Identification and characterization of Saccharomyces cerevisiae EXO1, a gene encoding an exonuclease that interacts with MSH2 (canceryhereditary nonpolyposis colorectal carcinomaymutagenesisymutS)

A two-hybrid screen was used to identify Saccharomyces cerevisiae genes encoding proteins that interact with MSH2. One gene was found to encode a homologue of Schizosaccharomyces pombe EXO1, a double-stranded DNA-specific 5*–3* exonuclease. S. cerevisiae EXO1 interacted with both S. cerevisiae and human MSH2 in two-hybrid and coimmunoprecipitation experiments. exo1 mutants showed a mutator phenotype, and epistasis analysis was consistent with EXO1 functioning in the MSH2-dependent mismatch repair pathway. exo1 mutations were lethal in combination with rad27 mutations, and overexpression of EXO1 suppressed both the temperature sensitive and mutator phenotypes of rad27 mutants. Genetic and biochemical studies have indicated eukaryotes contain a mismatch repair (MMR) pathway related to the bacterial MutHLS pathway (reviewed in refs. 1 and 2). However, recent evidence suggests that eukaryotic MMR is more complex. In Saccharomyces cerevisiae there are two MMR pathways that require MSH2, a MutS homologue that recognizes mispaired bases (3, 4). One is a single base substitution mispair pathway that requires a complex of MSH2 and the MutS homologue MSH6 (also called GTBP or p160 in humans) (1–3). There is also an insertionydeletion mispair pathway that requires either a complex of MSH2 and MSH6 or a complex of MSH2 and MSH3, a third MutS homologue (1–3). Additionally, four S. cerevisiae MutL homologues have been identified, PMS1 (PMS2 in humans) and MLH1–MLH3; PMS1 and MLH1 function in MMR and have been shown to form a heterodimer (1, 2). In vitro studies in Escherichia coli have shown that the excision step of MMR can occur either 59 to 39 or 39 to 59 of the initiating nick and requires the combination of a helicase (UvrD) and one of three single-stranded DNA exonucleases (Exo I, Exo VII or RecJ) (reviewed in ref. 2). In eukaryotic MMR, proteins involved in excising the mispair have not been identified, although some candidates have been suggested. These include S. cerevisiae RAD27 (RTH1, YKL510), a 59–39 exonuclease and flap endonuclease (5), and Schizosaccharomyces pombe EXO1 and its Drosophila homologue Tosca, which are members of the same family of endoand exonucleases as RAD27 (6, 7). The importance of determining the mechanism of MMR is underscored by its association with hereditary nonpolyposis colorectal carcinoma (HNPCC) (reviewed in refs. 2 and 8). HNPCC is associated primarily with germ-line mutations in two human MMR genes, MSH2 and MLH1, whereas mutations in other MMR genes are rare (ref. 9; reviewed in refs. 2 and 8). Somatic mutations in MMR genes have been found in some sporadic tumors, suggesting some sporadic cancers could be due to acquired mutations in MMR genes (reviewed in ref. 8, and see ref. 10). However, not all of HNPCC or sporadic cancers with mutator phenotypes can be accounted for by known MMR genes (9, 10). Consequently, there has been interest in identifying additional MMR genes. Here we describe the use of a two-hybrid screen to identify proteins that interact with MSH2 and function in MMR. MATERIALS AND METHODS Strains. Four series of isogenic strains were constructed by disrupting genes of interest using standard techniques. The first series of strains were in the MGD background (RKY2575-MATa, ade2, ura3-52, leu2-3, 112, trp1-289, his3D1, lys2-Bgl, hom3-10) with the following differences: RKY2537-MATa; RKY2587 and RKY2663-exo1D690::HIS3; RKY2558-msh2::hisG; RKY2588msh2::hisG, exo1D690::HIS3. The second series was in the S288c background (RKY2321 MATa, ura3-52, his3D200, leu2D1) and was RKY2662-exo1D690::HIS3. The third series was in the S288c background (RKY2672 MATa, ura3-52, his3D200, trp1D63, leu2D1, ade2D1, ade8, lys2-Bgl, hom3-10) and were RKY3044exo1D690::HIS3 and RKY2608-rad27::URA3. The fourth series was in the S288C background (RKY2664 MATa, ura3-52, his3D200, trp1D63 or RKY2666 MATa, ura3-52, his3D200, trp1D63) and were RKY3057-rad27::URA3 and RKY3056exo1D690::HIS3, respectively. The msh2 and rad27 mutations have been described (4, 11). General Genetic Techniques. Yeast extractypeptoney dextrose (YPD) media, sporulation media, synthetic drop-out (SD) media, 5-f luoroorotic acid, and canavanine media were as described (11). Mutation rates were calculated using the method of the median and at least five independent colonies as described (11). exo1D690::HIS3 deletion strains were constructed using an adaptation of a published method (12). A disruption construct was generated by amplifying the HIS3 gene present in pPS729 (from P. Silver, Dana–Farber Cancer Institute) by PCR using primers (HIS3 sequences are in lowercase) 22244 (59-A A AGGAGCTCGA A A A A ACTGA A AGGCGTAGAAAGGAATGGGTATCCAAGGTggcctcctctagtactc) and 21964 (59-CCTCCGATATGAAACGTGCAGTACTTAACTTTTATTTACCTTTATAAACAAATTGGGgcgcgcctcgttcagaatg) and Taq DNA polymerase. The resulting PCR product was used to transform RKY2321 (a S288c strain containing the hisD200 allele) resulting in strain RKY2662 in which the DNA sequence encoding amino acids 6–695 of the EXO1 open reading frame (ORF) was replaced with the HIS3 gene. This disruption was introduced into other strains by transformation with a DNA fragment containing the HIS3 gene bordered by The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y947487-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: SD medium, synthetic drop-out medium; MMR, mismatch repair; HA, hemagglutinin; y, Saccharomyces cerevisiae; h, human; Canr, canavanine resistant. ‡To whom reprint requests should be addressed.