Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Redundancy of Saccharomyces cerevisiae MSH3 and MSH6 in MSH2-dependent mismatch repair Gerald T. Marsischky, Nicole Filosi, Michael F. Kane, and Richard Kolodner I Charles A. Dana Division of Human Cancer Genetics, Dana-Farber Cancer Institute, Boston, Massachusetts 09.115 USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 USA Saccharomyces cerevisiae encodes six genes, MSH1-6, which encode proteins related to the bacterial MutS protein. In this study the role of MSH2, MSH3, and MSH6 in mismatch repair has been examined by measuring the rate of accumulating mutations and mutation spectrum in strains containing different combinations of rash2, rash3, and rash6 mutations and by studying the physical interaction between the MSH2 protein and the MSH3 and MSH6 proteins. The results indicate that S. cerevisiae has two pathways of MSH2-dependent mismatch repair: one that recognizes single-base mispairs and requires MSH2 and MSH6, and a second that recognizes insertion/deletion mispairs and requires a combination of either MSH2 and MSH6 or MSH2 and MSH3. The redundancy of MSH3 and MSH6 explains the greater prevalence of brash2 mutations in HNPCC families and suggests how the role of brash3 and hmsh6 mutations in cancer susceptibility could be analyzed. [Key Words: Cancer; mutagenesis; mismatch repair; routS; MSH2; MSH3; MSH6; Saccharomyces cerevisiae] Received October 17, 1995; revised version accepted January 17, 1996. DNA mismatch repair plays a number of roles in the cell al. 1993; Umar et al. 1994; Boyer et al. 1995; Liu et al. including the repair of mispaired bases produced as a 1995) have underscored the importance of understanding result of DNA replication errors, chemical damage to mismatch repair in detail. DNA and DNA precursors, processing of recombination The yeast Saccharomyces cerevisiae provides an ideal intermediates, and suppression or regulation of recombi- system for use in understanding mismatch repair be- nation between divergent DNA sequences (for review, cause of the availability of both genetic and biochemical see Modrich 1991, 1994; Kolodner 1995). DNA mis- methods for analyzing mismatch repair and the com- match repair is best understood in bacterial systems; plete S. cerevisiae genome sequence, soon to be avail- however a series of genetic and biochemical studies has able. Analysis of S. cerevisiae has led to the understand- shown that eukaryotes contain a mismatch repair sys- ing of at least three components of a bacterial MutHLS- tem that is similar to the bacterial MutHLS system in- like mismatch repair system. MSH2 is highly related to dicating evolutionary conservation: bf at least some of the bacterial MutS family of proteins, and as predicted by the components of mismatch repair (Bishop et al. 1987, this homology, MSH2 protein can bind to mispaired 1989; W. Kramer et al. 1989; Reenan and Kolodner bases, albeit with a higher affinity for insertion/deletion 1992a,b; Fishel et al. 1993; Leach et al. 1993; Bronner et mispair than for single-base mispairs (Reenan and Kolod- al. 1994; Nicolaides et al. 1994; Papadopoulos et al. net 1992a,b; Alani et al. 1995). PMS1 and MLHI are each 1994; Li and Modrich 1995). The recent observations homologs of MutL, and these two proteins form a com- that inherited mutations in mismatch repair genes cause plex that can bind to MSH2 when MSH2 is bound to a a common human cancer susceptibility syndrome mispaired base, similar to the interaction between Esch- (Fishel et al. 1993; Leach et al. 1993; Bronner et al. 1994; erichia coli MutL and MutS (Grilley et al. 1989; Kramer Kolodner et al. 1994, 1995; Liu et al. 1994; Nicolaides et et al. 1989; Prolla et al. 1994a, b). Human cells contain al. 1994; Papadopoulos et al. 1994], that acquired mis- homologs of the S. cerevisiae MSH2 (hMSH2), PMS1 match repair defective mutations occur in sporadic colon [hPMS2}, and MLH1 (hMLHI) proteins, and these pro- tumors (Borresen et al. 1995}, and that many human tu- teins appear to play roles that are similar to those of their mor cell lines are mismatch repair defective (Parsons et S. cerevisiae counterparts (Fishel et al. 1993; Leach et al. 1993; Bronner et al. 1994; Fishel et al. 1994; Nicolaides et al. 1994; Papadopoulos et al. 1994; Li and Modrich 1995). There is also evidence that the human mispair 1Correspondingauthor. recognition complex contains a second subunit in addi- GENES & DEVELOPMENT 10:407-420 © 1996 by Cold Spring Harbor LaboratoryPress ISSN 0890-9369/96 $5.00 407 Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Marsischky et al. tion to MSH2, GTBP/pl60, which is also a MutS ho- showing homology to the most conserved region of molog IDrummond et al. 1995; Palombo et al. 1995). MSH2 (Reenan and Kolodner 1992b; Fishel et al. 1993). S. cerevisiae also contains a number of other MSH One new gene encoding such a protein was identified genes whose function is generally less understood. and called MSH6. This gene was predicted to encode a MSH1 encodes a mispair binding protein that is im- 139,992 molecular weight protein, showed homology ported into mitochondria and appears to function in mi- along its entire length with E. coli MutS and S. cerevisiae tochondrial mismatch repair {Reenan and Kolodner MSH2 {Fig. 1AI, and had an amino acid identity of 18.5% 1992a, b; Chi and Kolodner 1994). Mutations in MSH3 and 18.1% with these two proteins, respectively. confer a weak nuclear mutator phenotype in some mu- After the studies described here had been partially tator assays {New et al. 1993; Alani et al. 1994; Strand et completed, we became aware of the identification of the al. 1995). However, the magnitude of these effects is sub- human GTBP gene by J. Jiricny (Instituto Di Richerche stantially less than those caused by mutations in MSH2, Di Biologia Moleculare P. Angeletti, Rome, Italy) and PMS1, or MLH1, suggesting that MSH3 may play a rel- collaborators. Regions of protein sequence identity be- atively minor role in mismatch repair (Williamson et al. tween S. cerevisiae and mouse MSH6 (G. Crouse and R. 1985; Reenan and Kolodner 1992a; New et al. 1993; Kolodner, unpubl.) were provided to J. Jincny, who found Alani et al. 1994; Prolla et al. 1994a; Strand et al. 1995). that they exactly matched regions of the human GTBP MSH4 and MSH5 do not appear to function in mismatch amino acid sequence. When larger amounts of the hu- repair but rather have a role in meiotic recombination man GTBP amino acid sequence became available iPa- (Ross-Macdonald and Roeder 1994; Hollingsworth et al. lombo et al. 1995), sequence alignments (Fig. 1B) dem- 1995). In this study we describe a sixth MSH gene, onstrated that S. cerevisiae MSH6 and human GTBP MSH6, and present results that demonstrate that MSH2, were related more closely to each other than to any other MSH3, and MSH6 all play important roles in mismatch MutS homolog 126.6% amino acid identity). repair. MSH6 is in volved in DNA repair Results To determine whether MSH6 is involved in DNA repair, MSH6 was disrupted in a diploid strain, which was then identification of MSH sporulated and analyzed by tetrad analysis. In all cases To identify additional MSH genes, the S. cerevisiae Ge- (27 spore clones analyzed)rash6 mutations cosegregated home Database was searched for protein sequences with a mutator phenotype as assessed in patch tests that A B MSH2 Human i i iiiii iii i iii i i iii iiiiii ii ii _,_2 ~ MSH2 Mouse 4 ................................................................................................... MUT~ MSH2 XenopuS 7 ~ MSH2 SPELl Drosophila I01 HSQE ~ SDTMI~SNTTEP KSTTT DEDL SSS Q SRRN}n~RVNYAE SDDDDS DTTPTAER/(~GKV%~SE3DEDE MLp D KNDG DEDD D rADDKED iK MSH6 ' MSH2 S. cerevis iae 4 ................................................................. II~tF I~I-IT ........................ I,IK~S MSH5 S, cerevisiae MUTS E.coli 201 ~DDD~LISLAETTSK~KFSYNTs~SS$PFTRNISRIA~qSK~SRP~AI~SRSYNPSHSQpSATSK~~WL~DERDAQF`RP~D~EYDPRT MSH6 MUTS S. typhimurium MUTS H. inf luenzae ~ MUTS A. vinlandii HEXA S. pne~moniae ~ ,~,,-,~N~t~,~v~i~ ..... - ............. ~i,~-----¢,~ ...... - ...... ~,~,~,-~ss~,~ ,~ MSHI S. cerevisiae ~ GTBP Human MSH6 Mouse ! MSH6 S. cerevisiae MSH3 Human REP3 Mouse SWI4 S .pombe MSH3 S. cerevislae MSH4 S. cerevisiae Figure 1. Sequence analysis of the S. cerevi- 760 ~T NAY~KAI~I I~ ..... ~I~RI~E~y~F .... ¢S N IQYKDS G~I ~E ~I SAT~V~ -WVQMAAN~ M~H6 siae MSH6 gene product. [A} Alignment of the amino acid sequences of E. coli MutS and S. cerevisiae MSH2 and MSH6. The amino acid identities with E. coli MutS are indicated by 658 S- - ~DP vm~a~-~Q MSH2 shaded boxes. {B] Phylogenetic tree of MutS- related proteins. The first 21 amino acids of 682 ......... DALEH~DT x ~TS 756 IEKNLKEQKHDOED M~2 the S. cerevisiae MSH1 sequence, encoding 1050 ~m~U~SJ~X~~S~~$SnUa4~Q~PU~S~LVO~AT~d~ ....... ~e~ MS~6 the mitochondrial targeting sequence, were not included in the analysis. All sequences were retrieved from GenBank except for the 853 MUTS mouse MSH6 sequence (G. Crouse and R. 955 YLEZ~K ~ MSH2 I~36 ZI~DL~ M~q6 Kolodner, unpubl.). 408 GENES & DEVELOPMENT Downloaded from genesdev.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press DNA mispair recognition detected the production of canavaninc-resistant mutants caused increases in the rate of accumulation of Can r mu- (Cant). To further analyze this mutator phenotype, a se- tations and reversion of hom3-IO and lys2-Bgl by 40-, ries of isogenic strains was constructed containing the 662- and 55-fold, respectively; compared with the wild- horn3-10 and Iys2-BgI alleles l+ 1 and +4 base frame- type control strain.