, and to give our hitherto published snm1 mutant alleles new pso2 allele numbers (cf. Table 2); this is done to minimize confusion with yet another SNM1 gene of S. cerevisiae, that encodes a protein component of this yeast’s RNase MRP [53] and that was originally isolated as a high-copy-number suppressor of the temperature sensitive nme1 mutant [54]. Depending on the mutagen used there may be large differences in stability of the DNA–ICL lesions, and

induced mutagenesis and mitotic recombination may be totally or partially blocked in pso2 mutants (cf. [7]). While 8-MOP + UVA induced thymine ICL are chemically stable, a fact which facilitated their exact quantification by biochemical methods [47], sulphur and nitrogen mustard N7-diguaninyl-ICL are not and have a half-life of approximately 2 h at 36◦ C [8]. The stability of an ICL may greatly influence its removability via DNA repair enzymes (once an ICL has lost one of its bonds to a base, or the ICL-bound base is excised, the resulting monoadduct may be readily repaired via NER) and may be directly correlated to its genotoxicity [8]. Among all ICL, those induced by 8-MOP + UVA are the most chemically stable, the hardest to repair by NER and thus amongst the most toxic [8,47]; their high stability apparently prevents repair via TLS as seen by absence of induced mutagenesis in a biologically realistic survival range in pso2/snm1 mutants [9], whereas pso2/snm1 has WT-like mutability after UVC irradiation [10]. Thermoconditional mutant snm1-2ts [55], now pso2-12ts (cf. Table 2) carries three point mutations, two silent ones and one that replaces glycine with arginine at a position 256 [56], thereby altering the hydrophilic ␤-lactamase domain of the protein. Temperature-shift experiments revealed that Pso2p functions within 5–6 h after ICL-inducing HN2 treatment [55]. Pso2p has a nuclear localization signal and has been localized in nuclear fraction with anti-Pso2p antibody. Pso2p has one putative zinc finger motif, that may, however, not be important for its function in ICL repair, since its removal by site-directed mutagenesis led to strains that still had WT-like resistance to HN2. With approximately 0.3 transcripts/cell PSO2 is poorly transcribed [57], but may be induced about four-fold by ICL-producing mutagens, including UVC, but not by the monofunctional alkylating agent MNNG or by the UV-mimeticum 4NQO [58]. Inducibility appears to depend on the presence of a DRE (damage response element) like motif in the PSO2 promoter [59], while a downstream silencer within the ORF of neighbor-gene CIN4 (formerly GTP1, [60]) is responsible for its low level constitutive expression. Analysis of DNA repair in 8-MOP + UVA-, HN2-, cisDDP-treated pso2-1 and pso2-11 mutants showed that incision near ICL, and also (partial) excision of the DNA damage proceeds WT-like but that a late step in ICL repair, reconstituting high m.w. DNA from low

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m.w. DNA generated by early incision/excision events is failing [48,51]. Sensitivity to bi- and poly-functional mutagens of NER-deficient mutants of the RAD3 epistasis group and the necessity of DSB repair was taken as indication that two modes of DNA repair, RAD3- and RAD51-like, are necessary to remove ICL [61]. In contrast to the repair of ICL in genomic DNA, Pso2p is not able to act on 8-MOP + UVA-induced plasmidial DNA-lesions [62]. A pso2-1 mutant incises near ICL-containing DNA sites in the transcriptionally active MATα locus but cannot do the same in the silent locus HMLα, indicating an association of Pso2p with NER of actively transcribed genes [63]. The blocking of recombinational, non-homologous end joining, and error-prone repair has influence on repair of HN2- and cisDDP-induced ICL indicating that all three modes of repair are involved [24]. However, contribution to repair of a particular pathway was cell cycle-dependent: ICL-containing dividing yeast cells generated DNA DSB as repair intermediates even in absence of any known incision mechanism, and the DSB were repaired via RAD52-dependent recombination and non-homologous end joining [24]. ICL in resting cells only led to few DSBs and the REV3-encoded polymerase ␨ activity appeared most important in resolving ICL repair intermediates in this cell stage. Treatment of isogenic pso2, rev3 and rad51 mutant strains in G1 with cisDDP with significant lethality did not hinder the cells from progressing through S-phase, but they permanently arrested in G2 , whereas likewise treated WT cells would re-enter the cell cycle. The three gene products thus seem to function after replication of the damaged DNA [64]. Protein–DNA cross-links were repaired WT-like in a pso2 mutant [65]. Fanconis anemia cells and snm1/pso2 yeast mutants (and Walker 256 rat tumor cells [66]) share a common phenotype: they are specifically sensitive to the bi-functional cross-linking agents cisDDP and HN2 [67]. Most Fanconi’s cell lines are capable of initiating repair processing of cross-linked DNA by incision [68] and thus, like snm1/pso2, must be defective in a later step of ICL removal. Attempts to enhance resistance of CHO cells to cross-linking agents by heterologous expression of the yeast SNM1 gene (amongst others), however, were not successful [69]. SNM1/PSO2 homologs have been found in mammals and humans. The putative human cross-link

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repair gene KIAA0086, based on homology with yeast SNM1 locus, contains no TATA region in its 5 -non-transcribed region but several consensus regions for transcription factors (each one site for NFkB and Sp1, three for AP1 and four for AP4 [70]. It is, therefore, thought inducible by a variety of stress situations, including ionizing radiation, UVC, HN1, cisDDP and reactive oxygen intermediates. Its putative regulation and the homology to the yeast SNM1/PSO2 locus make human KIAA0086 a likely candidate for a DNA repair gene. Mouse embryonic stem cells with a SNM1 (−/−) genotype are mitomycin C sensitive but, unlike the corresponding yeast mutant, not to other ICL-inducing chemicals. This was explained by the possible existence of further SNM1 homologs in mice [71]. Complementation of mitomycin C sensitivity could be seen after transfection with human SNM1 cDNA [71]. The recent linking of mutant alleles of the human ELAC2 gene, encoding a Snm1p-like protein, with hereditary pre-disposition for prostate cancer points to a contribution of human SNM1-like genes to DNA or chromosome stability and genome integrity in man [72]. The exact mechanism of ICL repair is still not understood, though it seems clear that the enzymes of NER start processing ICL-containing DNA and that the block in repair in pso2 is at a post-incision step (DNA degradation and no restoration of high m.w. DNA in the absence of Pso2p) [48,51]. Based on our knowledge on repair intermediates and mutant survival, two scenarios of repair events could be offered: (a) in resting cells, a not yet fully understood unilateral removal of one arm of the ICL (by NER of actively transcribed genes, BER, chemical instability), followed by gap filling TLS and, in a second step, removal of the remaining monofunctional lesion via NER [73]; and (b) in growing cells, by incision-induced ICL removal in both DNA strands (after DNA synthesis) in G2 with subsequent repair of the resulting DSB by non-homologous end joining or recombinational repair, the latter perhaps associated with mutation induction [74]. 2.3. Placing cross-link repair into perspective As stated above, the pso2 mutant’s role in the removal of ICL from yeast DNA is not well understood. The existence of Pso2-like proteins in mammals and

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their sensitivity to DNA–ICL-inducing treatments when these proteins are non-functional indicates some usefulness of Pso2p in repair of such lesions. In yeast, genes from all three epistasis groups of repair seem to encode proteins involved in this complex task [7]. When discussing repair of ICL one should, however, always keep in mind that 8-MOP + UVA-induced ICL are highly toxic and that about 20 of these ICL suffice for a lethal hit in a haploid WT cell [8]. Their repair, therefore, can be considered rather limited, especially when compared to the 14,000–18,000 UVC-induced CPDs per lethal hit for haploid WT yeast [75,76]. Since a pso2 mutant is about 10–20 times more sensitive to photoactivated 8-MOP than a WT cell, it may not be able to repair any of the induced ICL at all [8]. Though pso1 to pso4 mutants show epistatic interaction for ICL-inducing mutagens we do as yet have no evidence that repair proteins Pso1/Rev3 through Pso4/Prp19 would interact alone or in a complex (amongst themselves or with other repair proteins) for a putative sequential processing and removal of these DNA lesions in yeast. 2.4. PSO3 Mutant pso3-1 had the weakest psoralen sensitivity phenotype of the three initially isolated pso mutants [6] and showed blocked photoactivated psoralen-induced mutagenesis and mitotic gene conversion [9,10,12]. The pso3-1 mutant has nearly WT-resistance to alkylating agents, to UVC and to ␥-radiation [6,10], but still exhibits defective induced reverse and forward mutation [10] and induced mitotic recombination by specifically reducing induced gene conversion [12] whereas induction of reverse mutation in meiosis-committed pso3-1 diploids by photoactivated psoralens is WT-like [77]. A pso3-1 strain containing a duplication of the his4 gene had, depending on the orientation of the duplication a 2–40-fold decrease in the frequency of spontaneous recombination [78]. The pso3-1 mutant was also found sensitive to superoxide anion-generating paraquat [7,8,79], as well as to H2 O2 , to cadmium chloride, and to formaldehyde, suggesting an impaired repair of oxidative stress-related DNA-lesions. The original pso3-1 mutant and the WT from which it was derived exhibit a high resistance to MNNG and

generate a large number of spontaneously generated “petites”. This was shown to be related on low pools in glutathione (GSH) that were already present in the WT strain N123. After separation of pso3-1 from the low GSH-conferring gsh1-leaky allele that furnishes only about 15% of the normal GSH pool, some phenotypes, e.g. MNNG resistance, high petite induction, formaldehyde and cadmium sensitivity were lost [79]. Double mutants containing pso3-1 and selected rad mutant alleles exhibited epistatic interaction with rad3-12 and pso2-1 mutant alleles for sensitivity to 3-CPs and 8-MOP photoaddition [80], placing the PSO3 gene into the NER pathway (RAD3 type). Several attempts at molecular cloning PSO3 via complementation of one of the sensitivity or non-mutability phenotypes failed so far, though the screening of about 70,000 transformants (i.e. at least 10 times the number theoretically necessary to have all yeast genes from that gene bank represented) yielded several suppressor genes that, however, could only complement or partially complement the sensitivity phenotype of pso3-1 [81], while WT-like induced mutability or mitotic gene conversion was never fully restored in such transformants. Parallel to until now unsuccessful attempts at the cloning of PSO3 gene, we continued a detailed analysis of the physiological defects of the pso3-1 mutant. Since the sensitivity phenotype of pso3-1 suggests deficits in repair of oxidative DNA damage it might be defective in BER of oxidized bases. Heterologous expression of the E. coli nth gene that encodes exonuclease III, specific for removal of oxidized base damage, indeed could restore WT resistance and mutability in H2 O2 treated pso3-1 transformants [82]. Also, the high rate of H2 O2 treatment-induced DNA DSB in pso3-1 was significantly reduced in the nth transformants, clearly showing that introduction of a functional exonuclease III prevented DSB formation or, less likely, facilitated their repair [82]. Interestingly, the yeast nth homologs NTG1 and NTG2 [83,84] could not complement the pso3-1 mutation [85] although they were shown to be involved in repair a broad spectrum of oxidative damage in both mitochondrial and nuclear DNA [86]. However, one could think of other putative roles for Pso3p as other yeast genes may have a significant influence on DNA damage-induced error-free TLS and hence might influence induction of mutation and gene conversion [87].

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2.5. PSO4 Mutant pso4-1 is the only one of the nine pso mutants not derived from the originally mutagenized yeast culture [6]. The slightly X-ray sensitive haploid mutant xs9 was isolated by Benathen and Beam [88], and was re-named pso4-1 because of its high sensitivity to photoactivated psoralens [11]. The pso4-1 mutation confers a pleiotropic repair-defect phenotype (cf. [8]) and is a thermoconditional mutant with no viability at 36◦ C [89]. Isolation of a complementing clone yielded the sequence of gene PRP19 [89], an essential yeast gene encoding a spliceosomal complex-associated protein [90,91] and genetical crosses verified allelism of pso4-1 and PRP19. This suggested two reasons for the pleiotropic phenotype of pso4-1: (a) the Pso4p/Prp19p has more than one function, and one of these would directly affect DNA repair and recombination; or (b) non-effective pre-mRNA splicing of one to several of the 225 known intron-containing pre-mRNAs of yeast at permissive temperature would lead to partially impaired cell physiology. Thus, mutagen sensitivity, lower mutability, recombination, and sporulation found for pso4-1 and its homozygous diploid could all be the result of non-splicing (or partially non-splicing) of the respective pre-mRNAs. This second hypothesis can be tested by placing the respective null mutants into the pso4-1 background and assess for epistatic/synergistic

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interactions of the mutant alleles. With this approach we could show that the UVC sensitivity phenotypes of pso4-1 does not primarily result from faulty processing of RAD14-transcribed pre-mRNA [92]. Influence of temperature on splicing efficiency and on some phenotypes correlated to intron-containing genes has been proven (Table 3) [92]. The expressivity of non-splicing differs for the observed phenotypes: while pso4-1 displays the same high benomyl and NaCl sensitivity for each temperature tested, there is significantly higher resistance to caffeine and to calcofluor white at 23◦ C, where the mutant still has a splicing efficiency >70% (Table 3). The differential influence of splicing efficiency on benomyland osmo-sensitivity can be explained by the fact that benomyl resistance is controlled by at least four genes, one of them (TUB1) being essential [93], while osmosensitivity is controlled by at least seven genes, with three of them essential, including the ACT1 locus [94]. Theoretically, however, Pso4p/Prp19p could have more than one function, a feature not unknown for repair enzymes; its spliceosome-association thus would only be one of its functions, while the protein might also play a role in assembly or stability of, e.g. a DNA repair complex needed for function of error-prone TLS. Several two hybrid screenings using a Pso4p bait construct yielded 32 interacting isolates which could be allocated to 18 ORFs. Seven of these putative

Table 3 Temperature-dependent splicing and selected phenotypes in pso4-1a Temperature (◦ C) 23

30

33

0.73 ± 0.10 1

0.33 ± 0.09 1

0.10 ± 0.01 1

Splicing-related phenotypes LF1-1A (pso4-1) (treatment) NaCl (900 mM) SSSSS Benomyl (40 ␮g/ml) SSSSS Caffeine (0.5 mg/ml) SSS CFW (30 ␮g/ml) S

SSSSS SSSSS SSS S

SSSSS SSSSS SSSSS SSSSS

Sporulating efficiency (strain) W303 (WT) MG5128 MG5101

50 ± 3 0.5 ± 0.7 0.2 ± 0.2

n.d. n.d. n.d.

Splicing efficiency of ACT1–lacZ (%) (strain) LF1-1A (pso4-1) LF-2A (PSO4)

a

43 ± 2 13 ± 1 1.1 ± 0.3

Each S signifies one decade of cell killing, WT has 100% survival MG5128 (pso4-1/pso4-1), MG5101 (pso4-1/pso4::HIS3) [89], strain W303 contains the RAD5 WT allele.

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Pso4p interacting proteins are encoded by ORFs with as yet unknown functions. The remaining 11 interacting proteins can be roughly grouped into four functional classes: (a) DNA repair (1 ORF); (b) growth and cell cycle regulation (6 ORFs); (c) chromatin structure and chromosome dynamics (2 ORFs); and (d) pre-mRNA splicing (2 ORFs) [95]. The Prp19p-associated complex consists of at least eight proteins of which four have been identified [96–98]. They interact with each other to form the Prp19p-associated complex but not all seem essential for the splicing process. Perhaps one or more of the Pso4p interactors found might be members of this associated complex or might themselves react (bind to) proteins from the Prp19-associated complex. 2.6. PSO5 Stationary phase cells harbouring mutant allele pso5-1 are moderately sensitive to UVC, to the UV-mimeticum 4NQO and to the radiomimetica HN1 and HN2 [99]; they are also cross-sensitivite to 3-CPS + UVA, which produces either 4 –5 -furan-side pyrimidine monoadducts in DNA or singlet oxygen [100,101]. Further analysis also revealed a sensitivity phenotype to other oxidative stress-enhancing chemicals, such as H2 O2 and paraquat [99,102]. By molecular analysis pso5-1 was found it to be a mutant allele of RAD16 [102]. PSO5/RAD16 encodes a 91.3 kDa (790 aa) protein containing two putative zinc finger domains and several other helicase-typical regions that are also found in several other yeast proteins like Snf2, Rad54 and Mot1 [103,104], in Brm (Drosophila), and in Hsnf2, the human homolog of Snf2 [105]. The N-terminal part of Rad16p contains a high number of charged amino acids, a feature also found in yeast repair proteins Rad4, Rad6, and Rad7 and it also contains a signal sequence for nuclear localisation [103,104]. Rad16p has functions in global genome repair, a subpathway of NER that preferentially repairs CPD in either the HMLa or HMLα loci [104], in silent regions of DNA, and in the non-transcribed strands of active genes [103,104,106]. Rad16p combines with Rad7p and this complex binds UV-damaged DNA in an ATP-dependent manner [107,108]. Though yeast mutants rad16 and rad7 lack global genome repair, their transcription-coupled NER is not affected [106]. NER is carried out by

nucleotide-excision-repair factors NEF1 (Rad14p, Rad1p–Rad10p), NEF2 (Rad4p, Rad23p), NEF3 (Rad2, TFIH), and NEF4 (Rad7p–Rad16p), which function in incision and excision of CPD [107,109]. NEF4 may participate in postincision events during NER [110]. This, and the fact that Rad16p is a member of the SWI2/SNF2 superfamily of DNA-dependent ATPases [103], suggests that NEF4 may facilitate the remodeling of a protein–DNA structure generated by damage-specific incision (possibly including the NER machinery) that is uniquely for NER of transcriptionally repressed regions and the non-transcribed strand of transcriptionally active genes. This remodeling may facilitate oligonucleotide excision and repair synthesis. NER-dependent incision in regions such as HMLα is normal in rad7 and rad16 mutants [109,110] and Rad7p has been shown to physically interact with the UV-damaged DNA recognizing factor NEF2 [108], indicating that Rad7p may link NEF2 with NEF4 [111]. It thus appears that NEF4 plays a dual role in NER, i.e. in cooperation with NEF2 in the initial step of damage recognition, perhaps in the turnorver of the incision protein machinery, and in the recruitment of other NEF necessary for NER. By comparing the expression of ␤-galactosidase from DNA damage-inducible RNR2–lacZ and RNR3–lacZ fusion constructs in WT to that in a pso5/rad16 mutant, Paesi-Toresan et al. [112] have shown that the DNA damage-induced expression of RNR2 and RNR3 not only depends on a functional Pso5p/Rad16p but also on the type of DNA damage. While the mutagens UVC, 4NQO, and H2 O2 induced RNR2 and RNR3 via DNA damage, the oxidative stressors tBOOH and paraquat could not. Thus, the latter two agents may form DNA lesions unable to initiate the signal cascade of inducible NER [113] or the signals are not addressing the DRE motif-specific inducible genes like RNR2 and RNR3 [59,114]. For some mutagens, however, Pso5p/Rad16p seems to function in the signal transducing pathway controlling DNA damage-inducible components of NER and associated genes. Using the two-hybrid system, Rad16p/Pso5p was shown to interact with the SGS1-encoded protein Sgs1p [115] which in vitro displays 3 → 5 helicase as well as DNA-dependent ATPase activity [116,117]. Sgs1p also interacts with topoisomerases II and III [118,119] and is involved in premature aging in yeast

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[120]. It is homologous to the human Wrn, Blm, and RTS proteins, which are responsible for the Werner, Bloom, and Rothmund–Thomson syndroms that are related to premature aging and to pre-disposition to cancer phenotypes, respectively [118,121,122]. A rad16–sgs1 double mutants displays epistasis after treatment with several mutagens. The sgs1 mutant’s sensitivity to MMS, UVC, 4-NQO, and H2 O2 repair [115] and the sensitivity to UVC and ␥-ray irradiation of sgs1 [123] point to a function of Sgs1p in DNA repair. It appears that the helicase activity of Sgs1p is responsible for most elements of the sgs1 mutant phenotype, including its sensitivity to hydroxyurea [124]. Most of the sgs1 mutant alleles confer high MMS sensitivity [125] but in conjunction with a rad16 mutant allele that itself confers little MMS sensitivity, a significantly increased MMS resistance is observed [115]. Yeast mutants containing top3 mutant allele either alone or in conjunction with sgs1 or with sgs1 and rad16 mutant alleles are highly sensitive to MMS, 4-NQO, H2 O2 and UVC [115] again suggesting that Sgs1p, together with Pso5p/Rad16p and Top3p are involved in processing of DNA lesions. However, the interaction of sgs1 with the other mutant alleles is rather complex and varies with the mutagen used [115]. Mutations in SGS1 have been demonstrated to be synthetically lethal in combination with mutations in SRS2 [126]. Srs2p is also a 3 → 5 helicase that mediates DNA repair and recombination [127,128]. Absence of Rad51p and hence, absence of homologous recombination, will rescue a sgs1–srs2 double mutant from cell death [123]. Owing to the large number of helicases in a yeast cell one is tempted to speculate that there might exist specific helicases for the selective channeling into a correct repair pathway for certain types or groups of DNA lesions. In conjunction with the proposed Top3p–Sgs1p dependent repair of DNA single strand breaks [129] this would suggest a different networking of DNA repair. Deletion of a functional Rad16p significantly reduces the average life span of the mutants [115]. The rad16–sgs1 double mutant displayed a life span comparable to that of the sgs1 single mutant. This epistatic interaction of rad16 and sgs1 mutations suggests that RAD16/PSO5 and SGS1 share functions in a common pathway of life span control. Interestingly, loss of the NER-associated proteins Rad7p,

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Rad1p, and Rad26p [109] do not reduce life span in the respective mutants [130]. The life span reduction observed in pso5/rad16 mutants suggests that the RAD52 DNA repair pathway (Rad50p, Rad51p, Rad52p, Rad57p) [130], involved in DSB and homologous recombinational repair [14], is not solely responsible for life span control in yeast. 2.7. PSO8 Mutant pso8-1 was phenotypically characterized as sensitive to photoactivated 3-CPs and 8-MOP as well as to UVC, MNNG, 4NQO, and to DEO. It also showed reduced mutation induction (both reverse and forward) and a spontaneous mutator phenotype. Diploids homozygous for pso8-1 sporulated normally. In a pso8-1–rad4 double mutant the alleles interacted synergistically for UVC-sensitivity, so that the phenotypic characterization and the non-epistatic interaction with an allele of the NER (rad4) allowed allocation of the PSO8 locus to the RAD6 group of repair genes. The pso8-1 mutation was indeed complemented by a RAD6-containing single-copy vector [131]. The pso8-1 mutant allele contains a missense rad6–P64L mutation [131]. Together with the recently described phleomycin-sensitive mutant ph140 [132] it is yet another mutant allele of the RAD6 locus. Rad6p is responsible not only for DNA repair but also for ubiquitin conjugating activity that polyubiquitinates some histones and that depends on the integrity of its highly acidic 23 aa carboxyl terminus [133] for sporulation and telomere silencing [134]. Some mutant alleles, the rad6–P64L amongst them, still retain partial functionality in that they encode proteins still allowing sporulation of the respective homoallelic diploids [135]. 2.8. PSO9 Mutant pso9-1 defines a novel PSO gene as it is not only sensitive to photoactivated 3-CPs and 8-MOP but also is able to complement the psoralen sensitivity phenotypes when crossed to all hitherto defined pso mutants. While its molecular cloning is in progress, it has been characterized so far by its mutagen-sensitivity phenotype and its impaired mutability after treatment of resting cells with photoactivated psoralens and with UVC and MNNG. Mutant pso9-1 was not sensitive

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Table 4 Human homologs of yeast PSO genes Yeast

Human (aa identity) function

Reference

PSO1/REV3 PSO2/SNM1 PSO4/PRP19

REV3L (40%); repair protein, influences mutability KIAA0086 (27%); mutagen-, stress-inducible protein NMP200 (24%); common nuclear matrix protein; putative roles in structural support of RNA processing machinery and in regulation of splicing

[38] [70] [162]

PSO5/RAD16

SMARCA3 (29%); nuclear hydrolase, ATPase TTF2 (28%); ATP-dependent RNA polymerase II transcription termination factor

[163] [164]

PSO6/ERG3 PSO7/COX11

SC5DL (51%); putative C-5 sterol desaturase hCOX11 (55%); putative role in maturation of cytochrome c reductase

[165] [166]

PSO8/RAD6

UBE2B (70%); ubiquitin-conjugating enzyme UBE2A (69%); ubiquitin-conjugating enzyme

[167] [167]

to oxidative damage-inducing H2 O2 and paraquat exposure. Reverse mutation was significantly lower than in WT but not as low as in pso8-1 (rad6) mutant. Forward mutation in the CAN locus was severely inhibited as compared to the WT for all four tested mutagens. These phenotypic characteristics justify its association with the repair group of the pso mutants. 2.9. Putative human homologs of PSO genes Progress in sequencing, especially of mammalian genes, has revealed that many Pso-like proteins have been more or less conserved during evolution. Though some of the proteins only share identity of around 20% they are still thought of being putative homologs, mainly because of highly conserved functional domains that are also found in the corresponding yeast protein. In Table 4 we give a brief summary of the actual information of (putative) human homologs of the seven molecularly characterized yeast PSO genes.

3. PSO genes that are involved in mutagen metabolism and not in DNA repair The two mutants pso6-1 and pso7-1 are sensitive to the monofunctional 3-CPs + UVA and, to a much lesser degree, to photoactivated 8-MOP [99]. In addition to 3-CPs-thymine DNA monoadducts this treatment also generates singlet oxygen, an active oxygen species that has been shown to intensify oxidative DNA damage [100]. This latter activity may be suppressed in the presence of the singlet oxygen quencher

sodium azide [99] and the resulting near WT resistance indicates that the enhanced sensitivity of pso6-1 and pso7-1 may be the result of increased DNA damage induced by an activated oxygen species that is generated during photoactivation of 3-CPs. Lack of repair of 3-CPs + UVA induced DNA monoadducts, therefore, is probably not the reason for the mutants’ sensitivity. Since exposure to other mutagens, like HN2, HN1, MMS, UVC lead to WT-like survival and WT-like induced reverse mutation, these two mutants clearly differed from the above described other seven. 3.1. PSO6 The pronounced sensitivity to other treatments also generating oxidative stress, e.g. to paraquat and to H2 O2 clearly set pso6-1 apart from pso7-1 which displayed WT-resistance to these chemicals when cells were treated in phosphate buffer and only showed sensitivity to 3-CPs + UVA in resting cells [99]. The PSO6 gene was cloned via complementation of the paraquat sensitivity of pso6-1 and found to be allelic to the ERG3 locus [136] that encodes the enzyme sterol ∆5 -desaturase which introduces the C5=C6 double bond during ergosterol biosynthesis [137]. Ergosterol is the most prominent sterol in yeast membranes (90%), in contrast to other (higher) eukaryotic cells, which contain cholesterol as the main sterol. Presumably both sterols, due to their hydrophobic structure, can contribute to membrane stability. Anchoring amongst the polar long chain of fatty acids, they are thought to influence membrane fluidity thereby regulating flow, permeability,

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enzyme activity and as a consequence, also cell growth [138]. Lipid peroxidation (LP) is known to be one of the most toxic events related to oxidative stress. ROS, specially OH• and HOO• , can pull (extract) a bi-allylic hydrogen atom of unsaturated fatty acid (LH) to form lipid alkyl radical (L• ), which can be oxidized to a lipid peroxi radical (LOO• ), that in turn may attack adjacent LH and propagate the radical chain reaction [139,140]. Ergosterol is able to inhibit LP [141] and it was suggested [142] that this is due to the sterols of endoperoxide and hydroperoxide formed instead (only from ergosterol and not from episterol) which can protect membrane integrity. Ergosterol appears to play an important role in mediating the cytotoxic effects of singlet oxygen [136,143,144]. Subnormal content of membrane ergosterol in pso6/erg3 mutants would, therefore, explain their low efficiency of transformation (altered permeability to Li+ ), lack of protection from oxidative stress generated in respiratory metabolism of non-fermentable substrates like ethanol and glycerol (uncoupling of oxidative phosphorylation), poor mating (altered fusion ability) and maldistribution of chitin (presence of episterol either than ergosterol leading to overexpression of the enzyme Chs3, responsible for chitin membrane deposition) [136]. Since pso6/erg3 also shows WT-like resistance to HN1, HN2, 4NQO, and UVC, this mutant might have a normal endowment of DNA repair; oxidative stress, however, might be enhanced by mutagens that themselves generate ROS and this might ultimately lead to a higher number of oxidative base damages in DNA. Also, induced reverse mutation by UVC, HN1, and HN2 is WT-like in pso6/erg3 mutants, but is enhanced after treatment with 3-CPs + UVA, and to a lesser extent, after 8-MOP + UVA [99]. It thus seems unlikely that the PSO6/ERG3-encoded sterol ∆5 -desaturase is involved in any kind of DNA repair, but that its final product might perform a protective function to prevent oxidative stress in yeast cells. 3.2. PSO7 When in exponential phase of growth pso7-1 mutant cells are highly sensitive to 4NQO and this fact has been exploited to molecularly clone PSO7 via complementation [145]. Genetical and biochemical

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analysis of the complementing yeast gene have shown the allelism of PSO7 and COX11, a gene encoding a protein indispensable for the assembly of a functional cytochrome c oxidase (CcO) [146] which, located in the inner mitochondrial membrane (IMM), is the final electron acceptor of the respiratory chain (RC) responsible for reducing O2 –H2 O. The pso7-1 mutant allele is leaky, i.e. the mutants still contain less than 5% of the WT activity of CcO, whereas the cox11 construct shows no detectable activity of this enzyme [145]. This allows pso7-1 to still grow, though very slowly, on non-fermentable substrates [147], while a strain containing a cox11 allele is petite and will only grow in the presence of fermentable carbon sources [146]. The RC in S. cerevisiae grown on non-fermentable carbon sources behaves as one unit, implying that the different respiratory complexes physically interact [148], i.e. there is a coupling between the steady-state levels of the bc1 complex and CcO [149], indicating the existence of a regulatory mechanism that balances the stoichiometry of the RC complex. Therefore, the absence of a functional CcO in cox11 allele would not permit the RC to act as a single unit, albeit the leaky allele pso7-1 might still permit the RC to act as a supermolecular entity with a control coefficient of one for respiration [149]. The moderate to higher sensitivity to oxidative stress-generating treatments like 3-CPs + UVA [100] or 4NQO [150] has been explained by disturbed electron flows in the pso7/cox11 mutants that may lead to toxicity by a higher rate of LP [151] and to genotoxicity via a higher rate of oxidative DNA damage [100,151]. Also, in pso7/cox11 altered metabolization of certain mutagens (e.g. 4NQO, that is a pro-mutagen and carcinogen which undergoes a 4-electron reduction to become a nitro-radical anion [152] might lead to a higher-than-normal production of metabolites able to generate elevated intracellular oxidative stress that may have higher DNA damaging potential. Repair-proficient pso7/cox11 mutants were found highly sensitive to the mutagens N-nitrosodiethylamine (NDEA), an alkylating chemical that is metabolized via redox cycling to yield hydroxylamine radicals, ROS and LP [140,153–155] and to 8-hydroxyquinoline (8HQ), which may also be activated via altered oxygen metabolism [49] and possibly form diol-epoxide derivatives [156,157]. It should be noticed that pso6/erg3 mutants were also sensitive to NDEA, but

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not to 8HQ, most probably due to the inability of 8HQ to cause LP [49]. Construction of the double mutant pso2 cox11 (the former representing impaired DNA repair and the latter with a defect in mitochondrial function) confirmed that PSO7/COX11 is not involved in DNA repair. It exhibited additivity of 8HQ and NDEA sensitivities of the single mutants, indicating that two different repair/recovery systems are involved in resistance [16]. A slight protection could be detected as DEO (a DNA–ICL damage inducing mutagen [158,159]) sensitivity of the double mutant was equal or less than that of the single pso2 mutant. This suggests a slightly different DEO metabolism in the presence of cox11 (i.e. in the absence of respiring mitochondria) which might lower the number of mutagen-induced DNA–ICL lesions, thus allowing for a better survival in the absence of ICL repair [49]. By employing an established bacterial test specific for showing the formation of oxidative base damage [151] we could verify our results obtained with the pso mutants, which are thought to differentiate between ICL-damaged DNA (pso2) and oxidative base damage (pso6, pso7). After S9-mix biotransformation NDEA, and to a lesser extent 8HQ, lead to significantly higher mutagenesis in E. coli tester strain WP2-IC203 as compared to WP2, whereas DEO-induced mutagenicity remained unchanged, as expected. It thus is acceptable to state that the sensitivity response of the two non-repair genes PSO6 and PSO7 is strictly due to altered metabolism of some mutagens that is caused by intracellular macromolecular alterations (membrane lipids and RC complex IV), and that ultimately may lead to enhanced cell inactivation. Thus, although not participating in any role in DNA repair, ergosterol and CcO may be considered important factors in modulation of intracellular oxidative stress responses. Finally, these two essential metabolic components may be functionally closely related to each other. A perturbation of mitochondrial electron flow can indeed arise from a decrease in ergosterol levels in the IMM (extended oxidative stress if late stages of ergosterol biosynthesis are inhibited) or from a direct interaction between the chemical agents used and the mitochondrial enzyme complexes [160]. This would subsequently enhance permeability of membranes to, e.g. photosensitizers which in turn could lead to cel-

lular damage, including impairment of mitochondrial function and cell inactivation [144]. One could, therefore, assume that a pso6–pso7 double mutant had a much higher sensitivity to predominantly oxidative stress-causing agents. Should that be confirmed, these two mutants alleles, in conjunction with other specific DNA repair mutant alleles, could be employed for the construction of model strains for the typing of unknown mutagens. Such yeasts would detect and report, by changes in biological endpoints, specific and different reaction mechanisms with the applied chemicals: those capable of inducing direct DNA damage versus others inducing mainly LP or, when activated via redox cycle, enhancing intracellular oxidative stress.

4. Concluding remarks The study of pso mutants of S. cerevisiae clearly showed that PUVA-treatment of living cells produces mono- and di-adducts to DNA and that these genotoxic lesions are subject to DNA repair that may lead to mutagenesis and to recombination. Such genetical changes are known to enhance the probability for genetic instabilities in higher eukaryotes and may ultimately lead to cancer. Human PSO homologs, encoding proteins with Psop-like functions would, therefore, by virtue of their damage-eliminating function, act as tumor suppressors. Therefore, PUVA therapy in humans should only be considered in case of severe symptoms of skin disease and after verifying the normal functionality of repair of PUVA-induced DNA damage in such patients. We do not know the contribution of PUVA-treatment to the generation of skin cancer. As a preventive measure, however, one should avoid excessive intake of psoralen-containing food and beverages, especially in summer, as exposure to sunlight would constitute an involuntary PUVA-treatment. In this respect more information on psoralen-containing alimentation would be helpful.

Acknowledgements We thank L.F. Revers and M. Strauss for unpublished data and Drs. C. Pungartnik and M. Grey for critically reading of this manuscript and for helpful

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suggestions. The critical suggestions of two referees were very helpful. Research was supported by CNPq, CAPES and GENOTOX (to J.A.P.H.) and by the DFG and Qualitaetsweine D. Brendel (to M.B.). Travel Grants were furnished by DAAD and CNPq. References [1] T. Horio, Indications and action mechanisms of phototherapy, J. Dermatol. Sci. 23 (Suppl. 1) (2000) S17–S21. [2] L. Musajo, G. Rodighiero, F. Dall’Aqua, Evidences of a photoreaction of the photosensitizing furocoumarins with DNA and with pyrimidine nucleosides, Experientia 21 (1965) 24–25. [3] B.R. Scott, M.A. Pathak, G.R. Mohn, Molecular and genetic basis of furocoumarin reactions, Mutat. Res. 39 (1976) 29–74. [4] L. Musajo, G. Rodighiero, Mode of photosensitizing action of furocoumarins, Photophysiology 7 (1972) 115–147. [5] F. Dall’Acqua, S. Marciani, D. Vevaldi, G. Rodighiero, Skin photosensitization and crosslinking formation in native DNA by furocoumarins, Z. Naturforsch. 290 (1974) 635–636. [6] J.A.P. Henriques, E. Moustacchi, Isolation and characterization of pso mutants sensitive to photoaddition of psoralen derivatives in Saccharomyces cerevisiae, Genetics 95 (1980) 273–288. [7] J.A.P. Henriques, M. Brendel, The role of PSO and SNM genes in dark repair of the yeast Saccharomyces cerevisiae, Curr. Genet. 18 (1990) 387–393. [8] J.A.P. Henriques, J. Brozmanova, M. Brendel, Role of PSO genes in the repair of photoinduced interstrand cross-links and photooxidative damage in the DNA of the yeast Saccharomyces cerevisiae, J. Photochem. Photobiol. B 39 (1997) 185–196. [9] C. Cassier, R. Chanet, J.A.P. Henriques, E. Moustacchi, The effects of the three PSO genes on induced mutagenesis: a novel class of mutationally defective yeast, Genetics 96 (1980) 841–857. [10] C. Cassier, E. Moustacchi, Mutagenesis induced by monoand bi-functional alkylating agents in yeast mutants sensitive to photo-addition of furocoumarins (pso), Mutat. Res. 84 (1981) 37–47. [11] J.A.P. Henriques, E.J. Vicente, K.V.C.L. daSilva, A.C.G. Schenberg, PSO4: a novel gene involved in error-prone repair in Saccharomyces cerevisiae, Mutat. Res. 218 (1989) 111–124. [12] H.H.R. de Andrade, E. Moustacchi, J.A.P. Henriques, The PSO3 gene is involved in error-prone intragenic recombinational DNA repair in Saccharomyces cerevisiae, Mol. Gen. Genet. 219 (1989) 75–80. [13] H.H.R. de Andrade, E.K. Marques, A.C.G. Schenberg, J.A.P. Henriques, The PSO4 gene is responsible for an error-prone recombinational DNA repair pathway in Saccharomyces cerevisiae, Mol. Gen. Genet. 217 (1989) 419–426. [14] J.C. Game, The Saccharomyces repair genes at the end of the century, Mutat. Res. 451 (2000) 277–293.

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[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

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