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Evidence for Involvement of Yeast Proliferating Cell Nuclear Antigen in DNA Mismatch Repair

Evidence for Involvement of Yeast Proliferating Cell Nuclear Antigen in DNA Mismatch Repair THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 45, Issue of November 8, pp. 27987–27990, 1996 Communication © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. tein functions in conjunction with MSH3 or MSH6 protein in Evidence for Involvement mismatch recognition. Genetic and biochemical studies in both of Yeast Proliferating Cell yeast and humans have further indicated that the MSH2- MSH3 and MSH2-MSH6 complexes differ in substrate speci- Nuclear Antigen in DNA ficities. In yeast, mutations in MSH3 cause an increase in Mismatch Repair* instability of microsatellite tracts but have little effect on sin- gle-base mispairs, whereas mutations in MSH6 have a more (Received for publication, August 12, 1996, and in revised form, prominent effect on the incidence of single-base mispairs than September 13, 1996) on microsatellite tract instability (3–5). From these and other genetic observations, it has been inferred that MSH2-MSH3 Robert E. Johnson‡, Gopala K. Kovvali‡, Sami N. Guzder‡, Neelam S. Amin§, complex is more proficient in the removal of insertion-deletion Connie Holm§, Yvette Habraken‡, Patrick Sung‡, mismatches of two or more nucleotides (4), whereas MSH2- Louise Prakash‡, and Satya Prakash‡ MSH6 is better at removing single nucleotide mismatches (4, From the ‡Sealy Center for Molecular Science, 5). Human cell lines defective in the MSH6 component of the University of Texas Medical Branch, Galveston, Texas MSH2-MSH6 heterodimer hMutSa exhibit a selective loss in 77555-1061 and the §Department of Pharmacology, the repair of base-base and single-nucleotide insertion-deletion Division of Cellular and Molecular Medicine, mismatches; the repair of two-, three-, and four-nucleotide University of California, San Diego, insertion-deletion mismatches is reduced 2–4-fold in these cell La Jolla, California 92093-0651 lines (6, 7). Consistent with genetic observations, hMutSa DNA mismatch repair plays a key role in the mainte- binds a G/T mismatch or a one nucleotide insertion-deletion nance of genetic fidelity. Mutations in the human mis- mismatch with high efficiency (6). By contrast, the yeast match repair genes hMSH2, hMLH1, hPMS1, and hPMS2 MSH2-MSH3 heterodimer exhibits little affinity for a G/T mis- are associated with hereditary nonpolyposis colorectal match but binds insertion-deletion mismatches with high spec- cancer. The proliferating cell nuclear antigen (PCNA) is ificity (8). The manner by which PMS1 and MLH1 function in essential for DNA replication, where it acts as a proces- mismatch repair remains to be determined. sivity factor. Here, we identify a point mutation, pol30– The POL30 gene of Saccharomyces cerevisiae encodes 104, in the Saccharomyces cerevisiae POL30 gene encod- PCNA, an essential component of the DNA replication ma- ing PCNA that increases the rate of instability of simple chinery (9, 10). PCNA forms a homotrimer that acts as a sliding repetitive DNA sequences and raises the rate of sponta- clamp around the DNA duplex and increases the processivity of neous forward mutation. Epistasis analyses with muta- DNA polymerases d and e (Ref. 11 and references therein). tions in mismatch repair genes MSH2, MLH1, and PMS1 Here, we identify a mutation, pol30–104, that causes a dra- suggest that the pol30–104 mutation impairs MSH2/ MLH1/PMS1-dependent mismatch repair, consistent matic increase in the rate of microsatellite instability and spon- with the hypothesis that PCNA functions in mismatch taneous mutability. To examine if hypermutability in pol30– repair. MSH2 functions in mismatch repair with either 104 arises from a defect in mismatch repair, we compared tract MSH3 or MSH6, and the MSH2-MSH3 and MSH2-MSH6 instability and mutability of double mutants carrying pol30– heterodimers have a role in the recognition of DNA mis- 104 in combination with mismatch repair mutations with sin- matches. Consistent with the genetic data, we find spe- gle mutants of mismatch repair genes. From these and other cific interaction of PCNA with the MSH2-MSH3 studies, we suggest that hypermutability in pol30–104 derives heterodimer. 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 In both prokaryotes and eukaryotes, defects in DNA mis- MSH2-MSH3 heterodimer but not with MSH2. This observa- match repair cause elevated spontaneous mutation rates and tion is highly significant because genetic and biochemical stud- increased instability of simple repeat DNA sequences. Muta- ies have indicated that MSH2-MSH3 heterodimer and MSH2- tions in any of the human mismatch repair genes hMSH2, MSH6 heterodimer but not MSH2 are the biologically relevant hMLH1, hPMS1, and hPMS2 are associated with hereditary species in mismatch recognition. nonpolyposis colorectal cancer. Cell lines from these cancers MATERIALS AND METHODS are defective in DNA mismatch repair and display increased Generation of the pol30–104 Mutation—To generate new mutations levels of spontaneous mutations and frequent alterations of of yeast PCNA, PCR mutagenesis of the POL30 gene and plasmid microsatellite repeat sequences (1, 2). shuffle techniques were used. The POL30 gene in plasmid pCH1565 Epistasis analyses in yeast have suggested that MSH2 pro- was mutagenized by PCR amplification using pCH1565-specific prim- ers and the Perkin-Elmer Taq polymerase in the presence of 1 mM * This work was supported by Grant CA41261 from the NCI, Grants MgCl and 1 mM MnCl under standard conditions (10 mM Tris-HCl, pH 2 2 GM19261 and GM36510 from the National Institutes of Health, and 8.3, 50 mM KCl, 200 mm of dNTP). Mutagenized POL30 and an appro- Grant DE-FG03-93ER61706 from the Department of Energy. The costs priate pCH1565 fragment were then introduced into yeast strain of publication of this article were defrayed in part by the payment of CH2134 (pol30D::LEU2 pCH1511 [POL30 URA3]) to allow for recom- page charges. This article must therefore be hereby marked “advertise- bination of mutagenized POL30 into vector pCH1565. Transformants ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ¶ To whom correspondence should be addressed: Sealy Center for The abbreviations used are: PCNA, proliferating cell nuclear anti- Molecular Science, University of Texas Medical Branch, 6.104 Medical gen; PCR, polymerase chain reaction; 5-FOA, 5-fluoro-orotic acid; MMS, Research Bldg., 11th and Mechanic St., Galveston, TX 77555-1061. Tel.: methylmethane sulfonate; BSA, bovine serum albumin; MOPS, 3-(N- 409-747-8602; Fax: 409-747-8608; E-mail: [email protected]. morpholino)propanesulfonic acid; bp, base pair(s). This paper is available on line at http://www-jbc.stanford.edu/jbc/ 27987 This is an Open Access article under the CC BY license. 27988 Role of PCNA in Mismatch Repair TABLE I Effect of the pol30–104 mutation on the stability of poly(GT) tracts Tract length alterations were monitored in plasmid pSH91. All the strains are isogenic and differ only by the mutations indicated. Rate of Rate relative Strain Genotype tract instability to wild type (6 S.D.) MS71 Wild type 6.0 (6 0.7) 3 10 1 YPCNA1.14 pol30–104 4.6 (6 0.1) 3 10 80 YRP85 msh2D 1.4 (6 0.2) 3 10 230 YRP23 mlh1D 1.9 (6 0.4) 3 10 320 AMY101 pms1D 2.2 (6 0.5) 3 10 370 YPCNA1.19 pol30–104 msh2D 2.2 (6 0.2) 3 10 370 YPCNA32 pol30–104 mlh1D 1.3 (6 0.2) 3 10 220 YPCNA31 pol30–104 pms1D 1.5 (6 0.3) 3 10 250 TABLE II Types of poly(GT) tract alterations generated in pol30–104 and other mismatch repair defective strains Number of tracts with base pair Number of deletions (2) or additions (1) Strain Genotype tracts sequenced 24 22 12 14 Others MS71 Wild type 33 0 2 26 0 5 YPCNA1.14 pol30–104 58 1 24 3012 YRP85 msh2D 28 0 21 7 00 MS128 msh2D (77) (0) (52) (22) (3) (0) YRP23 mlh1D 46 1 28 17 00 YPCNA1.19 pol30–104 msh2D 50 2 32 16 00 YPCNA32 pol30–104 mlh1D 36 0 22 14 00 These data are taken from Strand et al. (3). b 2 The frequencies of the 22- and 12-bp alterations were similar in the msh2D and pol30–104 msh2D strains (x for 1 degree of freedom 5 0.6; p . 0.25). c 2 The frequencies of the 22- and 12-bp alterations were similar in the mlh1D and pol30–104 mlh1D strains (x for 1 degree of freedom 5 0.008; p . 0.90). Other alterations observed in the wild type strain were 216, 214, 210, 114, and 114. Other alterations observed in the pol30–104 strain were 210 and 210. were replica plated onto 5-FOA containing medium to select for loss of from Escherichia coli strain B834 containing the plasmid pBL228 as wild type PCNA plasmid pCH1511[POL30 URA3] and screened for cold described (10). PCNA (2 mg) and BSA (5 mg) were coupled to 1 ml of sensitivity (14 °C) and sensitivity to the alkylating agent methylmeth- Affi-Gel 15 in 0.1 M potassium MOPS, pH 7.5, following the instructions ane sulfonate (MMS). of the manufacturer (Bio-Rad). The coupling efficiency was greater than Construction of Mutant Strains—Isogenic mutant strains were gen- 95% for both PCNA and BSA, as determined by analyzing a sample erated by transformation of the respective wild type or mismatch repair before and after the coupling reaction by SDS-polyacrylamide gel defective strains with the appropriate plasmid. The pol30–104 muta- electrophoresis. tion was introduced into the yeast strain MS71 (MATa ade5–1 his7–2 Binding of MSH2 and MSH2-MSH3 Complex to PCNA Affinity Ma- s 2 trp1–289 ura3–52 CAN1 ) or its leu (leu2–3,-112) isogenic parental trix—MSH2 and MSH2-MSH3 heterodimer were purified to near ho- strain AMY125 by the gene replacement method. Plasmids pCH1577 mogeneity from a yeast strain carrying either the plasmid for overpro- (pol30–104:LEU2) or pBJ300 (pol30–104:hisG::URA3::hisG) were di- ducing MSH2 or the plasmids for overproducing both MSH2 and MSH3 gested with SacI and introduced into yeast. Genomic deletion muta- (8). Purified MSH2 protein (500 ng) and MSH2-MSH3 complex (1 mg) tions of MSH2 (4), MLH1, and PMS1 (12) were generated by the gene were mixed with 10 ml of the PCNA or BSA containing Affi-Gel beads in replacement method. All strains used in this study are isogenic and 140 ml of buffer A (20 mM Tris acetate, pH 7.0, 20% glycerol, 0.01% derived from AMY125. Integration of mutations were confirmed by Nonidet P-40, 100 mg/ml BSA, 1 mM dithiothreitol) containing 0.1 M KCl Southern (DNA) blot analyses. Loss of the URA3 gene by recombination at 4 °C for 1 h. The unbound proteins were removed by centrifugation, of the HisG sequences was selected for by plating on medium containing and the affinity matrix was washed with 0.2 ml of buffer A containing 5-FOA. 1 M KCl at 4 °C. Proteins were then eluted from the Affi-Gel beads by Rates of Microsatellite Instability—Wild type and isogenic mutant incubating in 25 ml of 2% SDS at 37 °C for 10 min. The supernatant (2 strains were transformed with plasmid pSH91. To monitor alterations ml) containing unbound proteins and the SDS eluates (0.5 ml) were in the pSH91 repeat tract, for each strain 19 independent 100-ml cul- subjected to immunoblot analysis to determine their content of MSH2 tures, each starting from ;10 5-FOA-sensitive cells, were grown at protein and the MSH2-MSH3 heterodimer. Immunoblot analysis of the 5 6 30 °C in yeast extract-peptone-dextrose (YPD) medium to ;10 –10 1 M KCl washes from all the binding reactions revealed that they did not cells before being plated onto medium containing 5-FOA. The rates of contain any MSH2 protein or MSH2-MSH3 complex. tract alterations were determined by the method of the median (13). RESULTS DNA Sequencing—Alterations in the pSH91 repeat tract were deter- mined by PCR analysis. Using plasmid DNA isolated from 5-FOA- Mutations of the POL30 gene were obtained using PCR mu- resistant cells, a single-stranded ;130-nucleotide region encompassing tagenesis and plasmid shuffle. A total of eight different pol30 the repeat tract was amplified by assymetric PCR using S-dATP mutations were identified by their sensitivity to MMS and by (Amersham Corp.) and the primers 59-CCATTCTAATGTCTGCCCC-39 their inability to grow at the restrictive temperature of 14 °C. and 59-GTTTTCCCAGTCACGAC-39. To determine the size of the alter- We screened these mutations for their effects on spontaneous ations, the products were compared with labeled PCR products of pre- mutability at the CAN1 locus and found that one of these, determined length on 6% polyacrylamide denaturing gels. Rates of Spontaneous Forward Mutation—For each strain, 19 inde- pol30–104, caused a dramatic increase in mutability at the pendent cultures were grown at 30 °C in 100 or 500 ml of YPD, each permissive temperature (30 °C), whereas the others had little starting from ;10 canavanine-sensitive cells. Cells were then plated or no effect. UV sensitivity is not affected by pol30–104. The onto arginine-deficient medium containing canavanine. Rates of spon- sequence of the entire coding region of the pol30–104 allele was taneous forward mutation at the CAN1 locus were determined from determined. The pol30–104 mutation isaCtoT transition at the number of canavanine-resistant colonies by the method of the nucleotide 752 of the coding sequence, which results in an median (13). PCNA Affi-Gel 15 Beads—Yeast PCNA was purified to homogeneity alanine to valine change at residue 251 of the PCNA protein. Role of PCNA in Mismatch Repair 27989 TABLE III Effect of the pol30–104 mutation on the rate of spontaneous forward mutation to canavanine resistance (can1 ) Strains were identical to those in Table I, except that they did not carry plasmid pSH91. Rate of forward Rate relative Strain Genotype mutation to can1 to wild type (6 S.D.) MS71 Wild type 3.8 (6 1.0) 3 10 1 YPCNA1.14 pol30–104 1.2 (6 0.2) 3 10 32 YRP23 mlh1D 9.6 (6 3.3) 3 10 25 YPCNA32 pol30–104 mlh1D 1.5 (6 0.5) 3 10 39 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 FIG.1. MSH2-MSH3 heterodimer but not MSH2 interacts with results in Table I show that tract instability in the pol30–104 PCNA. A, purified MSH2 protein (lane 1) was mixed with BSA Affi-Gel msh2D, pol30–104 mlh1D, and pol30–104 pms1D double mu- 15 and PCNA Affi-Gel 15 beads, which were treated with 2% SDS to tant strains was about the same as in the msh2D, mlh1D, and elute bound MSH2 protein. The supernatants containing unbound pms1D strains. These data are consistent with epistasis of MSH2 protein from the BSA Affi-Gel (lane 2) and the PCNA Affi-Gel (lane 3) and the SDS eluates containing MSH2 protein bound to the mutations in mismatch repair genes with pol30–104. Although BSA Affi-Gel (lane 4) and the PCNA Affi-Gel (lane 5) were subjected to the possibility of a role of PCNA in a parallel and independent immunoblot analysis with affinity purified anti-MSH2 antibodies (8). B, pathway cannot be excluded, these results suggest an involve- purified MSH2-MSH3 complex (lane 1) was mixed with BSA Affi-Gel 15 ment of PCNA in MSH2/MLH1/PMS1-dependent mismatch and PCNA Affi-Gel 15 beads, which were treated with 2% SDS to elute bound MSH2-MSH3 complex. The supernatants containing unbound repair. MSH2-MSH3 complex from the BSA Affi-Gel (lane 2) and the PCNA We determined the nature of tract alterations in the pol30– Affi-Gel (lane 3) and the SDS eluates containing MSH2-MSH3 complex 104 mutant by sequencing the tracts that had undergone bound to the BSA Affi-Gel (lane 4) and the PCNA Affi-Gel (lane 5) were changes in plasmid pSH91 (Table II). Additions or deletions of subjected to immunoblot analysis with affinity purified anti-MSH2 and anti-MSH3 antibodies (8). MSH3 protein migrates in SDS gels as a two base pairs represent the most common alterations in all the triplet. strains tested. In the wild type strain, most of the tract alter- ations are additions of 2 bp. In the msh2D and mlh1D mutants, there is about a 2-fold bias in favor of 2-bp deletions over 2-bp This residue is in a stretch of four amino acids that are con- additions, whereas in the pol30–104 mutant, 2-bp additions served in S. cerevisiae, Schizosaccharomyces pombe, Drosoph- and deletions occur about equally frequently (Table II). Thus, ila, mouse, and human PCNA. Because the pol30–104 muta- the pattern of tract alterations in pol30–104 resembles more tion differs from the other cold-sensitive and MMS-sensitive closely the pattern in the mismatch repair mutants than that pol30 mutations by increasing spontaneous mutability dramat- in the wild type. Because pol30–104 is a missense mutation, it ically, we examined the possibility that pol30–104 may confer is not surprising that it does not cause the same degree of tract a defect in mismatch repair. destabilization and bias as do null mutations in mismatch To examine the effect of the pol30–104 mutation on the repair genes. To verify that tract destabilization in the pol30– stability of simple DNA repeats, we used the centromeric plas- 104 mutant arose from inactivation of mismatch repair, we mid pSH91 that contains an in-frame 33-bp insertion of compared the pattern of tract alterations in the pol30–104 poly(GT) G in the coding sequence of a hybrid gene containing msh2D and pol30–104 mlh1D double mutant strains with that the yeast URA3 gene (12). The pSH91 repeat tract is in-frame in the msh2D and mlh1D single mutant strains. When our data with the URA3 gene, resulting in Ura cells. Alterations of the for the msh2D mutant are combined with those of Strand et al. tract that produce an out-of-frame mutation give rise to Ura (3) (Table II), then the numbers of 22 and 12 tracts in this cells that become resistant to 5-FOA. mutant are 73 and 29, respectively, compared with 32 and 16, Compared with wild type, the pol30–104 mutation caused an respectively, in the pol30–104 msh2D mutant (x for 1 degree of 80-fold increase in the rate of tract instability (Table I). Muta- freedom 5 0.70; p . 0.25). Thus, the pattern of tract alterations tions in the yeast mismatch repair genes MSH2, MLH1, and in these mutant strains is almost identical. The incidence of 22 PMS1 result in elevated rates of tract instability (12). To de- and 12 tracts was also the same in the pol30–104 mlh1D and termine whether the increased tract instability in the pol30– mlh1D mutants (Table II). 104 mutant could be due to a defect in mismatch repair, we We also examined the effect of the pol30–104 mutation on examined tract instability in the pol30–104 msh2D, pol30–104 spontaneous forward mutations of the CAN1 locus (Table III). mlh1D, and pol30–104 pms1D double mutant strains. If tract The pol30–104 mutation caused ;30-fold increase in the rate instability in the pol30–104 mutant had resulted from an in- of spontaneous mutability, which is similar to that observed in crease in slippage events during DNA replication rather than the mlh1D strain and the pol30–104 mlh1D strains (Table III). from a defect in mismatch repair, then the rate of tract insta- The results for can1 are consistent with the epistasis observed bility in these double mutant strains would have increased in a multiplicative fashion. In fact, a pms1 mutation, in combina- for microsatellite instability. To examine whether PCNA interacts physically with mis- tion with the pol3–01 mutation, which inactivates the 39 to 59 proof reading exonuclease function of DNA polymerase d, re- match repair proteins, we purified PCNA from E. coli cells sults in mutation rates that are the product of the relative rates expressing the protein and covalently coupled it to Affi-Gel 15 observed in the pms1 and pol3–01 single mutants (14). For beads for use as affinity matrix. The PCNA beads and control 27990 Role of PCNA in Mismatch Repair beads containing BSA were mixed with MSH2 protein and tive. The evidence implicating PCNA in mismatch repair would MSH2-MSH3 heterodimer that we had purified to near homo- suggest that Pold may be the polymerase involved in mismatch geneity from S. cerevisiae (8). After washing with a large vol- repair, because PCNA is an essential subunit of this DNA ume of 1 M KCl, the beads were incubated with 2% SDS to elute polymerase. Pole, however, could also be involved, because bound proteins followed by immunoblot analysis of the SDS PCNA also stimulates this DNA polymerase. One way by which eluates to determine the amount of MSH2 protein or of MSH2- PCNA might function in mismatch repair could be as follows. MSH3 heterodimer that was retained on the Affi-Gel beads in The mismatch repair proteins including MSH2-MSH3 or the each case. Interestingly, while MSH2 protein alone did not MSH2-MSH6 heterodimer and PMS1 and MLH1 could be tar- show any affinity for the PCNA beads (Fig. 1A), the majority geted to the mismatch via their affinity for the mismatch and (;80%) of the input MSH2-MSH3 heterodimer was retained on for one another, and PCNA, along with the DNA polymerase, the PCNA beads (Fig. 1B). This interaction between PCNA and could be loaded onto the nick remaining at the 59 side of the the MSH2-MSH3 complex is highly specific because (i) we mismatch prior to the removal of RNA primers and joining of observed no binding of MSH2-MSH3 heterodimer to the control nascent DNA fragments. Via DNA looping, perhaps catalyzed BSA Affi-Gel 15 beads (Fig. 1B) and (ii) the association of by the MSH proteins in a manner analogous to the MutS MSH2-MSH3 with the PCNA beads was stable to washing with catalyzed a-shaped loop structures in E. coli (17), the mismatch 1 M KCl, as indicated by the absence of the heterodimer in the bound proteins may become associated with the PCNA-DNA 1 M KCl wash (data not shown). polymerase complex, and these interactions could be important for the subsequent excision and repair synthesis reactions. The DISCUSSION pol30–104 mutation might impair any of these interactions. We show here that the pol30–104 mutation in PCNA causes The involvement of PCNA in mismatch repair suggests the an increase in the rate of instability of (GT) repeat sequences possibility that mutations in human PCNA that inactivate only and in the rate of forward mutations at the CAN1 locus. The its mismatch repair function may contribute to sporadic colo- effect of pol30–104 on tract alterations, however, is not quite as rectal cancers (18) and to other cancers, including those of the severe as that of null mutations in mismatch repair genes. This stomach, lung, breast, and pancreas, that are associated with is likely to be due to the fact that pol30–104 is a missense microsatellite instability (19–22). mutation and not a null mutation. Null mutations in POL30 Acknowledgment—We thank P. Burgers for plasmid pBL228. are lethal. 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Drummond, J. T., Li, G.-M., Longley, M. J., and Modrich, P. (1995) Science 268, 1909–1912 tract instability, the pattern of tract alterations, and sponta- 7. Papadopoulos, N., Nicolaides, N. C., Liu, B., Parson, R., Lengauer, C., neous can1 mutability were the same in double mutants of Palombo, F., D’Arrigo, A., Markowitz, S., Willson, J. K. V., Kinzler, K. W., pol30–104 with null mutations in mismatch repair genes and Jiricny, J., and Vogelstein, B. (1995) Science 268, 1915–1917 8. Habraken, Y., Sung, P., Prakash, L., and Prakash, S. (1996) Curr. Biol. 6, in single mismatch repair mutants. From these genetic obser- 1185–1187 vations, we infer that hypermutability in pol30–104 results 9. Bauer, G. A., and Burgers, P. M. J. (1990) Nucleic Acids Res. 18, 261–265 10. Ayyagari, R., Impellizzeri, K. J., Yoder, B. L., Gary, S. L., and Burgers, P. M. from a defect in mismatch repair. J. (1995) Mol. Cell. Biol. 15, 4420–4429 Our recent biochemical studies have indicated that while the 11. 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(1994) Cancer Res. 54, 41–44 had suggested that any of the three DNA polymerases, a, d,or 22. Merlo, A., Mabry, M., Gabrielson, E., Vollmer, R., Baylin, S. B., and Sidransky, e, might act in this process, as they are all aphidicolin-sensi- D. (1994) Cancer Res. 54, 2098–2101 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Evidence for Involvement of Yeast Proliferating Cell Nuclear Antigen in DNA Mismatch Repair

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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 45, Issue of November 8, pp. 27987–27990, 1996 Communication © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. tein functions in conjunction with MSH3 or MSH6 protein in Evidence for Involvement mismatch recognition. Genetic and biochemical studies in both of Yeast Proliferating Cell yeast and humans have further indicated that the MSH2- MSH3 and MSH2-MSH6 complexes differ in substrate speci- Nuclear Antigen in DNA ficities. In yeast, mutations in MSH3 cause an increase in Mismatch Repair* instability of microsatellite tracts but have little effect on sin- gle-base mispairs, whereas mutations in MSH6 have a more (Received for publication, August 12, 1996, and in revised form, prominent effect on the incidence of single-base mispairs than September 13, 1996) on microsatellite tract instability (3–5). From these and other genetic observations, it has been inferred that MSH2-MSH3 Robert E. Johnson‡, Gopala K. Kovvali‡, Sami N. Guzder‡, Neelam S. Amin§, complex is more proficient in the removal of insertion-deletion Connie Holm§, Yvette Habraken‡, Patrick Sung‡, mismatches of two or more nucleotides (4), whereas MSH2- Louise Prakash‡, and Satya Prakash‡ MSH6 is better at removing single nucleotide mismatches (4, From the ‡Sealy Center for Molecular Science, 5). Human cell lines defective in the MSH6 component of the University of Texas Medical Branch, Galveston, Texas MSH2-MSH6 heterodimer hMutSa exhibit a selective loss in 77555-1061 and the §Department of Pharmacology, the repair of base-base and single-nucleotide insertion-deletion Division of Cellular and Molecular Medicine, mismatches; the repair of two-, three-, and four-nucleotide University of California, San Diego, insertion-deletion mismatches is reduced 2–4-fold in these cell La Jolla, California 92093-0651 lines (6, 7). Consistent with genetic observations, hMutSa DNA mismatch repair plays a key role in the mainte- binds a G/T mismatch or a one nucleotide insertion-deletion nance of genetic fidelity. Mutations in the human mis- mismatch with high efficiency (6). By contrast, the yeast match repair genes hMSH2, hMLH1, hPMS1, and hPMS2 MSH2-MSH3 heterodimer exhibits little affinity for a G/T mis- are associated with hereditary nonpolyposis colorectal match but binds insertion-deletion mismatches with high spec- cancer. The proliferating cell nuclear antigen (PCNA) is ificity (8). The manner by which PMS1 and MLH1 function in essential for DNA replication, where it acts as a proces- mismatch repair remains to be determined. sivity factor. Here, we identify a point mutation, pol30– The POL30 gene of Saccharomyces cerevisiae encodes 104, in the Saccharomyces cerevisiae POL30 gene encod- PCNA, an essential component of the DNA replication ma- ing PCNA that increases the rate of instability of simple chinery (9, 10). PCNA forms a homotrimer that acts as a sliding repetitive DNA sequences and raises the rate of sponta- clamp around the DNA duplex and increases the processivity of neous forward mutation. Epistasis analyses with muta- DNA polymerases d and e (Ref. 11 and references therein). tions in mismatch repair genes MSH2, MLH1, and PMS1 Here, we identify a mutation, pol30–104, that causes a dra- suggest that the pol30–104 mutation impairs MSH2/ MLH1/PMS1-dependent mismatch repair, consistent matic increase in the rate of microsatellite instability and spon- with the hypothesis that PCNA functions in mismatch taneous mutability. To examine if hypermutability in pol30– repair. MSH2 functions in mismatch repair with either 104 arises from a defect in mismatch repair, we compared tract MSH3 or MSH6, and the MSH2-MSH3 and MSH2-MSH6 instability and mutability of double mutants carrying pol30– heterodimers have a role in the recognition of DNA mis- 104 in combination with mismatch repair mutations with sin- matches. Consistent with the genetic data, we find spe- gle mutants of mismatch repair genes. From these and other cific interaction of PCNA with the MSH2-MSH3 studies, we suggest that hypermutability in pol30–104 derives heterodimer. 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 In both prokaryotes and eukaryotes, defects in DNA mis- MSH2-MSH3 heterodimer but not with MSH2. This observa- match repair cause elevated spontaneous mutation rates and tion is highly significant because genetic and biochemical stud- increased instability of simple repeat DNA sequences. Muta- ies have indicated that MSH2-MSH3 heterodimer and MSH2- tions in any of the human mismatch repair genes hMSH2, MSH6 heterodimer but not MSH2 are the biologically relevant hMLH1, hPMS1, and hPMS2 are associated with hereditary species in mismatch recognition. nonpolyposis colorectal cancer. Cell lines from these cancers MATERIALS AND METHODS are defective in DNA mismatch repair and display increased Generation of the pol30–104 Mutation—To generate new mutations levels of spontaneous mutations and frequent alterations of of yeast PCNA, PCR mutagenesis of the POL30 gene and plasmid microsatellite repeat sequences (1, 2). shuffle techniques were used. The POL30 gene in plasmid pCH1565 Epistasis analyses in yeast have suggested that MSH2 pro- was mutagenized by PCR amplification using pCH1565-specific prim- ers and the Perkin-Elmer Taq polymerase in the presence of 1 mM * This work was supported by Grant CA41261 from the NCI, Grants MgCl and 1 mM MnCl under standard conditions (10 mM Tris-HCl, pH 2 2 GM19261 and GM36510 from the National Institutes of Health, and 8.3, 50 mM KCl, 200 mm of dNTP). Mutagenized POL30 and an appro- Grant DE-FG03-93ER61706 from the Department of Energy. The costs priate pCH1565 fragment were then introduced into yeast strain of publication of this article were defrayed in part by the payment of CH2134 (pol30D::LEU2 pCH1511 [POL30 URA3]) to allow for recom- page charges. This article must therefore be hereby marked “advertise- bination of mutagenized POL30 into vector pCH1565. Transformants ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ¶ To whom correspondence should be addressed: Sealy Center for The abbreviations used are: PCNA, proliferating cell nuclear anti- Molecular Science, University of Texas Medical Branch, 6.104 Medical gen; PCR, polymerase chain reaction; 5-FOA, 5-fluoro-orotic acid; MMS, Research Bldg., 11th and Mechanic St., Galveston, TX 77555-1061. Tel.: methylmethane sulfonate; BSA, bovine serum albumin; MOPS, 3-(N- 409-747-8602; Fax: 409-747-8608; E-mail: [email protected]. morpholino)propanesulfonic acid; bp, base pair(s). This paper is available on line at http://www-jbc.stanford.edu/jbc/ 27987 This is an Open Access article under the CC BY license. 27988 Role of PCNA in Mismatch Repair TABLE I Effect of the pol30–104 mutation on the stability of poly(GT) tracts Tract length alterations were monitored in plasmid pSH91. All the strains are isogenic and differ only by the mutations indicated. Rate of Rate relative Strain Genotype tract instability to wild type (6 S.D.) MS71 Wild type 6.0 (6 0.7) 3 10 1 YPCNA1.14 pol30–104 4.6 (6 0.1) 3 10 80 YRP85 msh2D 1.4 (6 0.2) 3 10 230 YRP23 mlh1D 1.9 (6 0.4) 3 10 320 AMY101 pms1D 2.2 (6 0.5) 3 10 370 YPCNA1.19 pol30–104 msh2D 2.2 (6 0.2) 3 10 370 YPCNA32 pol30–104 mlh1D 1.3 (6 0.2) 3 10 220 YPCNA31 pol30–104 pms1D 1.5 (6 0.3) 3 10 250 TABLE II Types of poly(GT) tract alterations generated in pol30–104 and other mismatch repair defective strains Number of tracts with base pair Number of deletions (2) or additions (1) Strain Genotype tracts sequenced 24 22 12 14 Others MS71 Wild type 33 0 2 26 0 5 YPCNA1.14 pol30–104 58 1 24 3012 YRP85 msh2D 28 0 21 7 00 MS128 msh2D (77) (0) (52) (22) (3) (0) YRP23 mlh1D 46 1 28 17 00 YPCNA1.19 pol30–104 msh2D 50 2 32 16 00 YPCNA32 pol30–104 mlh1D 36 0 22 14 00 These data are taken from Strand et al. (3). b 2 The frequencies of the 22- and 12-bp alterations were similar in the msh2D and pol30–104 msh2D strains (x for 1 degree of freedom 5 0.6; p . 0.25). c 2 The frequencies of the 22- and 12-bp alterations were similar in the mlh1D and pol30–104 mlh1D strains (x for 1 degree of freedom 5 0.008; p . 0.90). Other alterations observed in the wild type strain were 216, 214, 210, 114, and 114. Other alterations observed in the pol30–104 strain were 210 and 210. were replica plated onto 5-FOA containing medium to select for loss of from Escherichia coli strain B834 containing the plasmid pBL228 as wild type PCNA plasmid pCH1511[POL30 URA3] and screened for cold described (10). PCNA (2 mg) and BSA (5 mg) were coupled to 1 ml of sensitivity (14 °C) and sensitivity to the alkylating agent methylmeth- Affi-Gel 15 in 0.1 M potassium MOPS, pH 7.5, following the instructions ane sulfonate (MMS). of the manufacturer (Bio-Rad). The coupling efficiency was greater than Construction of Mutant Strains—Isogenic mutant strains were gen- 95% for both PCNA and BSA, as determined by analyzing a sample erated by transformation of the respective wild type or mismatch repair before and after the coupling reaction by SDS-polyacrylamide gel defective strains with the appropriate plasmid. The pol30–104 muta- electrophoresis. tion was introduced into the yeast strain MS71 (MATa ade5–1 his7–2 Binding of MSH2 and MSH2-MSH3 Complex to PCNA Affinity Ma- s 2 trp1–289 ura3–52 CAN1 ) or its leu (leu2–3,-112) isogenic parental trix—MSH2 and MSH2-MSH3 heterodimer were purified to near ho- strain AMY125 by the gene replacement method. Plasmids pCH1577 mogeneity from a yeast strain carrying either the plasmid for overpro- (pol30–104:LEU2) or pBJ300 (pol30–104:hisG::URA3::hisG) were di- ducing MSH2 or the plasmids for overproducing both MSH2 and MSH3 gested with SacI and introduced into yeast. Genomic deletion muta- (8). Purified MSH2 protein (500 ng) and MSH2-MSH3 complex (1 mg) tions of MSH2 (4), MLH1, and PMS1 (12) were generated by the gene were mixed with 10 ml of the PCNA or BSA containing Affi-Gel beads in replacement method. All strains used in this study are isogenic and 140 ml of buffer A (20 mM Tris acetate, pH 7.0, 20% glycerol, 0.01% derived from AMY125. Integration of mutations were confirmed by Nonidet P-40, 100 mg/ml BSA, 1 mM dithiothreitol) containing 0.1 M KCl Southern (DNA) blot analyses. Loss of the URA3 gene by recombination at 4 °C for 1 h. The unbound proteins were removed by centrifugation, of the HisG sequences was selected for by plating on medium containing and the affinity matrix was washed with 0.2 ml of buffer A containing 5-FOA. 1 M KCl at 4 °C. Proteins were then eluted from the Affi-Gel beads by Rates of Microsatellite Instability—Wild type and isogenic mutant incubating in 25 ml of 2% SDS at 37 °C for 10 min. The supernatant (2 strains were transformed with plasmid pSH91. To monitor alterations ml) containing unbound proteins and the SDS eluates (0.5 ml) were in the pSH91 repeat tract, for each strain 19 independent 100-ml cul- subjected to immunoblot analysis to determine their content of MSH2 tures, each starting from ;10 5-FOA-sensitive cells, were grown at protein and the MSH2-MSH3 heterodimer. Immunoblot analysis of the 5 6 30 °C in yeast extract-peptone-dextrose (YPD) medium to ;10 –10 1 M KCl washes from all the binding reactions revealed that they did not cells before being plated onto medium containing 5-FOA. The rates of contain any MSH2 protein or MSH2-MSH3 complex. tract alterations were determined by the method of the median (13). RESULTS DNA Sequencing—Alterations in the pSH91 repeat tract were deter- mined by PCR analysis. Using plasmid DNA isolated from 5-FOA- Mutations of the POL30 gene were obtained using PCR mu- resistant cells, a single-stranded ;130-nucleotide region encompassing tagenesis and plasmid shuffle. A total of eight different pol30 the repeat tract was amplified by assymetric PCR using S-dATP mutations were identified by their sensitivity to MMS and by (Amersham Corp.) and the primers 59-CCATTCTAATGTCTGCCCC-39 their inability to grow at the restrictive temperature of 14 °C. and 59-GTTTTCCCAGTCACGAC-39. To determine the size of the alter- We screened these mutations for their effects on spontaneous ations, the products were compared with labeled PCR products of pre- mutability at the CAN1 locus and found that one of these, determined length on 6% polyacrylamide denaturing gels. Rates of Spontaneous Forward Mutation—For each strain, 19 inde- pol30–104, caused a dramatic increase in mutability at the pendent cultures were grown at 30 °C in 100 or 500 ml of YPD, each permissive temperature (30 °C), whereas the others had little starting from ;10 canavanine-sensitive cells. Cells were then plated or no effect. UV sensitivity is not affected by pol30–104. The onto arginine-deficient medium containing canavanine. Rates of spon- sequence of the entire coding region of the pol30–104 allele was taneous forward mutation at the CAN1 locus were determined from determined. The pol30–104 mutation isaCtoT transition at the number of canavanine-resistant colonies by the method of the nucleotide 752 of the coding sequence, which results in an median (13). PCNA Affi-Gel 15 Beads—Yeast PCNA was purified to homogeneity alanine to valine change at residue 251 of the PCNA protein. Role of PCNA in Mismatch Repair 27989 TABLE III Effect of the pol30–104 mutation on the rate of spontaneous forward mutation to canavanine resistance (can1 ) Strains were identical to those in Table I, except that they did not carry plasmid pSH91. Rate of forward Rate relative Strain Genotype mutation to can1 to wild type (6 S.D.) MS71 Wild type 3.8 (6 1.0) 3 10 1 YPCNA1.14 pol30–104 1.2 (6 0.2) 3 10 32 YRP23 mlh1D 9.6 (6 3.3) 3 10 25 YPCNA32 pol30–104 mlh1D 1.5 (6 0.5) 3 10 39 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 FIG.1. MSH2-MSH3 heterodimer but not MSH2 interacts with results in Table I show that tract instability in the pol30–104 PCNA. A, purified MSH2 protein (lane 1) was mixed with BSA Affi-Gel msh2D, pol30–104 mlh1D, and pol30–104 pms1D double mu- 15 and PCNA Affi-Gel 15 beads, which were treated with 2% SDS to tant strains was about the same as in the msh2D, mlh1D, and elute bound MSH2 protein. The supernatants containing unbound pms1D strains. These data are consistent with epistasis of MSH2 protein from the BSA Affi-Gel (lane 2) and the PCNA Affi-Gel (lane 3) and the SDS eluates containing MSH2 protein bound to the mutations in mismatch repair genes with pol30–104. Although BSA Affi-Gel (lane 4) and the PCNA Affi-Gel (lane 5) were subjected to the possibility of a role of PCNA in a parallel and independent immunoblot analysis with affinity purified anti-MSH2 antibodies (8). B, pathway cannot be excluded, these results suggest an involve- purified MSH2-MSH3 complex (lane 1) was mixed with BSA Affi-Gel 15 ment of PCNA in MSH2/MLH1/PMS1-dependent mismatch and PCNA Affi-Gel 15 beads, which were treated with 2% SDS to elute bound MSH2-MSH3 complex. The supernatants containing unbound repair. MSH2-MSH3 complex from the BSA Affi-Gel (lane 2) and the PCNA We determined the nature of tract alterations in the pol30– Affi-Gel (lane 3) and the SDS eluates containing MSH2-MSH3 complex 104 mutant by sequencing the tracts that had undergone bound to the BSA Affi-Gel (lane 4) and the PCNA Affi-Gel (lane 5) were changes in plasmid pSH91 (Table II). Additions or deletions of subjected to immunoblot analysis with affinity purified anti-MSH2 and anti-MSH3 antibodies (8). MSH3 protein migrates in SDS gels as a two base pairs represent the most common alterations in all the triplet. strains tested. In the wild type strain, most of the tract alter- ations are additions of 2 bp. In the msh2D and mlh1D mutants, there is about a 2-fold bias in favor of 2-bp deletions over 2-bp This residue is in a stretch of four amino acids that are con- additions, whereas in the pol30–104 mutant, 2-bp additions served in S. cerevisiae, Schizosaccharomyces pombe, Drosoph- and deletions occur about equally frequently (Table II). Thus, ila, mouse, and human PCNA. Because the pol30–104 muta- the pattern of tract alterations in pol30–104 resembles more tion differs from the other cold-sensitive and MMS-sensitive closely the pattern in the mismatch repair mutants than that pol30 mutations by increasing spontaneous mutability dramat- in the wild type. Because pol30–104 is a missense mutation, it ically, we examined the possibility that pol30–104 may confer is not surprising that it does not cause the same degree of tract a defect in mismatch repair. destabilization and bias as do null mutations in mismatch To examine the effect of the pol30–104 mutation on the repair genes. To verify that tract destabilization in the pol30– stability of simple DNA repeats, we used the centromeric plas- 104 mutant arose from inactivation of mismatch repair, we mid pSH91 that contains an in-frame 33-bp insertion of compared the pattern of tract alterations in the pol30–104 poly(GT) G in the coding sequence of a hybrid gene containing msh2D and pol30–104 mlh1D double mutant strains with that the yeast URA3 gene (12). The pSH91 repeat tract is in-frame in the msh2D and mlh1D single mutant strains. When our data with the URA3 gene, resulting in Ura cells. Alterations of the for the msh2D mutant are combined with those of Strand et al. tract that produce an out-of-frame mutation give rise to Ura (3) (Table II), then the numbers of 22 and 12 tracts in this cells that become resistant to 5-FOA. mutant are 73 and 29, respectively, compared with 32 and 16, Compared with wild type, the pol30–104 mutation caused an respectively, in the pol30–104 msh2D mutant (x for 1 degree of 80-fold increase in the rate of tract instability (Table I). Muta- freedom 5 0.70; p . 0.25). Thus, the pattern of tract alterations tions in the yeast mismatch repair genes MSH2, MLH1, and in these mutant strains is almost identical. The incidence of 22 PMS1 result in elevated rates of tract instability (12). To de- and 12 tracts was also the same in the pol30–104 mlh1D and termine whether the increased tract instability in the pol30– mlh1D mutants (Table II). 104 mutant could be due to a defect in mismatch repair, we We also examined the effect of the pol30–104 mutation on examined tract instability in the pol30–104 msh2D, pol30–104 spontaneous forward mutations of the CAN1 locus (Table III). mlh1D, and pol30–104 pms1D double mutant strains. If tract The pol30–104 mutation caused ;30-fold increase in the rate instability in the pol30–104 mutant had resulted from an in- of spontaneous mutability, which is similar to that observed in crease in slippage events during DNA replication rather than the mlh1D strain and the pol30–104 mlh1D strains (Table III). from a defect in mismatch repair, then the rate of tract insta- The results for can1 are consistent with the epistasis observed bility in these double mutant strains would have increased in a multiplicative fashion. In fact, a pms1 mutation, in combina- for microsatellite instability. To examine whether PCNA interacts physically with mis- tion with the pol3–01 mutation, which inactivates the 39 to 59 proof reading exonuclease function of DNA polymerase d, re- match repair proteins, we purified PCNA from E. coli cells sults in mutation rates that are the product of the relative rates expressing the protein and covalently coupled it to Affi-Gel 15 observed in the pms1 and pol3–01 single mutants (14). For beads for use as affinity matrix. The PCNA beads and control 27990 Role of PCNA in Mismatch Repair beads containing BSA were mixed with MSH2 protein and tive. The evidence implicating PCNA in mismatch repair would MSH2-MSH3 heterodimer that we had purified to near homo- suggest that Pold may be the polymerase involved in mismatch geneity from S. cerevisiae (8). After washing with a large vol- repair, because PCNA is an essential subunit of this DNA ume of 1 M KCl, the beads were incubated with 2% SDS to elute polymerase. Pole, however, could also be involved, because bound proteins followed by immunoblot analysis of the SDS PCNA also stimulates this DNA polymerase. One way by which eluates to determine the amount of MSH2 protein or of MSH2- PCNA might function in mismatch repair could be as follows. MSH3 heterodimer that was retained on the Affi-Gel beads in The mismatch repair proteins including MSH2-MSH3 or the each case. Interestingly, while MSH2 protein alone did not MSH2-MSH6 heterodimer and PMS1 and MLH1 could be tar- show any affinity for the PCNA beads (Fig. 1A), the majority geted to the mismatch via their affinity for the mismatch and (;80%) of the input MSH2-MSH3 heterodimer was retained on for one another, and PCNA, along with the DNA polymerase, the PCNA beads (Fig. 1B). This interaction between PCNA and could be loaded onto the nick remaining at the 59 side of the the MSH2-MSH3 complex is highly specific because (i) we mismatch prior to the removal of RNA primers and joining of observed no binding of MSH2-MSH3 heterodimer to the control nascent DNA fragments. Via DNA looping, perhaps catalyzed BSA Affi-Gel 15 beads (Fig. 1B) and (ii) the association of by the MSH proteins in a manner analogous to the MutS MSH2-MSH3 with the PCNA beads was stable to washing with catalyzed a-shaped loop structures in E. coli (17), the mismatch 1 M KCl, as indicated by the absence of the heterodimer in the bound proteins may become associated with the PCNA-DNA 1 M KCl wash (data not shown). polymerase complex, and these interactions could be important for the subsequent excision and repair synthesis reactions. The DISCUSSION pol30–104 mutation might impair any of these interactions. We show here that the pol30–104 mutation in PCNA causes The involvement of PCNA in mismatch repair suggests the an increase in the rate of instability of (GT) repeat sequences possibility that mutations in human PCNA that inactivate only and in the rate of forward mutations at the CAN1 locus. The its mismatch repair function may contribute to sporadic colo- effect of pol30–104 on tract alterations, however, is not quite as rectal cancers (18) and to other cancers, including those of the severe as that of null mutations in mismatch repair genes. This stomach, lung, breast, and pancreas, that are associated with is likely to be due to the fact that pol30–104 is a missense microsatellite instability (19–22). mutation and not a null mutation. Null mutations in POL30 Acknowledgment—We thank P. Burgers for plasmid pBL228. are lethal. 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Published: Nov 1, 1996

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