Copyright  1999 by the Genetics Society of America

Characterization of the Repeat-Tract Instability and Mutator Phenotypes Conferred by a Tn3 Insertion in RFC1, the Large Subunit of the Yeast Clamp Loader

Yali Xie,* Chris Counter† and Eric Alani* *Section of Genetics and Development, Cornell University, Ithaca, New York 14853-2703 and †Department of Pharmacology and Cancer Biology, Department of Radiation Oncology, Duke University Medical Center, Durham, North Carolina 27710 Manuscript received August 3, 1998 Accepted for publication October 22, 1998

ABSTRACT The RFC1 encodes the large subunit of the yeast clamp loader (RFC) that is a component of eukaryotic DNA holoenzymes. We identified a mutant allele of RFC1 (rfc1::Tn3) from a large collection of Saccharomyces cerevisiae mutants that were inviable when present in a rad52 null mutation background. Analysis of rfc1::Tn3 strains indicated that they displayed both a mutator and repeat-tract instability phenotype. Strains bearing this allele were characterized in combination with mismatch repair (msh2⌬, pms1⌬), double-strand break repair (rad52), and DNA replication (pol3-01, pol30-52, rth1⌬/rad27⌬) mutations in both forward mutation and repeat-tract instability assays. This analysis indicated that the rfc1::Tn3 allele displays synthetic lethality with pol30, pol3, and rad27 mutations. Measurement of forward mutation frequencies in msh2⌬ rfc1:Tn3 and pms1⌬ rfc1:Tn3 strains indicated that the rfc1::Tn3 mutant displayed a mutation frequency that appeared nearly multiplicative with the mutation frequency exhibited by mismatch-repair mutants. In repeat-tract instability assays, however, the rfc1::Tn3 mutant displayed a tract instability phenotype that appeared epistatic to the phenotype displayed by mismatch-repair mutants. From these data we propose that defects in clamp loader function result in DNA replication errors, a subset of which are acted upon by the mismatch-repair system.

UTATIONS in that are involved in DNA A major type of chromosomal instability that has been M replication and repair often result in chromo- identified in yeast, in bacteria, and in cancer cells is somal instabilities such as substitutions and repeat-tract instability (reviewed in Crouse 1996; Kolod- frameshifts as well as insertion, deletion, and re- ner 1996; Modrich and Lahue 1996; Sia et al. 1997b). arrangement events (i.e., Schaaper and Dunn 1987; This instability is thought to result mainly from the Aguilera and Klein 1988; Strand et al. 1993; McAlear failure to repair DNA slippage events that occur during et al. 1996; Tran et al. 1996; Green and Jinks-Robert- the replication of repetitive DNA sequences such as son 1997). Mutations that confer these phenotypes have those that contain mono-, di-, tri-, and tetranucleotide been directly identified in Escherichia coli and Saccharo- repeats. In both yeast and mammalian cells, mutations myces cerevisiae using a variety of instability in mutS and mutL homolog mismatch-repair genes result assays (i.e., Feinstein and Low 1986; Aguilera and in a large increase (100- to 10,000-fold) in the rate of Klein 1988; Michaels et al. 1990; Jeyaprakash et al. repeat-tract instability (Strand et al. 1993; Tran et al. 1994). Such basic research approaches have also led to 1997; Umar et al. 1998). This increase is thought to be a better understanding of the underlying chromosome due to the inability of these mutants to repair small stability defects that have been observed in patients who loop insertion/deletion mutations that occur during suffer from particular inherited diseases. For example, DNA slippage (reviewed in Crouse 1996; Kolodner the phenotypes exhibited by mismatch-repair-defective 1996; Modrich and Lahue 1996; Sia et al. 1997b). In E. coli and S. cerevisiae strains provided evidence indi- yeast, mutations in DNA replication genes that encode cating that the underlying cause for a large percentage the polymerase factor PCNA (POL30) and of hereditary nonpolyposis colorectal cancers was a de- the flap endonuclease (RTH1/RAD27) have also been fect in mismatch repair (Levinson and Gutman 1987; shown to confer an increase in repeat-tract instability at Strand et al. 1993; Crouse 1996; reviewed in Kolodner a rate that is similar to that observed in mismatch-repair- 1996; Modrich and Lahue 1996). defective mutants (Johnson et al. 1995, 1996; Umar et al. 1996; Kokoska et al. 1998). Mutations in other DNA replication genes, such as those that encode the Pol␦ (POL3), and Polε (POL2) DNA , however, Corresponding author: Eric Alani, Section of Genetics and Develop- ment, Cornell University, 459 Biotechnology Bldg., Ithaca, NY 14853- resulted in only modest increases in repeat-tract instabil- 2703. E-mail: [email protected] ity (Johnson et al. 1995; Tran et al. 1997; Kokoska et

Genetics 151: 499–509 (February 1999) 500 Y. Xie, C. Counter and E. Alani

al. 1998). These data have led to the proposal that some man 1989; Burgers 1991; Fien and Stillman 1992; DNA replication factors function directly in the mis- Stillman 1994). match-repair pathway to repair loop insertion/deletions that result from DNA slippage events while others act In this study we tested strains bearing the rfc1::Tn3 at the level of DNA replication to prevent the formation allele alone or in combination with mismatch repair and of these events (Kokoska et al. 1998; reviewed in Sia et DNA replication mutations for defects in chromosome al. 1997b). stability. As described below, the rfc1::Tn3 mutation con- The above observations, in conjunction with the obser- ferred a mutator phenotype that appeared multipli- vation that many cancer cells display repeat-tract insta- cative with mismatch-repair mutations. In repeat-tract -10ف bilities that are unlinked to previously identified repair stability assays the rfc1::Tn3 mutation conferred an and replication genes, encouraged us to initiate screens fold increase in the frequency of dinucleotide repeat- in yeast to identify chromosomal instability mutants tract instability that appeared to be epistatic to the phe- (Liu et al. 1995). We hoped to identify additional factors notype observed in mismatch-repair-defective mutants. that are involved in preventing mutagenic replication Taken together, our data are consistent with the idea errors by preventing DNA slippage events or by facilitat- that defects in clamp loader function result in DNA ing the repair of these slippages through mismatch- replication errors, a subset of which are identified and repaired by the mismatch-repair system. repair mechanisms. In one screen we searched for mu- tants that displayed an increase in the frequency of both repeat-tract insertion/deletion and base pair substitu- MATERIALS AND METHODS tion/frameshift events; unfortunately, this screen only identified mutants that displayed mutations in the pre- Media and chemicals: E. coli strains were grown in Luria- viously characterized mutS (MSH2) and mutL (PMS1, Bertani (LB) broth or on LB agar, which was supplemented ␮ MLH1) homolog genes and the RAD27 gene (Y. Xie, with 100 g/ml ampicillin when required (Miller 1972). Yeast strains were grown in either YPD or minimal selective L. Schvaneveldt and E. Alani, unpublished data). In media (Rose et al. 1990). Selective media contained 0.7% yeast a second screen that is the focus of this article, we nitrogen base, 2% agar, 2% glucose, and 0.09% of a drop-out searched for mutants that were inviable in a double- mix that lacks the amino acid used for selection. Sporulation strand break-repair-deficient (rad52) background and media (SPM) were prepared as described previously (Detloff also displayed a mutator phenotype (Counter et al. et al. 1991). When required, canavanine was included in mini- mal selective media at 60 mg/liter (Rose et al. 1990). 5-fluoro- 1997). This screen was conducted on the basis of two orotic acid (5-FOA) was purchased from U.S. Biologicals and observations made in E. coli: used as described previously (Boeke et al. 1984). When re- quired, methyl-methane sulfonate (MMS; Aldrich Chemical, 1. Certain DNA replication mutants display chromo- Milwaukee) was included in YPD media at 0.017% (v/v). some instability defects such as chromosomal break- E. coli strains: DH5␣ [FЈ phi80, dLacZ⌬ (lacZYA-argF), U169, Ϫ ϩ Ϫ ages that are lethal in recombination-deficient back- recA1, endA1, hsdr17 (r K, m K), lambda , thi1, gyrA, relA1] and KC8 (gammax1486Ϫ, MK12ϩ, leuB600, trpC9830, pyrF::Tn5, grounds (Michel et al. 1997). hisB463, ⌬lacx74, StrA, galU, K) were used to amplify and 2. Strains that lack dam methylase, and are thus defec- manipulate all plasmids described in this article. tive in strand discrimination during mismatch re- S. cerevisiae strains: The genotypes of all strains used in pair, display a mutator phenotype and are inviable these studies are shown in Table 1. With the exception of in recombination deficient (recAϪ) backgrounds NKY1068, EAY561, and DNR53, all strains were derived from the isogenic FY strain background (Winston et al. 1995). (McGraw and Marinus 1980). This inviability, DNR53 is also an S288C-derived strain that contains the which can be rescued by mutations in the mutS, rfc1::Tn3 allele (Burns et al. 1994; Counter et al. 1997). This mutL, and mutH mismatch-repair genes, is thought strain also contains a rad52::HIS3 mutation and is viable be- to be caused by unrepairable double-strand breaks cause it contains an ARS1, CEN4, URA3, plasmid bearing the in damϪ recAϪ strains that form as the result of MutH wild-type RAD52 gene (Counter et al. 1997). NKY1068 is an SK1-derived strain (Kane and Roth 1974) that was kindly incising both template and newly replicated strands provided by Douglas Bishop and EAY561 is a rfc1::Tn3 deriva- (McGraw and Marinus 1980; Au et al. 1992). Us- tive of NKY1068. ing this second screen we identified and character- The msh2⌬::TRP1, msh2⌬::hisG, pms1⌬::hisG, rad52⌬::URA3, ized a transposon Tn3 insertion allele of RFC1,a rad52⌬::LEU2, and rad27⌬::HIS3 alleles contain complete or gene that encodes the large subunit of the highly nearly compete coding region deletions of their respective genes and were introduced into FY23 and FY86 by single-step conserved RFC complex that functions in eukaryo- transplacement. The primer sequences that were used to make tic DNA replication and repair. During DNA repli- polymerase chain reaction (PCR)-amplified DNA fragments cation, RFC interacts with the sliding clamp PCNA containing the rad27⌬::HIS3 allele were described by Tish- at the replication fork primer terminus in steps that koff et al. (1997) and the rad52⌬::URA3 and rad52⌬::LEU2 require adenosine 5Ј-triphosphate (ATP). Forma- disruption plasmids were kindly provided by Todd Milne and Dennis Livingston, respectively. All of the other disruption tion of the RFC-PCNA-primer terminus complex plasmids were made in the Alani laboratory and are available then promotes efficient DNA synthesis by both the upon request. Double mutant combinations of the alleles de- ␦ and ε DNA polymerases (Tsurimoto and Still- scribed in Table 1 were made by standard crosses (Rose et al. RFC1 and Repeat-Tract Instability 501

1990). The pol3-01 and pol30-52 alleles were introduced by Nucleic acid and techniques: All restriction endonu- two-step transplacement (Rose et al. 1990). Vectors used to cleases were purchased from New England Biolabs (Beverly, introduce the pol3-01 (YIPAM26) and pol30-52 (pBL241-52) MA) and used according to manufacturers’ specifications. Taq alleles into the FY strain background were kindly provided by and Expand polymerases were purchased from Perkin-Elmer Akio Sugino and Peter Burgers, respectively. The rfc1::Tn3 Cetus (Norwalk, CT) and Boehringer Mannheim (Indianapo- allele was introduced into the FY and NKY strain backgrounds lis), respectively. Plasmid DNA was isolated by alkaline lysis by single-step transplacement using DNA that had been PCR and all DNA manipulations were performed as described pre- amplified from DNR53 chromosomal DNA using RFC1 spe- viously (Maniatis et al. 1982). Yeast chromosomal DNA was cific primers. The phenotype of rfc1::Tn3 in the different strain prepared as described by Holm et al. (1986). Preparations backgrounds was identical with respect to mutator phe- were made from 5-ml yeast cultures grown to saturation in notype and cold, MMS, and UV sensitivity. All of the alleles YPD. The purified chromosomal DNA was dissolved in 50 ␮l that were introduced by single-step transplacement were con- of double-distilled water and stored at Ϫ20Њ prior to use. firmed by PCR analysis of chromosomal DNA isolated from Polymerase chain reaction (PCR) was performed as de- the transformed strains (primers available upon request). scribed previously (Saiki et al. 1985) and amplification condi- The introduction of the pol30-52 mutation into the FY strain tions and primer sequences for the different reactions are was confirmed by sequencing PCR-amplified DNA frag- available upon request. The basic reaction was performed for ments containing the POL30 open reading frame. The pres- 30 cycles using a denaturation step of 30 sec at 94Њ, an anneal- ence of the pol3-01 mutation was confirmed by digesting the ing step of 30 sec at 58Њ, and a polymerization step of 2 min PCR-amplified POL3 gene from candidate strains with EcoRV at 72Њ. Reactions were performed using Taq polymerase in 25 ␮g of yeast DNA. The 1ف recognition site is lost) or BstUI (recognition site is gained). ␮l with 5 pmol of each primer and) Detailed information about these restriction enzyme digestion DNA primer synthesis and DNA sequencing were performed protocols is available upon request. at the Cornell Biotechnology Analytical/Synthesis facility. Genetic techniques: Yeast were transformed with DNA us- ing the lithium acetate method as described by Geitz and Schiestl (1991). Tetrads were dissected on YPD plates imme- diately after zymolyase treatment using previously established RESULTS methods (Rose et al. 1990). In the tetrad analysis described Identification of a rfc1 allele containing a Tn3::LEU2 in Table 3, all tetrads that yielded four, three, and sometimes two and one viable spores were examined for relevant genetic insertion: We screened strains mutagenized with Tn3 markers by PCR, by segregation of a linked marker (i.e., transposon insertions for those that were inviable in a rad27⌬::HIS3), or by phenotype (i.e., mutator phenotype, double-strand break-repair-deficient rad52 null back- s MMS ). ground (Burns et al. 1994; Howell et al. 1994; Counter Mutation frequencies shown in Table 2 were determined by measuring the frequency of forward mutation to canavanine et al. 1997). A collection of 836 yeast mutants that were colonies was examined for 300,000ف resistance (i.e., Reenan and Kolodner 1992). Repeat-tract identified from instability frequencies (Tables 2 and 4) were determined by mutator phenotypes on canavanine plates and a single measuring frameshift events that resulted in resistance to candidate (DNR53, Table 1) was identified (Rose et al. 5-FOA in strains containing the plasmid pSH44[(GT)16T-URA3, 1990; Counter et al. 1997). Sequencing of the DNA ARSH4, CEN6, TRP1](Henderson and Petes 1992). In both the mutator and repeat-tract instability studies, tested strains that flanked the Tn3 insertion in this candidate revealed were streaked to form single colonies on selective minimal that the Tn3 element was located at bp 756 in the RFC1 plates containing 2% glucose. Eleven independent colonies open reading frame (ORF) between homology boxes I were suspended in water and appropriate dilutions were then (ligase homology domain) and II (Figure 1; Cullmann plated onto minimal media with or without canavanine or 5-FOA. The median frequency of canavanine and 5-FOA resis- et al. 1995). The allele created by the insertion is referred tance was determined for each experiment and the average to as rfc1::Tn3. Previously, Howell et al. (1994) showed of three-to-six independent experiments is presented for each that the RFC1 gene product is required for cell viability; strain. however, plasmids containing a deletion variant of the The length of GT repeat tracts was determined by sequenc- RFC1 gene that lacks the entire aminoterminal ligase ing pSH44-derived plasmids recovered from independently isolated 5-FOAr colonies (Rose et al. 1990). Plasmids were homology domain (⌬1-273) can weakly complement the sequenced using the Ϫ40 sequencing primer described by cold-sensitive phenotype of rfc1-1 mutants (Howell et Henderson and Petes (1992). To examine large insertion/ al. 1994; Figure 1). Consistent with this observation was deletion alterations in the CAN1 gene, the complete open the finding that the ligase homology domain of the reading frame of the CAN1 gene was amplified by PCR from chromosomal DNA isolated from independently identified human homolog of RFC1 is not required for PCNA canr colonies. Amplified DNA was digested with Hph1 and interactions or replication functions in vitro (Uhlmann then electrophoresed on a 2% TAE-agarose gel. et al. 1997). On the basis of this information we hypothe- Mitotic recombination frequencies were determined by mea- size that in rfc1::Tn3 strains a cryptic promoter within the ϩ suring the frequency of His colonies in wild-type (NKY1068) Tn3 element is driving expression of an aminoterminal- and rfc1:Tn3 (EAY561) strains bearing a his4X-ADE2-his4B cas- sette (Bishop et al. 1992). From each strain 13 independent truncated version of the RFC1. colonies were plated using the appropriate dilutions onto Characterization of the rfc1::Tn3 phenotype: The minimal media with or without histidine. The median fre- rfc1::Tn3 allele that was identified in DNR53 was intro- ϩ quency of His recombinants was determined. duced into the FY and SK-1 strain backgrounds (Table The genetic data presented in Table 2 and in the text were analyzed using the Mann-Whitney test statistic where P values 1; materials and methods) and tested in DNA repair Ͻ0.05 are considered significant (Pfaffenberger and Pat- and mutator assays. This strain displayed a 19-fold in- terson 1977). crease in the frequency of forward mutations to canr 502 Y. Xie, C. Counter and E. Alani

TABLE 1 Strains used in this study

Strain Genotype Source DNR53 MATa leu2-3, 112 lys2⌬201 trp1⌬1 ura3-52 his3⌬200 rad52::HIS3 C. Counter rfc1::Tn3::LEU2 pRAD52 (ARS-CEN, URA3) FY 23 MATa ura3-52 leu2⌬1 trp1⌬63 F. Winston FY 86 MAT␣ ura3-52 leu2⌬1 his3⌬200 F. Winston EAY 255 MAT␣ ura3-52 leu2⌬1 his3⌬200 rad52⌬::LEU2 Lab collection EAY 265 MAT␣ ura3-52 leu2⌬1 his3⌬200 msh2⌬::TRP1 rad52⌬::LEU2 Lab collection EAY 281 MATa ura3-52 leu2⌬1 trp1⌬63 msh2⌬::hisG Lab collection EAY 310 MATa ura3-52 leu2⌬1 trp1⌬63 pms1⌬::hisG Lab collection EAY 432 MATa ura3-52 leu2⌬1 trp1⌬63 rad52⌬::URA3 Lab collection EAY 545 MAT␣ ura3-52 leu2⌬1 his3⌬200 rad27⌬::HIS3 This study EAY 546 MATa ura3-52 leu2⌬1 trp1⌬63 rfc1::TN3::LEU2 This study EAY 547 MAT␣ ura3-52 leu2⌬1 his3⌬200 rfc1::Tn3::LEU2 This study EAY 549 MAT␣ ura3-52 leu2⌬1 his3⌬200 pol30-52 This study EAY 550 MAT␣ ura3-52 leu2⌬1 trp1⌬63 pol30-52 This study EAY 552 MATa ura3-52 leu2⌬1 trp1⌬63 his3⌬200 pol30-52 msh2⌬::hisG This study EAY 554 MAT␣ ura3-52 leu2⌬1 trp1⌬63 his2⌬200 rfc1::Tn3::LEU2 msh2⌬::hisG This study EAY 556 MATa ura3-52 leu2⌬1 trp1⌬63 his3⌬200 rfc1::Tn3::LEU2 pms1⌬::hisG This study EAY 565 MATa ura3-52 leu2⌬ trp1⌬63 pol3-01 Lab collection NKY 1068 MAT␣ho::LYS2 lys2 ura3 leu2::hisG ade2::LK his4X-ADE2-his4B D. Bishop EAY 561 MAT␣ho::LYS2 lys2 ura3 leu2::hisG ade2::LK his4X-ADE2-his4B This study rfc1::Tn3::LEU2

(P ϭ 0.034), was sensitive to UV and the alkylating agent repair mutants, suggesting that these two functions act MMS, was cold sensitive for growth at 14Њ, and displayed in series in the repair of replication errors. The PCNA a 9.5-fold higher median frequency of mitotic Hisϩ re- (POL30) and RAD27 replication factor genes have also combinants compared to wild type (4.3 ϫ 10Ϫ4 for wild been implicated in mismatch avoidance and/or correc- type vs. 4.1 ϫ 10Ϫ3 for rfc1::Tn3) in an intrachromosomal tion, as rad27⌬ and certain pol30 (pol30-52 and pol30-104) gene conversion assay (Table 2; materials and meth- mutants display a mutator/slippage phenotype that is ods; data not shown; Bishop et al. 1992). The replication similar to that observed in msh2⌬ mutants (Strand et and repair defects that were observed in rfc1::Tn3 strains al. 1993; Johnson et al. 1996; Umar et al. 1996; Kokoska were similar to those described for rfc1 conditional mu- et al. 1998). Finally, genetic analyses of RFC1 and the tants (Howell et al. 1994; McAlear et al. 1996). flap endonuclease RAD27 have revealed that alleles of The rfc1::Tn3 mutation is synthetically lethal with both are lethal in a rad52 null background and are also rad52⌬, rad27⌬, pol30-52, and pol3-01 mutations: Several synthetically lethal with each other (Table 3; Tishkoff lines of genetic evidence have begun to reveal the com- et al. 1997; Merrill and Holm 1998). This information, plex interplay of replication and repair functions in in conjunction with recent data from the Holm labora- both the generation and repair of mutagenic replication tory, where they showed that rfc1 mutants accumulate errors (misincorporation or repeat-tract insertion/dele- small single-stranded DNA fragments (Merrill and tion events). Studies of Morrison et al. (1993) revealed Holm 1998), supports the idea that rad27⌬ and rfc al- that a polymerase ␦ proofreading mutant (pol3-01) dis- leles are both defective in the maturation of Okazaki played multiplicative mutator defects with mismatch- fragments.

Figure 1.—The rfc1::Tn3 allele contains a mini-Tn3:: LACZ::LEU2 (Burns et al. 1994) transposon insertion at bp 756 in the RFC1 open reading frame. The transposon inser- tion in RFC1 is located be- tween box I (ligase homology domain) and II in the RFC1 ORF. Boxes II–VIII are de- fined as containing short mo- tifs that are conserved among the five subunits of the yeast and human clamp loader complex. Boxes III and V contain conserved sequences characteristic of nucleotide-binding ; the functions of the other boxes are unknown (Cullmann et al. 1995). RFC1 and Repeat-Tract Instability 503

TABLE 2 Median frequency of forward mutations and dinucleotide repeat-tract instability in wild type, rfc1::Tn3, pms1⌬, msh2⌬, rad27⌬, and pol30-52 strains

Forward mutation frequency to canr (ϫ10Ϫ7) Tract instability frequency (ϫ10Ϫ5) Relevant genotype Average (individual expt.) Relative Average (individual expt.) Relative Wild type 6.4 (10.1, 9.0, 4.0, 2.4) 1.0 2.4 (3.0, 2.3, 2.0) 1.0 rfc1::Tn3::LEU2 120 (140, 120, 110) 19 24 (33, 22, 16) 10 msh2⌬::hisG 320 (360, 310, 300) 50 660 (880, 600, 500) 275 pms1⌬:hisG 350 (540, 360, 150) 55 1,200 (1300, 1200, 1100) 500 rfc1::Tn3::LEU2 msh2⌬::hisG 3,000 (5100, 3300, 2600, 2200, 1800) 469 940 (1500, 1200, 1000, 760, 670, 500) 392 rfc1::Tn3::LEU2 pms1⌬::hisG 2,700 (2900, 2700, 2600) 422 1,100 (1300, 1200, 670) 458 rad27⌬::HIS3 900 (1070, 860, 780) 141 not tested pol30-52 790 (960, 820, 580) 123 820 (1250, 1050, 750, 640, 630, 620) 342 pol30-52 msh2⌬::hisG 3,000 (4600, 2900, 1600) 469 1,900 (2500, 2000, 1300) 792 Wild type (FY23), rfc1::Tn3:: LEU2 (EAY546), msh2⌬::hisG (EAY281), pms1⌬::hisG (EAY310), rfc1::Tn3::LEU2 msh2⌬::hisG (EAY554), rfc1::Tn3::LEU2 pms1⌬::hisG (EAY556), rad27⌬::HIS3 (EAY545), pol30-52 (EAY550), and pol30-52 msh2⌬::hisG (EAY552) strains were tested in the canr and 5-FOAr assays as described in materials and methods. For each row the indicated strain was directly tested in the forward mutation assay and then transformed with pSH44 for testing in the repeat-tract instability assay. In each experiment, 11 independent colonies were tested. The average median frequency in each assay is also presented relative to the wild-type frequency.

The above information encouraged us to explore the mutant strains that were analyzed in the mutator and interplay of rfc1::Tn3 with mutants in these unlinked repeat-tract instability assays described below. Double replication and repair functions. Haploid strains con- mutant combinations (i.e., crosses 1–5, Table 3) were taining the rfc1::Tn3, rad27⌬, rad52⌬, msh2⌬, pms1⌬, classified as inviable on the the basis of the following: pol3-01, and pol30-52 mutations were mated to each (1) The segregation patterns of tetrads bearing four other and tetrads from the resulting diploids were exam- (PD), three (TT), and two (NPD) viable spores fit the ined for spore viability, segregation of markers, and expected pattern for double mutant lethality in the case mutator and repeat-tract instability phenotypes (Tables where two genes are segregating independently of each 2–4). Double mutant combinations (i.e., crosses 6–9, other (1 PD: 4 TT: 1 NPD). This pattern is also mani- Table 3) were classified as viable on the basis of the fested in reduced spore viability. (2) The inviability of following: (1) The majority of tetrads dissected con- double mutant combinations was confirmed by genotyp- tained four viable spores (91–99% spore viability). (2) ing all spore clones in tetrads containing four and three Genotyping analysis of several four-spore viable tetrads viable spores, and in some cases tetrads that contained from each cross resulted in the identification of double two or one viable spores were genotyped. In cases of

TABLE 3 Tetrad analysis of strains containing rfc1::Tn3::LEU2, pol30-52, pol3-01, rad27⌬, rad52⌬, msh2⌬, and pms1⌬ mutations

No. of tetrads Spore viability Cross Relevant genotype 4:0 3:1 2:2 1:3 0:4 Total (%) 1. EAY546 ϫ EAY549 rfc1::Tn3 ϫ pol30-52 6 15 7 0 1 29 71.6 2. EAY546 ϫ EAY545 rfc1::Tn3 ϫ rad27⌬::HIS3 12 23 6 0 0 41 78.2 3. EAY547 ϫ EAY565 rfc1::Tn3 ϫ pol3-01 3 19 5 0 0 27 73.1 4. EAY549 ϫ EAY565 pol30-52 ϫ pol3-01 3 5 5 3 1 17 58.8 5. EAY546 ϫ EAY265 rfc1::Tn3 ϫ msh2⌬::TRP1 rad52⌬::URA3 6 36 20 1 3 66 66.0 6. FY 86 ϫ EAY432 Wild type ϫ rad52⌬::URA3 23 1 5 0 0 29 90.5 7. EAY547 ϫ EAY281 rfc1::Tn3 ϫ msh2⌬::hisG 37 1 0 0 0 38 99.3 8. EAY547 ϫ EAY310 rfc1::Tn3 ϫ pms1⌬::hisG 36 2 0 0 0 38 98.7 9. EAY549 ϫ EAY281 pol30-52 ϫ msh2⌬::hisG 16 1 1 0 0 18 95.0 Strains in crosses 1–9 were mated and tetrads dissected and scored for the relevant genotypes as described in materials and methods. In crosses 1–5, all spore clones from the 4Ϻ0 and 3Ϻ1 tetrads were scored for the genotype of the relevant markers to determine whether double and triple mutant combinations were viable; none of these combinations were found in viable spores. 504 Y. Xie, C. Counter and E. Alani

TABLE 4 Distribution of poly(GT) tract alterations in wild type, rfc1:Tn3::LEU2, msh2⌬, rad27⌬, pol3-t, and pol30 strains

No. of bp deletions (Ϫ) or additions (ϩ) Relevant tracts genotype sequenced ϩ2 Ϫ2 ϩ4 Ϫ4 Ϫ10 ϩ14 Ϫ14 ϩ16 Ϫ16 ϩ20 Ϫ20 Ϫ22 Wild type 11 9 1 1 0 00000000 rfc1::Tn3 342051000113210 Wild typea 332620012101000 msh2⌬a 287210000000000 pol30-104a 5830241120000000 rad27⌬a 352905000100000 pol3-t b 25350300105021 a Data from Johnson et al. (1995, 1996). b Data from Kokoska et al. (1998). In this data set five other alterations were observed in the URA3 coding region. synthetic lethality, no spore clones were identified that mutations. This observation was also confirmed by show- contained both mutations. ing that a msh2⌬ derivative of DNR53 (relevant genotype In control crosses, genotyping and spore viability anal- rfc1::Tn3, msh2⌬, rad52⌬, pRAD52 ARS-CEN URA3) was ysis demonstrated that rfc1::Tn3 rad52⌬, rad27⌬ rad52⌬, not viable on 5-FOA media that selected for the loss of and pol30-52 rad52⌬ double mutants were inviable (data the pRAD52 plasmid (data not shown). not shown); this was expected because rad27⌬ rad52⌬ The rfc1::Tn3 mutant displays a repeat-tract instability strains were previously shown to be inviable (Tishkoff phenotype: The phenotype of the rfc1::Tn3 allele, as et al. 1997), and different mutant alleles of the RFC1 well as previous studies indicating that mismatch repair (rfc1-1; Merrill and Holm 1998) and POL30 (pol30- and DNA replication mutants displayed an increased 104; Merrill and Holm 1998) genes were shown to be frequency of repeat-tract instability, encouraged us to synthetically lethal with rad52 null mutations. pol3-01 test whether the rfc1::Tn3 mutation confers a similar rad52⌬ double mutants were found to be viable (data defect (Table 2; Strand et al. 1993; Johnson et al. 1996; not shown); this result was also expected because previ- Kolodner 1996; Umar et al. 1996; Tran et al. 1997). ous analysis indicated that the rad52 null mutation did We measured the frequency of tract instability in an not exhibit synthetic lethality with mutations in the assay that detects frameshift events resulting in resis- pol␣, pol␦, and polε polymerase genes (Merrill and tance to 5-FOA. These tests were performed in FY23- Holm 1998). rfc1::Tn3 strains were also mated to strains or FY86-derived strains containing the GT repeat-tract bearing the pol30-52, rad27⌬, and pol3-01 mutations. As plasmid pSH44 [(GT)16T-URA3, ARSH4, CEN6, TRP1] shown in Table 3, rfc1::Tn3 pol30-52 (Cross 1), rfc1::Tn3 (Henderson and Petes 1992). As shown in Table 2, rad27⌬::hisG (Cross 2), and rfc1::Tn3 pol3-01 (Cross 3) the rfc1::Tn3 allele displayed a moderate repeat-tract double mutants were inviable because poor spore viabil- instability phenotype (10-fold increased, P ϭ 0.049) that ity (59–78%) was observed in tetrad analysis and no was lower than that observed in mismatch-repair mu- spore clones were obtained that contained both muta- tants but was similar to that observed in strains bearing tions. the DNA polymerase mutations pol3-01 and pol3-t (Tran As shown in Tables 2 and 3, rfc1::Tn3 msh2⌬::hisG (Cross et al. 1997; Kokoska et al. 1998). 7) and rfc1::Tn3 pms1⌬::hisG (Cross 8) double mutants pSH44-derived plasmids obtained from independent were viable and displayed the MMSs and colds phenotype 5-FOAr rfc1::Tn3 colonies were sequenced to examine conferred by the rfc1::Tn3 allele and a mutator pheno- the repeat-tract sequence changes that had occurred type that was nearly equivalent to the product of the (Table 4). The majority (20/34) of tract alterations in mutator frequencies of the individual mutants. The via- rfc1::Tn3 strains were 1-repeat insertion mutations. The bility of these double mutants encouraged us to test remaining alterations comprised one group (6/34) con- whether, analogous to the suppression of damϪ recAϪ sisting of 1- or 2-repeat insertion/deletion mutations lethality by mutS Ϫ mutations, a msh2 mutation could and another group (8/34) consisting of larger 7- to 10- suppress the lethality observed in rfc1::Tn3 rad52 double repeat insertion/deletion mutations. This spectrum of mutants (McGraw and Marinus 1980). In crosses be- tract alterations was somewhat similar to that observed tween a rfc1::Tn3 strain and rad52⌬ and msh2⌬ rad52⌬ in wild-type and rad27⌬ strains in that the majority of strains (Table 3, Cross 5; data not shown), no spore tract alterations in all three backgrounds were single clones were recovered that contained both (rfc1::Tn3 repeat insertion mutations. However, compared to the and rad52⌬ ) or all three (msh2⌬ rfc1::Tn3, and rad52⌬) rad27⌬ strain, the rfc1::Tn3 strain displayed a higher RFC1 and Repeat-Tract Instability 505

Figure 2.—The rfc1::Tn3 mutation does not result in large insertion/deletion muta- tions in the CAN1 gene. Inde- pendent canr colonies were ob- tained from rad27⌬::HIS3 (EAY 545) and rfc1::Tn3 (EAY 547) strains, and a DNA frag- ment containing the CAN1 open reading frame was PCR amplified from each of these strains and incubated with HphI. Restriction enzyme-digested DNA fragments were electrophoresed in a 2% TAE-agarose gel. Lanes marked M contain a DNA marker and lanes 11 and 22 display DNA fragments from wild-type cans strains. Digestion of the amplified CAN1 DNA with HphI resulted in 490-, 411-, 314-, 252-, 249-, and 207-bp fragments that could be detected by gel electrophoresis. Two smaller bands of 87 and 46 bp could not be detected. Lanes 1–10 and 12–21 display HphI-digested CAN1 DNA from canr rad27⌬::HIS3 and rfc1::Tn3 strains, respectively. number of larger tract insertions/deletions (8/34 for Lanes 12–21) or 8 canr pol30-52 strains (data not shown). rfc1::Tn3 vs. 1/35 for rad27⌬). This finding is also consistent with results of McAlear The spectrum of repeat-tract insertion/deletion events et al. (1996), which showed that the rfc1-1 mutation in the rfc1::Tn3 strain differed from that observed in conferred a mutator phenotype that resulted principally mismatch repair (msh2⌬) and other DNA replication from an elevated frequency of base pair substitution (pol30-104 and pol3-t) defective strains (Table 4 and mutations. Johnson et al. 1995, 1996; Kokoska et al. 1998). In Double mutant analysis indicated a synergistic - msh2⌬ strains only single repeat insertion/deletions tionship between rfc1 and mismatch-repair mutations: were observed, with the majority of these events con- To test whether RFC1 is required during mismatch re- sisting of deletions (Johnson et al. 1996). In pol30-104 pair, we examined the mutation frequency of rfc1::Tn3 strains, which display a mutator and repeat-tract instabil- strains in combination with mismatch repair and other ity phenotype similar to that observed in mismatch- replication mutations in both forward mutation and repair mutants, the vast majority (56/58) of tract al- tract instability assays. As shown in Table 2, in the for- terations were one- or two-repeat insertion/deletion ward mutation assay, the frequency of mutations in mutations, with a similar number of insertions and dele- rfc1::Tn3 msh2⌬ (469-fold increase) and rfc1::Tn3 pms1⌬ tions (Johnson et al. 1996). In pol3-t strains, which con- (422-fold increase) double mutant strains appeared to tain a mutation in polymerase ␦ that is thought to reduce nearly equal the product of the mutation frequencies the rate of DNA elongation, there was an approximately of the individual mutations (msh2⌬, 50-fold increase; equal distribution of small and large repeat insertion/ pms1⌬, 55-fold increase; rfc1::Tn3, 19-fold increase). deletion mutations and a smaller number of alterations While results in the forward mutation assay indicated a that were presumably not due to repeat-tract instability nearly multiplicative relationship for defects in the (Kokoska et al. 1998). clamp loader and mismatch-repair genes, the results Recent analysis of rad27⌬ strains revealed that, in from the tract instability assay were less clear. As shown addition to repeat-tract length instability, these strains in Table 2, frequency of tract instability in msh2⌬, pms1⌬, also displayed a high frequency of insertion/deletion and pol30-52 strains (275- to 500-fold) was much higher events at the CAN1 and LYS2 loci (Ͼ14 bp, the majority than in rfc1::Tn3 strains (10-fold). The frequencies of of which were duplications) (Johnson et al. 1995; Tish- tract instability in rfc1::Tn3 msh2⌬ and rfc1::Tn3 pms1⌬ koff et al. 1997; Kokoska et al. 1998). The similarity in double mutants, however, were similar to those observed the spectrum of tract instability events in rfc1::Tn3 and in msh2⌬ or pms1⌬ strains (rfc1::Tn3 msh2⌬ vs. msh2⌬, rad27⌬ mutants and the fact that rfc1::Tn3 rad27⌬, P ϭ 0.25; rfc1::Tn3 pms1⌬ vs. pms1⌬, P ϭ 0.66). rfc1::Tn3 rad52⌬ and rad27⌬ rad52⌬ strains are inviable Double mutant analysis also indicated that rfc1::Tn3 (Table 3 and Tishkoff et al. 1997) encouraged us to pms1⌬ and rfc1::Tn3 msh2⌬ mutants were viable but examine whether similar types of chromosomal re- rfc1::Tn3 pol30-52 double mutants were inviable (Table arrangements could be detected in rfc1::Tn3 strains. We 3). We were interested in understanding this result be- examined rfc1::Tn3, rad27⌬::HIS3, and pol30-52 strains cause pol30-52 mutants display a strong mismatch-repair for the presence of large insertion/deletion mutations defect that appears to be epistatic to the defect observed in the CAN1 (Figure 2). As predicted, a difference in msh2 and pms1 mutants (Umar et al. 1996). However, in the size of at least one CAN1-derived fragment was given the known role of PCNA and RFC in DNA replica- observed in CAN1 genes amplified from 9 out of 10 tion, this result suggests that pol30-52 strains display canr rad27⌬ strains (Figure 2, lanes 1–10). However, defects in cellular functions such as DNA replication no changes were observed in the size of CAN1 gene that are in addition to or are different from those involv- fragments amplified from 10 canr rfc1::Tn3 (Figure 2, ing mismatch repair. Analysis of msh2⌬ pol30-52 double 506 Y. Xie, C. Counter and E. Alani mutants in the forward mutation and repeat-tract in- RFC and mismatch-repair functions act in series in a stability assays also supported this conclusion. In both single repair pathway, as proposed for pol3-01 and pms1 assays, the msh2⌬ pol30-52 mutant displayed a mutator double mutants in Morrison et al. (1993), or act in and repeat-tract instability phenotype that appeared to distinct DNA repair pathways that compete for the same be roughly additive when compared to the mutator phe- substrates (Haynes and Kunz 1981). On the basis of notype observed for each of the single mutations (Table the known function of RFC as a clamp loader in DNA 2). This observation supports the idea that the mutator replication, we favor the idea that defects in the clamp phenotype exhibited by pol30-52 and msh2⌬ mutants loader result in replication errors that are acted upon reflects the action of gene products functioning in paral- by mismatch repair (acting in series; Morrison et al. lel, noncompeting pathways (Haynes and Kunz 1981; 1993). The types of mutations found in a rfc1-1 strain Morrison et al. 1993). In such a scenario, the pol30- that confers repair and replication defects similar to 52 mutation confers replication errors as the result of those observed in rfc1::Tn3 strains provides further sup- defects in both mismatch repair and DNA replication. port for this idea (McAlear et al. 1996). The rfc1-1 The finding that the pol30-52 mutation, unlike mis- mutation was shown to confer an increase in base pair match-repair mutations, confers sensitivity to DNA-dam- substitutions that are likely to have formed from base aging agents and, like rad27⌬::HIS3 and rfc1::Tn3,is pair mismatches that are substrates for mismatch repair synthetically lethal with rad52 null mutations, supports (reviewed in Kolodner 1996). this idea; moreover, pol30-52 is defective in homotri- A similar relationship between mismatch repair and meric interactions and is also defective in in vitro DNA RFC functions was not observed in the repeat-tract insta- replication reactions (Table 3; Ayyagari et al. 1995). bility assay because the rfc1::Tn3 msh2 or rfc1::Tn3 pms1 Further support is provided from the work of Kokoska double mutants displayed a mutator phenotype that was et al. (1999, accompanying article) in which they ana- indistinguishable from that observed in msh2 or pms1 lyzed the effect of the pol30-52 mutation on micro- and single mutants. The different phenotypes in these two minisatellite instability and found that, in addition to assays were not unexpected considering that RFC1, conferring a defect in mismatch repair, the pol30-52 MSH2, and PMS1 are likely to be functioning in multi- mutation conferred a minisatellite instability pheno- subunit DNA replication and repair machines and it is type. They hypothesized that this phenotype was the difficult to determine which effects are direct and which result of the pol30-52 mutation affecting DNA replica- are indirect. Three possible explanations for these dif- tion by increasing the rate of DNA polymerase slippage. ferences are as follows: 1. Repeat-tract instability in msh2 and pms1 mutants, as DISCUSSION measured by the (GT)14-T-URA3 detection assay, is We identified the rfc1::Tn3 allele in a screen for muta- already occurring at a saturating level and so an in- tions that are lethal in a rad52 null background. This crease in these events could not be detected in dou- analysis indicated the rfc1::Tn3 allele conferred a muta- ble mutant combinations. We believe that this is not tor phenotype, an elevated recombination frequency, the case because higher frequencies of repeat-tract sensitivity to UV and MMS, cold sensitivity, and synthetic instabilities have been observed in pol30-52 msh2⌬ lethality with rad52, rad27⌬, and pol30 mutations. The strains and even higher frequencies have been re- phenotypes conferred by the rfc1::Tn3 allele were similar ported in msh2⌬ strains containing mononucleotide to those reported for previously characterized rfc1 alleles repeat tracts. (Table 2; Sia et al. 1997a). (Moir et al. 1982; Howell et al. 1994; McAlear et al. 2. The increase in repeat-tract instability in rfc1::Tn3 1994). In addition, we showed that rfc1::Tn3 displays strains resulted not from DNA polymerase slippage a repeat-tract instability phenotype. This observation events but from an increase in unequal sister chroma- encouraged us to test genetic interactions between the tid exchanges (Strand et al. 1993). The fact that rfc1::Tn3 mutation and mutations in other replication the rfc1::Tn3 allele confers a hyper-recombination (RAD27, POL3) and repair (MSH2, PMS1) genes. phenotype provides support for this idea. In such a In the canavanine mutator assay, a nearly multiplica- scenario one would expect the repeat-tract instability tive effect on mutation frequency was observed when observed in rfc1::Tn3 strains to be dependent on the rfc1::Tn3 mutation was analyzed in combination with RAD52 function. Unfortunately, this hypothesis can- msh2⌬ and pms1⌬ mutations. A multiplicative relation- not be tested as rfc1::Tn3 mutants are lethal in rad52 ship was previously observed in pol3-01 pms1 double null backgrounds. mutants; this observation led Morrison et al. (1993) 3. The repeat-tract instability phenotype in rfc1::Tn3 to propose that DNA replication errors resulting from strains could result from an increase in DNA slippage defects in polymerase ␦ proofreading functions are re- events that are not recognized by, or occur indepen- paired by the mismatch-repair pathway. Because our dently of, the mismatch-repair system. The fact that of the tract alterations in rfc1::Tn3 strains were %25ف data displayed a relationship that was almost, but not exactly, multiplicative, we cannot distinguish whether larger in size (Ͼ14 bp) than would be expected to RFC1 and Repeat-Tract Instability 507

be repaired by the mismatch-repair pathway provides ent from that observed in rad27⌬ and pol3-t mutants some support for this idea (Sia et al. 1997a), as do where the effect of these mutations on repeat-tract insta- recent observations suggesting that the pol30-52 mu- bility was dependent on the size of the repeat unit tation increases the rate of repeat-tract instability (Kokoska et al. 1998). (2) Larger DNA rearrangements, through mechanisms that appear independent of as analyzed by DNA sequencing or by restriction digest mismatch repair [Table 2; Kokoska et al. (1999)]. analysis of the CAN1 gene, occurred at high frequency in rad27⌬ strains but were not observed in rfc1::Tn3 Synthetic lethality analysis and chromosome instabil- or pol30-52 strains (Figure 2; Tishkoff et al. 1997). In ity assays indicate that the rfc1, pol30, and rad27 muta- summary, while each of these mutations appears to de- tions interact genetically but display unique chromo- crease the efficiency of DNA replication in ways that some instability phenotypes: Genetic analysis of rfc1, are likely to stall the DNA replication fork, their overall pol30, and rad27 mutations indicated that they were all effect on chromosomal instability appears to be unique synthetically lethal with mutations in the RAD52 recom- for each mutation and is not well understood at this binational repair pathway and that rfc1 pol30 and rfc1 time. rad27 double mutants were also inviable (Merrill and In this study we observed that the pol3-01 mutation, Holm 1998). Additional analysis also showed that mu- which causes a defect in the polymerase-␦ exonuclease tations in all three of these genes resulted in a muta- proofreading function, was synthetically lethal with the tor phenotype as well as sensitivity to DNA-damaging rfc1::Tn3 mutation (Table 3). Previous analysis by Mor- agents. One hypothesis that is consistent with the above rison et al. (1993) showed that haploid pol3-01 pms1 data is that mutations in each of these genes result in double mutants are inviable because of the accumula- the stalling of the replication fork, thus leading to the tion of a catastrophic number of mutations. While the formation of double-strand breaks that are repaired by inviability of pol3-01 rfc1::Tn3 mutants might be ex- the RAD52 double-strand break repair system. This hy- plained in this manner, we believe that this is not the pothesis is partly based on the findings in E. coli that case because msh2, pms1, and pol3-01 single mutants all mutations that disrupt replication fork movement result display similar mutation frequencies and rfc1::Tn3 msh2 in the formation of double-strand breaks that are lethal and rfc1::Tn3 pms1 double mutants are viable (Mor- in recombination-deficient backgrounds and the obser- rison et al. 1993; Kolodner 1996; Umar et al. 1996). vation that rfc1 mutants grow slowly and are delayed A similar conclusion was reached by Kokoska et al. in progressing through S phase (Howell et al. 1994; (1998) to explain why pol3-01 rad27⌬ double mutants McAlear et al. 1996; Michel et al. 1997). In addition, are inviable. They hypothesized that the inviability of recent studies by the Holm laboratory (Merrill and pol3-01 rad27⌬ mutants was not due to mutational load Holm 1998) indicated that rad27, pol30, and rfc1 mu- because diploids homozygous for both mutations were tants accumulate small single-stranded DNA fragments also inviable. In cases where mutational load was sus- during DNA replication in vivo, suggesting that they are pected as the cause of haploid inviability, diploids homo- defective in Okazaki fragment maturation. zygous for the mutational load mutations were found to On the basis of the phenotypes of the mutants de- be viable, presumably because recessive lethal mutations scribed above and the observation that Pol30p physically occur less frequently in diploid cells (Morrison et al. interacts with the RFC and Rad27p, one might have 1993). On the basis of the observation that pol3-01 expected the spectrum of chromosomal instability de- rad27⌬ double mutants are inviable and the fact that the fects in these mutants to be similar (Burgers 1991; Li RAD27 gene product is required for Okazaki fragment et al. 1995; Uhlmann et al. 1997). However, as outlined processing, Kokoska et al. (1998) proposed that the below, the chromosome instability profiles of the pol30, proofreading exonuclease function of POL3 was some- rad27, and rfc1 mutants are different. (1) The frequency how required for Okazaki fragment processing. The fact of repeat-tract instability in rfc1::Tn3 strains was much that rfc1, pol30, and rad27⌬ mutants all display Okazaki lower than was observed in rad27⌬ or pol30-52 mutants fragment processing defects and rfc1 and rad27⌬ muta- (Table 2; Johnson et al. 1995, 1996; Umar et al. 1996; tions are inviable in a pol3-01 background is consistent Tishkoff et al. 1997; Kokoska et al. 1998; Merrill and with this idea (Merrill and Holm 1998). Holm 1998) and the spectrum of repeat-tract changes RFC is unlikely to play a direct role in mismatch was unique for each strain (Table 4). In an independent repair: A major question that remains to be answered study, P. Greenwell, R. J. Kokoska and T. D. Petes in eukaryotic mismatch repair is how strand discrimina- (personal communication) found that the rfc1-1 allele, tion is accomplished so that the newly replicated strand which displays a phenotype similar to the rfc1::Tn3 al- containing the replication error is removed. This ques- lele, confers a repeat-tract instability phenotype that is tion is of interest because in the yeast genome no strand independent of the size of the repeat unit; for each discrimination homologs based on the dam/mutH sys- repeat unit examined (1–20 bp), the rfc1-1 mutation tem of E. coli have been identified (reviewed in Kolod- fold increase in the rate of repeat- ner 1996). As outlined in the Introduction, the rfc1::Tn3-10ف conferred an tract instability. This spectrum of changes was very differ- mutation was isolated in a screen designed to identify 508 Y. Xie, C. Counter and E. Alani strand discrimination candidates. However, double mu- viability but are specifically defective in mismatch repair. tant analysis of the mismatch-repair mutations msh2 and It is unlikely, for example, that we would have detected pms1 and the rfc1::Tn3 mutation indicated that the RFC mutations in the POL30 gene, the product of which is was unlikely to play a role in mismatch repair because a strand discrimination candidate, using Tn3 or UV the mutator phenotype of rfc1 was not epistatic to, but mutagenesis screens (i.e., pol30-52) because POL30 is was in series with, the mutator phenotypes conferred essential for viability and only rare mutations in POL30 by msh2 and pms1 mutations. The repeat-tract instability that were found by targeted mutagenesis display both phenotype conferred by the rfc1::Tn3 mutation was weak a strong tract instability and mutator phenotype (Ayya- relative to the mutator phenotype as judged by canavan- gari et al. 1995). This information suggests that fraction- ine papillation assays and in comparison with the repeat- ation of cell extracts that catalyze mismatch repair in tract instability phenotype observed in msh2⌬ and pol30- vitro might be a more effective way to identify new mis- 52 strains (Strand et al. 1993; Umar et al. 1996). A match-repair components. mutant defective in strand discrimination would be ex- We thank Elizabeth Evans, Todd Milne, Lori Schvaneveldt, Tanya pected to function downstream of mismatch recogni- Sokolsky, Daniel Smith, and Barbara Studamire for providing advice, tion steps and would therefore be expected to display an reagents, and/or technical assistance, Elizabeth Evans, Tom Petes, equally strong mutator and tract instability phenotype Daniel Smith, Tanya Sokolsky, and Barbara Studamire for helpful (reviewed in Kolodner 1996). In addition, the spec- discussions and comments on the manuscript, and Tom Petes and Michael Liskay for sharing unpublished data. E.A. and Y.X. were trum of repeat-tract insertion and deletion events ob- supported by National Institutes of Health grant GM53085 and U.S. served in rfc1::Tn3 strains was different from that ob- Department of Agriculture Hatch Grant NYC-186424. served in msh2 strains because a large number of tract alterations in rfc1::Tn3 strains involved Ͼ14 bp inser- tion/deletions: such alterations were not observed in LITERATURE CITED mismatch-repair mutants (Table 4; Strand et al. 1993; Johnson et al. 1995). Finally, unlike in E. coli where Aguilera, A., and H. L. Klein, 1988 Genetic control of intrachro- mosomal recombination in Saccharomyces cerevisiae. I. Isolation Ϫ Ϫ dam recA double mutant lethality can be suppressed and genetic characterization of hyper-recombination mutations. by a mutation in mutS, in the analogous situation in Genetics 119: 779–790. yeast the msh2⌬ mutation was unable to suppress the Au, K. G., K. Welsh and P. Modrich, 1992 Initiation of methyl- directed mismatch repair. J. Biol. Chem. 267: 12142–12148. lethality of rfc1::Tn3 rad52 double mutants (see results; Ayyagari, R., K. J. Impellizzeri, B. L. Yoder, S. L. Gary and P. M. McGraw and Marinus 1980). This observation pro- Burgers, 1995 A mutational analysis of the yeast proliferating vides further support that the RFC is not playing a strand cell nuclear antigen indicates distinct roles in DNA replication and DNA repair. Mol. Cell. Biol. 15: 4420–4429. discrimination role analogous to that of dam methylase. Bishop, D. K., D. Park, L. Xu and N. Kleckner, 1992 DMC1: a As described in the Introduction, we performed an- meiosis-specific yeast homolog of E. coli recA required for recom- other screen aimed at identifying new mutations in mis- bination, synaptonemal complex formation, and cell cycle pro- gression. Cell 69: 439–456. match-repair genes. We screened 70,000 cells mutagen- Boeke, J. D., F. Lacroute and G. R. Fink, 1984 A positive selection ized with ultraviolet light for those that displayed both for mutants lacking orotidine-5Ј-phosphate decarboxylase activity a repeat-tract instability and a mutator phenotype. The in yeast: 5-fluoro-orotic acid resistance. Mol. Gen. Genet. 197: 345–346. 51 mutants that were identified all contained mutations Burgers, P. M., 1991 Saccharomyces cerevisiae . II. in either the MLH1, PMS1,orMSH2 genes. In a subse- Formation and activity of complexes with the proliferating cell quent screen involving 220,000 UV-mutagenized cells nuclear antigen and with DNA polymerases delta and epsilon. J. Biol. Chem. 266: 22698–22706. that was designed to avoid detection of these three Burns, N., B. Grimwade, P. B. Ross-Macdonald, E. Y. Choi, K. genes, only mutations in RAD27 were identified (Y. Xie, Finberg et al., 1994 Large-scale analysis of , L. Schvaneveldt and E. Alani, unpublished data). protein localization, and gene disruption in Saccharomyces cerevis- iae. Genes Dev. 8: 1087–1105. Why were mutations in genes that are likely to play Counter, C. M., M. Meyerson, E. N. Eaton and R. A. Weinberg, a role in mismatch repair (i.e., , single-strand 1997 The catalytic subunit of yeast . Proc. Natl. Acad. binding proteins, and exonucleases) not identified? Sci. USA 94: 9202–9207. One possibility is that mismatch-repair functions are Crouse, G. F., 1996 Mismatch repair systems in Saccharomyces cerevis- iae, pp. 411–448 in DNA Damage and Repair: Biochemistry, Genetics overlapping or are redundant because studies in E. coli and Cell Biology, edited by J. Nickoloff and M. Hoekstra. Hu- have suggested that at least three exonucleases partici- mana Press, Clifton, NJ. pate in mismatch repair (Harris et al. 1998). Another Cullmann, G., K. Fien, R. B. Kobayashi and B. Stillman, 1995 Characterization of the five replication factor C genes of Saccharo- possibility is that any remaining uncharacterized mis- myces cerevisiae. Mol. Cell. Biol. 15: 4661–4671. match-repair factors also play essential roles during Detloff, P., J. Sieber and T. D. Petes, 1991 Repair of specific base DNA replication: in such a scenario, a transposon muta- pair mismatches formed during meiotic recombination in the yeast Saccharomyces cerevisiae. Mol. Cell. Biol. 11: 737–745. genesis scheme might not allow for the identification Feinstein, S. I., and K. B. Low, 1986 Hyper-recombining recipient of an essential mismatch-repair protein such as a single- strains in bacterial conjugation. Genetics 113: 13–33. strand binding protein or a DNA , while a ge- Fien, K., and B. Stillman, 1992 Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand nome-wide UV mutagenesis might not have been effi- DNA replication complex. Mol. Cell. Biol. 12: 155–163. cient enough to detect mutations that are essential for Geitz, R. D., and R. H. Schiestl, 1991 Applications of high effi- RFC1 and Repeat-Tract Instability 509

ciency lithium acetate transformation of intact yeast cells using late small single-stranded DNA fragments during DNA synthesis. single-stranded nucleic acids as carrier. Yeast 7: 253–263. Genetics 148: 611–624. Green, C. N., and S. Jinks-Robertson, 1997 Frameshift intermedi- Michaels, M. L., C. Cruz and J. H. Miller, 1990 mutA and mutC: ates in homopolymer runs are removed efficiently by yeast mis- two mutator loci in Escherichia coli that stimulate transversions. match repair proteins. Mol. Cell. Biol. 17: 2844–2850. Proc. Natl. Acad. Sci. USA 87: 9211–9215. Harris, R. S., K. J. Ross, M. J. Lombardo and S. M. Rosenberg, Michel, B., S. D. Erlich and M. Uzest, 1997 DNA double-strand 1998 Mismatch repair in Escherichia coli cells lacking single- breaks caused by replication arrest. EMBO J. 16: 430–438. strand exonucleases ExoI, ExoVII, and RecJ. J. Bacteriol. 180: Miller, J., 1972 Experiments in Molecular Genetics. Cold Spring Harbor 989–993. Laboratory Press, Cold Spring Harbor, NY. Haynes, R. H., and B. A. Kunz, 1981 DNA repair and mutagenesis, Modrich, P., and R. S. Lahue, 1996 Mismatch repair in replication pp. 371–414 in The Molecular and Cellular Biology of the Yeast Sacchar- fidelity, genetic recombination and cancer biology. Annu. Rev. omyces: Life Cycle and Inheritance, edited by J. N. Strathern, E. W. Biochem. 65: 101–133. Jones and J. R. Broach. Cold Spring Harbor Laboratories, Cold Moir, D., S. E. Stewart, B. C. Osmond and D. Botstein, 1982 Cold- Spring Harbor, NY. sensitive cell-division-cycle mutants of yeast: isolation, properties Henderson, S. T., and T. D. Petes, 1992 Instability of simple se- and pseudo-revertant studies. Genetics 100: 547–563. quence DNA in Saccharomyces cerevisiae. Mol. Cell. Biol. 12: 2749– Morrison, A., A. L. Johnson, L. H. Johnston and A. Sugino, 1993 2757. Pathway correcting DNA replication errors in Saccharomyces cerevis- Holm, C., D. W. Meeks-Wagner, W. L. Fangman and D. Botstein, iae. EMBO J. 12: 1467–1473. 1986 A rapid, efficient method for isolating DNA from yeast. Pfaffenberger, R. C., and J. H. Patterson, 1977 Statistical Methods Gene 42: 169–173. for Business and Economics. Richard D. Irwin Inc., Homewood, IL. Howell, E. A., M. A. McAlear, D. Rose and C. Holm, 1994 CDC44: Reenan, R. A. G., and R. D. Kolodner, 1992 Characterization of a putative nucleotide-binding protein required for cell cycle pro- insertion mutations in the Saccharomyces cerevisiae MSH1 and gression that has homology to subunits of replication factor C. MSH2 genes: evidence for separate mitochondrial and nuclear Mol. Cell. Biol. 14: 255–267. functions. Genetics 132: 975–985. Jeyaprakash, A., J. W. Welch and S. Fogel, 1994 Mutagenesis of Rose, M. D., F. Winston and P. Hieter, 1990 Methods in Yeast Genet- yeast MW104-1B strain has identified the uncharacterized PMS6 ics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, DNA mismatch repair gene locus and additional alleles of existing NY. PMS1, PMS2 and MSH2 genes. Mutat. Res. 325: 21–29. Saiki, R. K., S. Scharf, F. Faloona, K. B. Mullis, G. T. Horn et al., Johnson, R. E., G. K. Kovvali, L. Prakash and S. Prakash, 1995 1985 Enzymatic amplification of beta-globin genomic se- Requirement of the yeast RTH1 5Ј to 3Ј exonuclease for the quences and restriction site analysis for diagnosis of sickle cell stability of simple repetitive DNA. Science 269: 238–240. anemia. Science 230: 1350–1354. Johnson, R. E., G. K. Kovvali, S. N. Guzder, N. S. Amin, C. Holm Schaaper, R. M., and R. L. Dunn, 1987 Spectra of spontaneous et al., 1996 Evidence for involvement of yeast proliferating cell mutations in Escherichia coli strains defective in mismatch correc- nuclear antigen in DNA mismatch repair. J. Biol. Chem. 271: tion: the nature of in vivo DNA replication errors. Proc. Natl. 27987–27990. Acad. Sci. USA 84: 6220–6224. Kane, S. M., and R. Roth, 1974 Carbohydrate metabolism during Sia, E. A., R. J. Kokoska, M. Dominska, P. Greenwell and T. D. ascospore development in yeast. J. Bacteriol. 118: 8–14. Petes, 1997a Microsatellite instability in yeast: dependence on Kokoska, R. J., L. Stefanovic, H. T. Tran, M. A. Resnick, D. A. repeat unit size and DNA mismatch repair genes. Mol. Cell. Biol. Gordenin et al., 1998 Destabilization of yeast micro- and minisa- 17: 2851–2858. tellite DNA sequences by mutations affecting a nuclease involved Sia, E. A., S. Jinks-Robertson and T. D. Petes, 1997b Genetic in Okazaki fragment processing (rad27) and DNA polymerase ␦ control of microsatellite stability. Mutat. Res. 383: 61–70. (pol3-t). Mol. Cell. Biol. 18: 2779–2788. Stillman, B., 1994 Smart machines at the DNA replication fork. Kokoska, R. J., L. Stefanovic, A. B. Buermeyer, R. M. Liskay and Cell 78: 725–728. T. D. Petes, 1999 A mutation of the yeast gene encoding PCNA Strand, M., T. A. Prolla, R. M. Liskay and T. D. Petes, 1993 Desta- (pol30-52) destabilizes both microsatellite and minisatellite DNA bilization of tracts of simple repetitive DNA in yeast by mutations sequences. Genetics 151: 511–519 affecting DNA mismatch repair. Nature 365: 274–276. Kolodner, R., 1996 Biochemistry and genetics of eukaryotic mis- Tishkoff, D. X., N. Filosi, G. M. Gaida and R. D. Kolodner, 1997 match repair. Genes Dev. 10: 1433–1442. A novel mutation avoidance mechanism dependent on S. cerevisiae Levinson, G., and G. A. Gutman, 1987 High frequencies of short RAD27 is distinct from DNA mismatch repair. Cell 88: 253–263. frameshifts in poly-CA/TG tandem repeats borne by bacterio- Tran, H. T., D. A. Gordenin and M. A. Resnick, 1996 The preven- phage M13 in Escherichia coli K-12. Nucleic Acids Res. 15: 5313– tion of repeat-associated deletions in Saccharomyces cerevisiae by 5338. mismatch repair depends on size and origin of deletions. Genetics Li, X., J. Li, J. Harrington, M. R. Lieber and P. M. Burgers, 1995 143: 1579–1587. Lagging strand DNA synthesis at the eukaryotic replication fork Tran, H. T., J. D. Keen, M. Kricker, M. A. Resnick and D. A. Gorde- involves binding and stimulation of FEN-1 by proliferating cell nin, 1997 Hypermutability of homonucleotide runs in mis- nuclear antigen. J. Biol. Chem. 270: 22109–22112. match repair and DNA polymerase proofreading yeast mutants. Liu, B., N. C. Nicolaides, S. Markowitz, J. K. Willson, R. E. Par- Mol. Cell. Biol. 17: 2859–2865. sons et al., 1995 Mismatch repair gene defects in sporadic colo- Tsurimoto, T., and B. Stillman, 1989 Purification of a cellular rectal cancers with microsatellite instability. Nat. Genet. 9: 48–55. replication factor, RF-C, that is required for coordinated synthesis Maniatis, T., E. F. Fritsch and J. Sambrook, 1982 Molecular Clon- of leading and lagging strands during simian virus 40 DNA replica- ing: A Laboratory Manual. Cold Spring Harbor Laboratory Press, tion in vitro. Mol. Cell. Biol. 9: 609–619. Cold Spring Harbor, NY. Uhlmann, F., J. Cai, E. Gibbs, M. O’Donnell and J. Hurwitz, 1997 McAlear, M. A., E. A. Howell, K. K. Espenshade and C. Holm, 1994 Deletion analysis of the large subunit p140 in human replication Proliferating cell nuclear antigen (pol30) mutations suppress rfc1 factor C reveals regions required for complex formation and mutations and identify potential regions of interaction between replication activities. J. Biol. Chem. 272: 10058–10064. the two encoded proteins. Mol. Cell. Biol. 14: 4390–4397. Umar, A., A. B. Buermeyer, J. A. Simon, D. C. Thomas, A. B. Clark McAlear, M. A., K. M. Tuffo and C. Holm, 1996 The large subunit et al., 1996 Requirement for PCNA in DNA mismatch repair at of replication factor C (Rfc1p/Cdc44p) is required for DNA a step preceding DNA resynthesis. Cell 87: 65–73. replication and DNA repair in Saccharomyces cerevisiae. Genetics Umar, A., J. I. Risinger, W. E. Glaab, K. R. Tindall, J. C. Barrett 142: 65–78. et al., 1998 Functional overlap in mismatch repair by human McGraw, B. R., and M. G. Marinus, 1980 Isolation and characteriza- MSH3 and MSH6. Genetics 148: 1637–1646. ϩ tion of Dam revertants and suppressor mutations that modify Winston, F., C. Dollard and S. L. Ricupero-Hovasse, 1995 Con- secondary phenotypes of dam-3 strains of Escherichia coli K-12. struction of set of convenient Saccharomyces cerevisiae strains that Mol. Gen. Genet. 178: 309–315. are isogenic to S288C. Yeast 11: 53–55. Merrill, B. J., and C. Holm, 1998 The RAD52 recombinational repair pathway is essential in pol30 (PCNA) mutants that accumu- Communicating editor: P. L. Foster