Evidence for involvement of yeast proliferating cell nuclear antigen in DNA mismatch repair.

DNA mismatch repair plays a key role in the maintenance of genetic fidelity. Mutations in the human mismatch repair genes hMSH2, hMLH1, hPMS1, and hPMS2 are associated with hereditary nonpolyposis colorectal cancer. The proliferating cell nuclear antigen (PCNA) is essential for DNA replication, where it acts as a processivity factor. Here, we identify a point mutation, pol30-104, in the Saccharomyces cerevisiae POL30 gene encoding PCNA that increases the rate of instability of simple repetitive DNA sequences and raises the rate of spontaneous forward mutation. Epistasis analyses with mutations in mismatch repair genes MSH2, MLH1, and PMS1 suggest that the pol30-104 mutation impairs MSH2/MLH1/PMS1-dependent mismatch repair, consistent with the hypothesis that PCNA functions in mismatch repair. MSH2 functions in mismatch repair with either MSH3 or MSH6, and the MSH2-MSH3 and MSH2-MSH6 heterodimers have a role in the recognition of DNA mismatches. Consistent with the genetic data, we find specific interaction of PCNA with the MSH2-MSH3 heterodimer.

In both prokaryotes and eukaryotes, defects in DNA mismatch repair cause elevated spontaneous mutation rates and increased instability of simple repeat DNA sequences. Mutations in any of the human mismatch repair genes hMSH2, hMLH1, hPMS1, and hPMS2 are associated with hereditary nonpolyposis colorectal cancer. Cell lines from these cancers are defective in DNA mismatch repair and display increased levels of spontaneous mutations and frequent alterations of microsatellite repeat sequences (1,2).
Epistasis analyses in yeast have suggested that MSH2 pro-tein functions in conjunction with MSH3 or MSH6 protein in mismatch recognition. Genetic and biochemical studies in both yeast and humans have further indicated that the MSH2-MSH3 and MSH2-MSH6 complexes differ in substrate specificities. In yeast, mutations in MSH3 cause an increase in instability of microsatellite tracts but have little effect on single-base mispairs, whereas mutations in MSH6 have a more prominent effect on the incidence of single-base mispairs than on microsatellite tract instability (3)(4)(5). From these and other genetic observations, it has been inferred that MSH2-MSH3 complex is more proficient in the removal of insertion-deletion mismatches of two or more nucleotides (4), whereas MSH2-MSH6 is better at removing single nucleotide mismatches (4,5). Human cell lines defective in the MSH6 component of the MSH2-MSH6 heterodimer hMutS␣ exhibit a selective loss in the repair of base-base and single-nucleotide insertion-deletion mismatches; the repair of two-, three-, and four-nucleotide insertion-deletion mismatches is reduced 2-4-fold in these cell lines (6,7). Consistent with genetic observations, hMutS␣ binds a G/T mismatch or a one nucleotide insertion-deletion mismatch with high efficiency (6). By contrast, the yeast MSH2-MSH3 heterodimer exhibits little affinity for a G/T mismatch but binds insertion-deletion mismatches with high specificity (8). The manner by which PMS1 and MLH1 function in mismatch repair remains to be determined. The POL30 gene of Saccharomyces cerevisiae encodes PCNA, 1 an essential component of the DNA replication machinery (9,10). PCNA forms a homotrimer that acts as a sliding clamp around the DNA duplex and increases the processivity of DNA polymerases ␦ and ⑀ (Ref. 11 and references therein). Here, we identify a mutation, pol30 -104, that causes a dramatic increase in the rate of microsatellite instability and spontaneous mutability. To examine if hypermutability in pol30 -104 arises from a defect in mismatch repair, we compared tract instability and mutability of double mutants carrying pol30 -104 in combination with mismatch repair mutations with single mutants of mismatch repair genes. From these and other studies, we suggest that hypermutability in pol30 -104 derives from a defect in mismatch repair. We have purified the MSH2-MSH3 complex and the MSH2 protein to near homogeneity from yeast (8) and show that PCNA interacts strongly with the MSH2-MSH3 heterodimer but not with MSH2. This observation is highly significant because genetic and biochemical studies have indicated that MSH2-MSH3 heterodimer and MSH2-MSH6 heterodimer but not MSH2 are the biologically relevant species in mismatch recognition.

MATERIALS AND METHODS
Generation of the pol30 -104 Mutation-To generate new mutations of yeast PCNA, PCR mutagenesis of the POL30 gene and plasmid shuffle techniques were used. The POL30 gene in plasmid pCH1565 was mutagenized by PCR amplification using pCH1565-specific primers and the Perkin-Elmer Taq  were replica plated onto 5-FOA containing medium to select for loss of wild type PCNA plasmid pCH1511[POL30 URA3] and screened for cold sensitivity (14°C) and sensitivity to the alkylating agent methylmethane sulfonate (MMS).
Construction of Mutant Strains-Isogenic mutant strains were generated by transformation of the respective wild type or mismatch repair defective strains with the appropriate plasmid. The pol30 -104 mutation was introduced into the yeast strain MS71 (MAT␣ ade5-1 his7-2 trp1-289 ura3-52 CAN1 s ) or its leu Ϫ (leu2-3,-112) isogenic parental strain AMY125 by the gene replacement method. Plasmids pCH1577 (pol30 -104:LEU2) or pBJ300 (pol30 -104:hisG::URA3::hisG) were digested with SacI and introduced into yeast. Genomic deletion mutations of MSH2 (4), MLH1, and PMS1 (12) were generated by the gene replacement method. All strains used in this study are isogenic and derived from AMY125. Integration of mutations were confirmed by Southern (DNA) blot analyses. Loss of the URA3 gene by recombination of the HisG sequences was selected for by plating on medium containing 5-FOA.
Rates of Microsatellite Instability-Wild type and isogenic mutant strains were transformed with plasmid pSH91. To monitor alterations in the pSH91 repeat tract, for each strain 19 independent 100-l cultures, each starting from ϳ10 5-FOA-sensitive cells, were grown at 30°C in yeast extract-peptone-dextrose (YPD) medium to ϳ10 5 -10 6 cells before being plated onto medium containing 5-FOA. The rates of tract alterations were determined by the method of the median (13).
DNA Sequencing-Alterations in the pSH91 repeat tract were determined by PCR analysis. Using plasmid DNA isolated from 5-FOAresistant cells, a single-stranded ϳ130-nucleotide region encompassing the repeat tract was amplified by assymetric PCR using 35 S-dATP (Amersham Corp.) and the primers 5Ј-CCATTCTAATGTCTGCCCC-3Ј and 5Ј-GTTTTCCCAGTCACGAC-3Ј. To determine the size of the alterations, the products were compared with labeled PCR products of predetermined length on 6% polyacrylamide denaturing gels.
Rates of Spontaneous Forward Mutation-For each strain, 19 independent cultures were grown at 30°C in 100 or 500 l of YPD, each starting from ϳ10 canavanine-sensitive cells. Cells were then plated onto arginine-deficient medium containing canavanine. Rates of spontaneous forward mutation at the CAN1 s locus were determined from the number of canavanine-resistant colonies by the method of the median (13).
PCNA Affi-Gel 15 Beads-Yeast PCNA was purified to homogeneity from Escherichia coli strain B834 containing the plasmid pBL228 as described (10). PCNA (2 mg) and BSA (5 mg) were coupled to 1 ml of Affi-Gel 15 in 0.1 M potassium MOPS, pH 7.5, following the instructions of the manufacturer (Bio-Rad). The coupling efficiency was greater than 95% for both PCNA and BSA, as determined by analyzing a sample before and after the coupling reaction by SDS-polyacrylamide gel electrophoresis.
Binding of MSH2 and MSH2-MSH3 Complex to PCNA Affinity Matrix-MSH2 and MSH2-MSH3 heterodimer were purified to near homogeneity from a yeast strain carrying either the plasmid for overproducing MSH2 or the plasmids for overproducing both MSH2 and MSH3 (8). Purified MSH2 protein (500 ng) and MSH2-MSH3 complex (1 g) were mixed with 10 l of the PCNA or BSA containing Affi-Gel beads in 140 l of buffer A (20 mM Tris acetate, pH 7.0, 20% glycerol, 0.01% Nonidet P-40, 100 g/ml BSA, 1 mM dithiothreitol) containing 0.1 M KCl at 4°C for 1 h. The unbound proteins were removed by centrifugation, and the affinity matrix was washed with 0.2 ml of buffer A containing 1 M KCl at 4°C. Proteins were then eluted from the Affi-Gel beads by incubating in 25 l of 2% SDS at 37°C for 10 min. The supernatant (2 l) containing unbound proteins and the SDS eluates (0.5 l) were subjected to immunoblot analysis to determine their content of MSH2 protein and the MSH2-MSH3 heterodimer. Immunoblot analysis of the 1 M KCl washes from all the binding reactions revealed that they did not contain any MSH2 protein or MSH2-MSH3 complex.

RESULTS
Mutations of the POL30 gene were obtained using PCR mutagenesis and plasmid shuffle. A total of eight different pol30 mutations were identified by their sensitivity to MMS and by their inability to grow at the restrictive temperature of 14°C. We screened these mutations for their effects on spontaneous mutability at the CAN1 s locus and found that one of these, pol30 -104, caused a dramatic increase in mutability at the permissive temperature (30°C), whereas the others had little or no effect. UV sensitivity is not affected by pol30 -104. The sequence of the entire coding region of the pol30 -104 allele was determined. The pol30 -104 mutation is a C to T transition at nucleotide 752 of the coding sequence, which results in an alanine to valine change at residue 251 of the PCNA protein. This residue is in a stretch of four amino acids that are conserved in S. cerevisiae, Schizosaccharomyces pombe, Drosophila, mouse, and human PCNA. Because the pol30 -104 mutation differs from the other cold-sensitive and MMS-sensitive pol30 mutations by increasing spontaneous mutability dramatically, we examined the possibility that pol30 -104 may confer a defect in mismatch repair.
To examine the effect of the pol30 -104 mutation on the stability of simple DNA repeats, we used the centromeric plasmid pSH91 that contains an in-frame 33-bp insertion of poly(GT) 16 G in the coding sequence of a hybrid gene containing the yeast URA3 gene (12). The pSH91 repeat tract is in-frame with the URA3 gene, resulting in Ura ϩ cells. Alterations of the tract that produce an out-of-frame mutation give rise to Ura Ϫ cells that become resistant to 5-FOA.
Compared with wild type, the pol30 -104 mutation caused an 80-fold increase in the rate of tract instability (Table I). Mutations in the yeast mismatch repair genes MSH2, MLH1, and PMS1 result in elevated rates of tract instability (12). To determine whether the increased tract instability in the pol30 -104 mutant could be due to a defect in mismatch repair, we examined tract instability in the pol30 -104 msh2⌬, pol30 -104 mlh1⌬, and pol30 -104 pms1⌬ double mutant strains. If tract instability in the pol30 -104 mutant had resulted from an increase in slippage events during DNA replication rather than from a defect in mismatch repair, then the rate of tract instability in these double mutant strains would have increased in a multiplicative fashion. In fact, a pms1 mutation, in combination with the pol3-01 mutation, which inactivates the 3Ј to 5Ј proof reading exonuclease function of DNA polymerase ␦, results in mutation rates that are the product of the relative rates observed in the pms1 and pol3-01 single mutants (14). For example, compared with wild type, although forward mutation rates of URA3 are elevated 40-and 130-fold in the pms1 and pol3-01 single mutants, respectively, the mutation rate in the pms1 pol3-01 double mutant increases 19,000-fold (14). On the other hand, if tract instability in the pol30 -104 mutant had resulted from a defect in mismatch repair, then double mutant strains carrying the pol30 -104 mutation in combination with the mismatch repair mutations will exhibit the same level of tract instability as the msh2, mlh1, and pms1 mutants. The results in Table I show that tract instability in the pol30 -104 msh2⌬, pol30 -104 mlh1⌬, and pol30 -104 pms1⌬ double mutant strains was about the same as in the msh2⌬, mlh1⌬, and pms1⌬ strains. These data are consistent with epistasis of mutations in mismatch repair genes with pol30 -104. Although the possibility of a role of PCNA in a parallel and independent pathway cannot be excluded, these results suggest an involvement of PCNA in MSH2/MLH1/PMS1-dependent mismatch repair.
We determined the nature of tract alterations in the pol30 -104 mutant by sequencing the tracts that had undergone changes in plasmid pSH91 (Table II) (Table II). Thus, the pattern of tract alterations in pol30 -104 resembles more closely the pattern in the mismatch repair mutants than that in the wild type. Because pol30 -104 is a missense mutation, it is not surprising that it does not cause the same degree of tract destabilization and bias as do null mutations in mismatch repair genes. To verify that tract destabilization in the pol30 -104 mutant arose from inactivation of mismatch repair, we compared the pattern of tract alterations in the pol30 -104 msh2⌬ and pol30 -104 mlh1⌬ double mutant strains with that in the msh2⌬ and mlh1⌬ single mutant strains. When our data for the msh2⌬ mutant are combined with those of Strand et al. (3) (Table II), then the numbers of Ϫ2 and ϩ2 tracts in this mutant are 73 and 29, respectively, compared with 32 and 16, respectively, in the pol30 -104 msh2⌬ mutant ( 2 for 1 degree of freedom ϭ 0.70; p Ͼ 0.25). Thus, the pattern of tract alterations in these mutant strains is almost identical. The incidence of Ϫ2 and ϩ2 tracts was also the same in the pol30 -104 mlh1⌬ and mlh1⌬ mutants (Table II).
We also examined the effect of the pol30 -104 mutation on spontaneous forward mutations of the CAN1 locus (Table III). The pol30 -104 mutation caused ϳ30-fold increase in the rate of spontaneous mutability, which is similar to that observed in the mlh1⌬ strain and the pol30 -104 mlh1⌬ strains (Table III). The results for can1 r are consistent with the epistasis observed for microsatellite instability.
To examine whether PCNA interacts physically with mismatch repair proteins, we purified PCNA from E. coli cells expressing the protein and covalently coupled it to Affi-Gel 15 beads for use as affinity matrix. The PCNA beads and control  beads containing BSA were mixed with MSH2 protein and MSH2-MSH3 heterodimer that we had purified to near homogeneity from S. cerevisiae (8). After washing with a large volume of 1 M KCl, the beads were incubated with 2% SDS to elute bound proteins followed by immunoblot analysis of the SDS eluates to determine the amount of MSH2 protein or of MSH2-MSH3 heterodimer that was retained on the Affi-Gel beads in each case. Interestingly, while MSH2 protein alone did not show any affinity for the PCNA beads (Fig. 1A), the majority (ϳ80%) of the input MSH2-MSH3 heterodimer was retained on the PCNA beads (Fig. 1B). This interaction between PCNA and the MSH2-MSH3 complex is highly specific because (i) we observed no binding of MSH2-MSH3 heterodimer to the control BSA Affi-Gel 15 beads (Fig. 1B) and (ii) the association of MSH2-MSH3 with the PCNA beads was stable to washing with 1 M KCl, as indicated by the absence of the heterodimer in the 1 M KCl wash (data not shown). DISCUSSION We show here that the pol30 -104 mutation in PCNA causes an increase in the rate of instability of (GT) n repeat sequences and in the rate of forward mutations at the CAN1 s locus. The effect of pol30 -104 on tract alterations, however, is not quite as severe as that of null mutations in mismatch repair genes. This is likely to be due to the fact that pol30 -104 is a missense mutation and not a null mutation. Null mutations in POL30 are lethal. The strong mutator phenotype of pol30 -104, which is observed at the permissive temperature, distinguishes this mutation from the other cold-sensitive and MMS-sensitive pol30 mutations that we have identified. This suggested to us that hypermutability in pol30 -104 may arise from a defect in mismatch repair rather than from a defect in DNA replication. To test the validity of this hypothesis, we carried out epistasis analyses of pol30 -104 with null mutations in mismatch repair genes MSH2, MLH1, and PMS1. We found that the rates of tract instability, the pattern of tract alterations, and spontaneous can1 r mutability were the same in double mutants of pol30 -104 with null mutations in mismatch repair genes and in single mismatch repair mutants. From these genetic observations, we infer that hypermutability in pol30 -104 results from a defect in mismatch repair.
Our recent biochemical studies have indicated that while the MSH2-MSH3 heterodimer binds insertion/deletion mismatches with high affinity but does not bind a G/T mismatch, MSH2 by itself does not recognize any of these mismatches (8). We now show that PCNA interacts with the MSH2-MSH3 complex but not with MSH2 protein, and the interaction between PCNA and MSH2-MSH3 is strong, being stable to challenge with 1 M KCl, further emphasizing the significance of the MSH2-MSH3 heterodimer in mismatch repair. Besides binding the MSH2-MSH3 complex, PCNA may also interact with additional components of the mismatch repair machinery, such as the MSH2-MSH6 heterodimer, PMS1, and MLH1.
Our findings have implications regarding which DNA polymerase may function in mismatch repair. The aphidicolin sensitivity of mismatch repair-associated DNA synthesis (15,16) had suggested that any of the three DNA polymerases, ␣, ␦, or ⑀, might act in this process, as they are all aphidicolin-sensi-tive. The evidence implicating PCNA in mismatch repair would suggest that Pol␦ may be the polymerase involved in mismatch repair, because PCNA is an essential subunit of this DNA polymerase. Pol⑀, however, could also be involved, because PCNA also stimulates this DNA polymerase. One way by which PCNA might function in mismatch repair could be as follows. The mismatch repair proteins including MSH2-MSH3 or the MSH2-MSH6 heterodimer and PMS1 and MLH1 could be targeted to the mismatch via their affinity for the mismatch and for one another, and PCNA, along with the DNA polymerase, could be loaded onto the nick remaining at the 5Ј side of the mismatch prior to the removal of RNA primers and joining of nascent DNA fragments. Via DNA looping, perhaps catalyzed by the MSH proteins in a manner analogous to the MutS catalyzed ␣-shaped loop structures in E. coli (17), the mismatch bound proteins may become associated with the PCNA-DNA polymerase complex, and these interactions could be important for the subsequent excision and repair synthesis reactions. The pol30 -104 mutation might impair any of these interactions.
The involvement of PCNA in mismatch repair suggests the possibility that mutations in human PCNA that inactivate only its mismatch repair function may contribute to sporadic colorectal cancers (18) and to other cancers, including those of the stomach, lung, breast, and pancreas, that are associated with microsatellite instability (19 -22).