Mol Gen Genet (2001) 264: 702±708 DOI 10.1007/s004380000358

O R I GI N A L P A P E R

F. Onoda á M. Seki á A. Miyajima á T. Enomoto

Involvement of SGS1 in DNA damage-induced heteroallelic recombination that requires RAD52 in Saccharomyces cerevisiae

Received: 10 December 1999 / Accepted: 6 July 2000 / Published online: 21 October 2000 Ó Springer-Verlag 2001

Abstract The SGS1 gene of Saccharomyces cerevisiae is homologous to the genes that are mutated in Bloom's syndrome and Werner's syndrome in humans. Disruption of SGS1 results in high sensitivity to methyl methanesulfonate (MMS), poor sporulation, and a hyper-recombination phenotype including recombination between heteroalleles. In this study, we found that SGS1 forms part of the RAD52 epistasis group when cells are exposed to MMS. Exposure to DNA-damaging agents causes a striking, Rad52-dependent, increase in heteroallelic recombination in wild-type cells, but not in sgs1 disruptants. However, in the absence of DNA damage, the frequency of heteroallelic recombination in sgs1 disruptants was several-fold higher than in wild-type cells, as described previously. These results imply a function for Sgs1: it acts to suppress spontaneous heteroallelic recombination, and to promote DNA damage-induced heteroallelic recombination. Key words sgs1 Disruptants á Heteroallelic recombination á Methyl methanesulfonate á Rad52 á Saccharomyces cerevisiae

Communicated by H. Ikeda F. Onoda á M. Seki á T. Enomoto (&) Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan E-mail: [email protected] Tel.: +81-22-2176874 Fax: +81-22-2176873 A. Miyajima Division of Pharmacology, Biological Safety Research Center, National Institute of Health Sciences, Setagaya-ku, Tokyo 158-8501, Japan

Introduction Escherichia coli RecQ helicase is a multifunctional protein, and null mutations in recQ, in conjunction with mutations on other genes, result in a reduction in the incidence of homologous recombination, as well as an increase in sensitivity to UV irradiation (Nakayama et al. 1985). In addition, illegitimate recombination is increased in recQ mutants relative to wild-type cells (Hanada et al. 1997). In human cells, ®ve genes encoding RecQ homologues have been identi®ed (Seki et al. 1994; Puranam et al. 1994; Ellis et al. 1995; Yu et al. 1996; Kitao et al. 1998). Two of the ®ve human genes are BLM and WRN, mutations in which are responsible for Bloom's syndrome (BS) and Werner's syndrome (WS), respectively (Ellis et al. 1995; Yu et al. 1996). In BS cells, the incidence of interchanges between homologous chromosomes is increased and an abnormally high number of sister chromatid exchanges (SCEs) occur (Chaganti et al. 1974; German 1993). Cells derived from WS patients show chromosome instability, a shorter life span in in vitro culture, and accelerated telomere shortening (Martin 1977; Schulz et al. 1996). Saccharomyces cerevisiae has only one RecQ homologue, Sgs1. A mutant allele of SGS1 was identi®ed as a suppressor of the slow-growth phenotype of top3 mutants (Ganglo€ et al. 1994). The sgs1 mutants display pleiotropic phenotypes, showing defects in the ®delity of chromosome segregation during mitosis and meiosis (Watt et al. 1995), increases in several kinds of spontaneous recombination (Watt et al. 1996; Yamagata et al. 1998), poor sporulation (Watt et al. 1995), and premature aging of yeast mother cells (Sinclair and Guarente 1997; Sinclair et al. 1997). Thus sgs1 disruptants seem to be a good model for BS and WS. Although the helicases of the RecQ family, such as E. coli RecQ, Sgs1, and BLM, act as anti-recombination proteins, genetic and biochemical evidence indicates that E. coli RecQ helicase acts to initiate homologous

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recombination (Nakayama et al. 1985; Harmon and Kowalczykowski 1998). However, no evidence has been obtained for the involvement of Sgs1 or human RecQ homologues in the initiation of homologous recombination. In Schizosaccharomyces pombe, loss of function of the rqh1+/hus2+/rad12+ gene, which is also a recQ homologue in these yeast cells, confers sensitivity to UV light and hydroxyurea, and hyper-recombination phenotypes (Murray et al. 1997; Stewart et al. 1997; Davey et al. 1998). An epistasis analysis of the UV sensitivity of rqh1 mutants indicated that rqh1 belongs to the recombinational repair pathway (Murray et al. 1997). However, nothing is known about the precise role of Rqh1 in this pathway. In this study, we found that SGS1 belongs to the RAD52 recombinational repair pathway by an epistasis analysis of the methyl methanesulfonate (MMS) sensitivity of SGS1 disruptants. In addition, we found that the frequency of interchromosomal recombination, which increased in wild-type cells treated with MMS, was not increased in sgs1 disruptants, indicating the involvement of Sgs1 in homologous recombination.

Materials and methods Yeast strains The yeast strains used in this study are listed in Table 1. The rad52D mutants were constructed by one-step gene replacement using the EcoRI-SalI fragment of plasmid HT19. The rad14 and apn1 mutants were constructed by one-step gene replacement using the SpeI-EcoRV fragment of plasmid rad14 and the BamHI-EcoRI fragment of plasmid pSCP108, respectively. Plasmids The full-length SGS1 gene was isolated by PCR. The YCp vector pRS314 was used as the single-copy vector (Sikorski et al. 1989). pYCp1305 and SA7850 were constructed by inserting the XhoI ()207 bp)-SacI (5398 bp) fragment of SGS1 into pRS314 and pBluescript SK+, respectively. pH14 was constructed by subcloning an 0.7-kb (2258±3048 bp) fragment of SGS1 into pBluescript SK+. pA12 contained the 1.1-kb SmaI URA3 fragment inserted in the StuI site in the SGS1 coding region of pH14, and was used to construct the sgs1::URA3 mutant strain. The SGS1 gene was disrupted by the one-step gene substitution method (Rothstein 1983). The resultant transformants were selected on SC plates lacking uracil. Gene disruption was con®rmed by PCR or Southern analysis. pHA12 was constructed by inserting the 2.5-kb MscI-SmaI fragment of the AUR1-C gene from pAUR101 (TaKaRa) in the StuI site of pHA14, and was used to construct the sgs1::AUR1 mutant strain. Transformants were selected in YPAD medium containing 0.5 lg/ml Aureobasidin A (TaKaRa). SA9051 was constructed by inserting the KpnI ()1352 bp)XhoI ()207 bp) fragment of SGS1 into SA7850 after creating a KpnI site by PCR. The XhoI-EheI fragment containing most of the SGS1 ORF in SA9051 was replaced with the 2.5-kb MscISmaI fragment of AUR1-C, to generate pSN1-27. The sgs1D:: AUR1 fragment from pNS1-27 was digested with KpnI and SacI and used to create the sgs1D:: AUR1 mutant strain. Transformants were selected in YPAD medium containing 0.5 lg/ml Aureobasidin A.

Table 1 Yeast strains used Strain

Genotype

MR101

MATa/MATa ura3-52/ura3-52 leu2-3,112/ leu2-3,112 trp1-289/trp1-289 his1-7/his1-1 MATa/MATa ura3-52/ura3-52 leu2-3,112/ leu2-3,112 trp1-289/trp1-289 his1-7/his1-1 sgs1::URA3/sgs1::LEU2 MATa/MATa ura3-52/ura3-52 leu2-3,112/ leu2-3,112 trp1-289/trp1-289 his1-7/his1-1 sgs1D::AUR1-C/sgsID::AUR1-C MATa/MATa ura3-52/ura3-52 leu2-3,112/ leu2-3,112 trp1-289/trp1-289 his1-7/his1-1 rad52 D::hisG-URA3-hisG/rad52D::hisG-URA3-hisG MATa/MATa ura3-52/ura3-52 leu2-3,112/ leu2-3,112 trp1-289/trp1-289 his1-7/his1-1 sgs1D::AUR1-C/sgsID::AUR1-C rad52D::hisGURA3-hisG/rad52D::hisG-URA3-hisG MATa ura3-52 leu2-3,112 trp1-289 his1-7 MATa ura3-52 leu2-3,112 trp1-289 his1-1 MATa ura3-52 leu2-3,112 trp1-289 his1-7 sgs1::URA3 MATa ura3-52 leu2-3,112 trp1-289 his1-1 sgs1::LEU2 MATa ura3-52 leu2-3,112 trp1-289 his1-7 sgs1D::AUR1-C MATa ura3-52 leu2-3,112 trp1-289 his1-1 sgs1D::AUR1-C MATa ura3 leu2 trp1 his3 MATa ura3 leu2 trp1 his3 sgs1::AUR1-C MATa ura3 leu2 trp1 his3 rad14::HIS3 MATa ura3 leu2 trp1 his3 sgs1::AUR1-C rad14::HIS3 MATa ura3 leu2 trp1 his3 apn1::URA3 MATa ura3 leu2 trp1 his3 sgs1::AUR1-C apn1::URA3 MATa/MATa lys2/lys1 ho::LYS2/ho::hisG ura3/ ura3 leu2::hisG/leu2::hisG his4B::LEU2/ his4X::LEU2 (BamHI)-URA3 MATa/MATa lys2/lys1 ho::LYS2/ho::hisG ura3/ ura3 leu2::hisG/leu2::hisG his4B::LEU2/ his4X::LEU2 (BamHI)-URA3 sgs1::AUR1-C/ sgs1::AUR1-C

MR202 ds1 dr52 ds1r52

MR966 MR93-28C MRQ966 MRQ93 966NS-1 93NS-25 7830-2-4a F364s1 F364r14 F364s1r14 F364a1 F364s1a1 NKY2856 RKH225

Epistasis analysis of SGS1 Cells were cultured at 30 °C for 1 day in YPAD medium, diluted with distilled water, and inoculated onto YPAD plates, or YPAD plates that contained various concentrations of MMS. After a 3-day incubation at 30 °C, colonies were counted. Alternatively, cells were cultured at 30 °C for 1 day in YPAD medium, diluted to 5 ´ 106 cells/ml, and exposed to 0.1% MMS for the indicated periods. The MMS-treated cells were washed with a medium containing 10% sodium thiosulfate, diluted and inoculated onto YPAD plates. After 3 days at 30 °C, the colonies were counted. Frequency of recombination between heteroalleles Strains were constructed such that recombination between heteroalleles, his1-1/his1-7 or his4X-LEU2 URA3/his4B-LEU2, in a diploid could be detected by the restoration of histidine prototrophy. Cells treated with various DNA-damaging agents were inoculated onto SC plates lacking histidine. After 3 days incubation at 30 °C, the colonies were counted.

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Fig. 1A±D Epistasis analysis of sgs1 disruptants with respect to MMS sensitivity. A Relationship between SGS1 and RAD14. Wildtype (7830-2-4a, ®lled squares), sgs1 (F364s1, ®lled circles), rad14 (F364r14, ®lled triangles), and rad14-sgs1 (F364s1r14, open triangles) cells were cultured in YPAD medium at 30 °C, diluted and inoculated on YPAD plates containing the indicated concentration of MMS. Colonies were counted after 3 days. B Relationship between SGS1 and APN1. Wild-type (7830-2-4a, ®lled squares), sgs1 (F364s1, ®lled circles), apn1 (F364a1, ®lled triangles), and apn1-sgs1 (F364s1a1, open triangles) cells were cultured in YPAD medium at 30 °C, diluted and inoculated on YPAD plates containing the indicated concentration of MMS. C Relationship between SGS1 and RAD52. Wild-type (MR101, ®lled squares), sgs1 (ds1, ®lled circles), rad52 (dr52, open squares) and rad52-sgs1 (ds1r52, open circles) cells were cultured in YPAD medium at 30 °C, diluted and inoculated on YPAD plates, containing the indicated concentration of MMS. D Wild-type (MR101, ®lled squares), sgs1 (ds1, ®lled circles), rad52 (dr52, open squares) and rad52-sgs1 (ds1r52, open circles) cells were treated with 0.1% MMS for 0, 10, 20, 30, or 40 min as described in Materials and methods

Results and discussion SGS1 belongs to the RAD52 epistasis group Mutants in which the SGS1 gene was disrupted showed high sensitivity to MMS. We therefore investigated the relationship between SGS1 and known DNA repair mechanisms. Double mutants carrying a sgs1 mutation and either a nucleotide excision repair (NER) mutation (rad14) or a base excision repair (BER) mutation (apn1) showed signi®cantly greater sensitivity to MMS than the

respective single mutants (Fig. 1A and B). The survival curves for sgs1 rad52 double mutants and the rad52 single mutant following exposure to MMS, however, were essentially indistinguishable (Fig. 1C and D), indicating that SGS1 belongs to the RAD52 repair epistasis group. Sgs1 is involved in heteroallelic recombination induced by MMS or UV As epistasis analyses had indicated that SGS1 belongs to the RAD52 group, we next examined heteroallelic recombination (interchromosomal recombination) between his1-1/his1-7 in diploid sgs1 disruptants (ds1). The frequency of spontaneous recombination was elevated several-fold relative to that in wild-type cells (Fig. 2A), in agreement with the result reported by Watt et al. (1996). When wild-type cells were exposed to 0.1% MMS for 45 min, the number of His+ colonies increased by about 50-fold. In contrast, the frequency of MMSinduced interchromosomal recombination in sgs1 disruptants was markedly reduced relative to that in wild-type cells. To determine whether the increase in the number of His+ colonies observed following exposure to MMS was due to reversion mutations, we assayed rates of reversion of the tested auxotrophic markers in haploid wild-type and sgs1 cells. The rates of reversion were not a€ected by the presence of MMS in either the wildtype or the sgs1 strain.

705

Fig. 2A±D Heteroallelic recombination increases after exposure of wild-type, but not sgs1, cells to MMS. A, B Heteroallelic recombination is not enhanced by exposure of sgs1 disruptants to MMS. Wild-type (MR101, squares) and sgs1 (ds1, circles) cells were treated with 0.1% MMS for 0, 15, 30 or 45 min as described under Materials and methods, and then inoculated on SC plates lacking His (106 cells/plate), and on YPAD plates (103 cells/plate) to monitor viability. The plates were incubated at 30 °C for 3 days and the colonies that appeared were counted. The open symbols (A) indicate the numbers of His+ colonies per 106 survivors, and percentage survival is indicated by the ®lled symbols (B). C, D Involvement of SGS1 in MMS-induced heteroallelic recombination. Wild-type cells (MR101) were transformed with pRS314 (circles) or YCp1305 (squares) . The sgs1 disruptants (ds1) were transformed with pRS314 (diamonds) or YCp1305 (triangles). The cells were treated with MMS as described above. Cells (106) were inoculated on SC plates lacking His and Trp, and 103 cells were inoculated on SC plates lacking Trp to monitor viability. After 3 days at 30 °C, colonies were counted. The open symbols (C) indicate the numbers of His+ colonies per 106 survivors, the ®lled symbols (D) percentage survival

Introduction of the wild-type SGS1 gene, but not an empty vector, into sgs1 disruptants suppressed the spontaneous hyperrecombination and restored MMSinduced heteroallelic recombination (Fig. 2C), indicating that suppression of spontaneous recombination and induction of recombination after exposure to MMS are intrinsic functions of SGS1. To test the generality of the above observation, we measured heteroallelic recombination at di€erent loci (his4X-LEU2 URA3/ his4BLEU2) in di€erent strains. The numbers of His+ recombinants found after treatment of 106 viable cells with 0.1% MMS for 30 min were 1002 for wild-type cells

(NKY2856) and 28.0 for sgs1 disruptants (RKH225), respectively, while the numbers of spontaneous His+ recombinants were 27.8 for wild-type cells and 173 for sgs1 disruptants. The reduction in the frequency of MMS-induced interchromosomal recombination in sgs1 disruptants is unique, because loss of the function of other eukaryotic RecQ proteins so far reported results in a hyper-recombination phenotype under conditions that induce DNA damage. Since the viability of sgs1 disruptants exposed to MMS for 40 min was reduced to nearly 10% that of unexposed cells (Fig. 2B), it was possible that recombination occurred in the cells that eventually died. To exclude such a possibility, we examined the recombination frequency of his1-1/his1-7 heteroalleles by exposing cells to lower concentrations of MMS which had little e€ect on the viability of sgs1 disruptants (Fig. 3). Under these conditions, the frequency of recombination in wild-type cells was increased by exposure to MMS, but no increase was observed in sgs1 disruptants. We next examined the induction of interchromosomal recombination by another DNA-damaging agent, UV light. The sgs1 disruptants showed a slightly higher sensitivity to UV irradiation than wild-type cells. A considerable increase in recombination frequency was observed in wild-type cells following UV irradiation, but little induction of recombination was observed in sgs1 disruptants (Fig. 4).

706

Fig. 3A, B Induction of heteroallelic recombination by low concentrations of MMS which have little e€ect on viability. The wild-type (MR101, squares) and sgs1 (ds1, circles) cells were inoculated on YPAD plates or SC plates lacking His, which contained the indicated concentrations of MMS, and incubated at 30 °C for 3 days. The colonies that appeared on the plates were then counted. The numbers of His+ colonies observed per 106 survivors are inidcated by the open symbols (A), and the percentage survival by the ®lled symbols (B)

RAD52 is involved in UV-induced heteroallelic recombination We next examined the involvement of RAD52 in UVinduced and spontaneous heteroallelic recombination. Since rad52 disruptants showed very low viability after exposure to MMS, we used UV instead of MMS as the DNA-damaging agent. As shown in Table 2, the frequency of spontaneous heteroallelic recombination in sgs1 disruptants was drastically decreased by disruption of RAD52, as reported previously (Watt et al. 1996). The increase in the frequency of heteroallelic recombination following UV irradiation ± which requires Sgs1 function ± was also diminished by disruption of RAD52, indicating a dependency of Sgs1 on Rad52 for UV-induced heteroallelic recombination. Thus, this result demonstrates, for the ®rst time, a link between the function of a eukaryotic RecQ helicase, Sgs1, and the Rad52-dependent recombination pathway. In E. coli, genetic data

show that RecQ function is required for recombination in a recBC sbcB background (Nakayama et al. 1985). In addition, it has been shown biochemically that RecQ protein initiates DNA recombination in concert with RecA and SSB proteins in vitro (Harmon et al. 1998). Thus the involvement of RecQ family proteins in DNA recombination seems to be evolutionarily conserved. In this context, it is interesting to note that WRN has nuclease activity as well as helicase activity, like RecBCD protein (Huang et al. 1998; Kamath-Loeb et al. 1998; Suzuki et al. 1999). The results presented here clearly show that Sgs1 is a multifunctional protein. The next key question is how Sgs1 performs di€erent functions depending on the situation. It is conceivable that the function of Sgs1 is regulated by modi®cation. If Sgs1 is modi®ed in response to DNA damage, the modi®ed Sgs1 could play a role in the homologous recombination that requires

Fig. 4A, B Induction of heteroallelic recombination by irradiation with UV light. Wild-type (MR101, squares) and sgs1 (ds1, circles) cells cultured in YPAD medium were diluted with distilled water and inoculated on YPAD plates or SC plates lacking His, to monitor viability and heteroallelic recombination, respectively. The cells were irradiated with the indicated doses of UV light and incubated at 30 °C for 3 days. The numbers of His+ colonies observed per 106 survivors are indicated by the open symbols (A), and the percentage survival by the ®lled symbols (B)

707 Table 2 Involvement of RAD52 in UV-induced homologous recombination Strain

Genotype

MR101

SGS1/SGS1; RAD52/ RAD52 sgs1/sgs1 rad52/rad52 sgs1/sgs1 rad52/rad52

ds1 dr52 ds1r52 a b

Induced recombination ratea, b

Cell survival (%)b

12 ‹ 4.2

126 ‹ 24

88.6 ‹ 16.4

120 ‹ 42 0.64 ‹ 0.20 0.98 ‹ 0.42

109 ‹ 53 1.0 ‹ 1.66 0.89 ‹ 0.42

70.9 ‹ 21.2 38.2 ‹ 19.0 50.7 ‹ 16.8

Spontaneous recombination ratea

The values indicate the numbers of recombinants per 106 viable cells The cells were exposed to 5 J/m2 UV light

Rad52 function, as in Escherichia coli, where RecQ is suggested to be involved in the initiation of recombination together with RecA and SSB (Harmon and Kowalczykowski 1998). Unmodi®ed Sgs1 would act to suppress recombination, eliminating recombination intermediates as reported by Bennett et al. (1999). Alternatively, Sgs1 may recognize di€erent types of DNA substrates, the processing of which results in the suppression or promotion of recombination, depending on the DNA substrates. These possibilities must be addressed in future studies. Although many experiments have been done on heteroallelic recombination, it remains to be clari®ed how cells initiate heteroallelic recombination. Therefore, the ®nding in this study that Sgs1 is involved in MMSand UV-induced heteroallelic recombination provides a clue as to the mechanism that underlies damage-induced heteroallelic recombination. Acknowledgments We thank Dr. Hideyuki Ogawa for helpful discussion and for his generous gift of the plasmid HT19. We also thank Dr. Bruce Demple and Dr. Errol Friedberg for their generous gifts of the plasmids pSCP108 and rad14, respectively, and also thank Dr. Nancy Kleckner and Dr. Leland Hartwell for their gifts of the yeast strains NKY2856 and 7830-2-4a, respectively. This work was supported by Grants-in-Aid for Scienti®c Research, and for Scienti®c Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan, Health Sciences Research Grants from the Ministry of Health and Welfare of Japan, and a grant from the Mitsubishi Foundation.

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