MSHl and MSH2 Genes: Evidence for Separate Mitochondrial and Nuclear Functions

The MSHl and MSH2 genes of Saccharomyces cerevisiae are predicted to encode proteins that are homologous to the Escherichia coli MutS and Streptococcus pneumoniae HexA proteins and their homologs. Disruption of the MSHl gene caused a petite phenotype which was established rapidly. A functional MSHl gene present on a single-copy centromere plasmid was incapable of rescuing the established mshl petite phenotype. Analysis of mshl strains demonstrated that mutagenesis and largescale rearrangement of mitochondrial DNA had occurred. 4’,6-Diamidino-2-phenylindole (DAPI) staining of mshl yeast revealed an aberrant distribution of mtDNA. Haploid msh2 mutants displayed an increase of 85-fold in the rate of spontaneous mutation to canavanine resistance. Sporulation of homozygous msh2/msh2 diploids gave rise to a high level of lethality which was compounded during increased vegetative growth prior to sporulation. msh2 mutations also affected gene conversion of two HIS4 alleles. The his4x mutation, lying near the 5’ end of the gene, was converted with equal frequency in both wild-type and msh2 strains. However, many of the events in the msh2 background were post-meiotic segregation (PMS) events (46.4%) while none (<0.25%) of the aberrant segregations in wild type were PMS events. The his4b allele, lying 1.6 kb downstream of his4x, was converted at a 10-fold higher frequency in the msh2 background than in the corresponding wild-type strain. Like the his4x allele, his46 showed a high level of PMS (30%) in the msh2 background compared to the corresponding wild-type strain where no (<0.26%) PMS events were observed. These results indicate that MSHl plays a role in repair or stability of mtDNA and MSH2 plays a role in repair of 4-bp insertion/deletion mispairs in the nucleus. M ISMATCH repair has been detected in a wide variety of organisms from procaryotes to mammalian systems. Two sets of studies suggest that the components and mechanism of mismatch repair have been highly conserved throughout evolution. First, homologs of proteins encoded by the Escherichia coli m u t H L S , u v r D mismatch repair genes, whose products have been characterized in the greatest biochemical detail, have been identified in a broad range of organisms. Gram-negative bacteria, gram-positive bacteria, mouse and human homologs of the E. coli MutS protein have been identified (FUJII and SHIMADA 1989; HABER et al. 1988; LINTON et al. 1989; PRIEBE et al. 1988) and homologs of the mutL and u v r D genes are known in gram-negative bacteria, gram-positive bacteria and yeast (ABOUSSEKHRA et al. 1989; KRAMER et al . 1989b; PRUDHOMME et al. 1989). Secondly, mutations in some of the genes encoding these homologs have proven to affect mismatch repair in other organisms besides E. coli. Mutations of the hexA and hexB genes of Streptococcus pneumoniae (homologs of E. coli mutS and m u t L genes) drastically affect mismatch repair in this organism (LACKS 1970; TIRABY (;enetics 132 975-985 (December, 1992) and SICARD 1973; LACKS, DUNN and GREENBERG 1982; GASC, SICARD and CLAVERYS 1989). Mutations in the P M S l gene of yeast (the yeast mutL homolog) similarly affect mismatch repair (BISHOP et al. 1987; BISHOP, ANDERSON and KOLODNER 1989; WILLIAMSON, GAME and FOCEL 1985; KRAMER et al. 1989a), and several less well characterized mutations cause phenotypes that could possibly be explained by an effect on mismatch repair (WILLIAMSON, GAME and FOGEL 1985). The identification of a Saccharomyces cerevisiae homolog of mutL/hexB that functions in mismatch repair provided the rational for searching for mutS homologs and suggested that such homologs may function in mismatch repair. In a companion study (REENAN and KOLODNER 1992), we reported the cloning and sequence analysis of two yeast homologs of mutS/hexA, M S H l and M S H 2 . In this report, we describe the isolation of mutations in the M S H l and M S H 2 genes and a preliminary genetic analysis to determine the role that M S H l and M S H 2 play in repair. The results suggest that M S H l and M S H 2 are involved in mismatch repair in the mitochondrion and nucleus, respectively. R. A. G. Reenan and R. D. Kolodner 976 MATERIALS AND METHODS Enzymes and chemicals: Chemicals, enzymes and oligonucleotides are as described in a companion study (REENAN and KOLODNER 1992). Strains and media: The S. cerevisiae strains used in this study are derived from SK1 and were the gift of NANCY KLECKNER (Harvard University, Cambridge, Massachusetts). Methods for the construction and manipulation of these strains have been described elsewhere (TISHKOFF, JOHNSON and KOLODNER 199 1 ;CAO, ALANI and KLECKNER 1990). The two strain combinations NK859: MATu ho::LYS2 lys2 ura3 leu2::hisC his4x and NK860: MATa ho::LYS2 lys2 uru3 leu2::hisC his4b or NK858: MATa ho::LYS2 lys2 ura3 leu2::hisC his4x and NK861: MATa ho::LYS2 lys2 ura3 leu2::hisC his46 were crossed to construct the diploids used for all MSH gene disruptions. Haploid strains bearing the MSH gene insertion mutations in combination with a particular HIS4 allele were generated as needed from the disruption heterozygotes and used for phenotypic characterization or constructing diploids homozygous for the insertion mutations. This was done as a precaution, assuming the disruption mutants might be mutators. The his4b and his4x alleles used in these studies are four base insertion mutations (CAO, ALANI and KLECKNER 1990). Wild-type HIS4 alleles were generated from the above mentioned strains by selection on media lacking histidine. All strains described in this work are derived from these starting strains by transformation and are therefore isogenic. Canavanine plates lacked arginine and contained 30 pg/ml canavanine. The nonfermentable carbon source plates used here were both YPAcetate (YPAc) and YPGlycerol (YPgly) formulated as described by SHERMAN, FINK and HICKS (1986). Other yeast and E. coli media were as described in a companion study (REENAN and KOLODNER 1992). The E. coli strain RK1400 (SYMINGTON, FOCERTY and KOLODNER 1983) was used for all plasmid constructions. Strains used for transposon mutagenesis are described below. Plasmids: Plasmids were constructed using the materials and standard procedures outlined in a companion study (REENAN and KOLODNER 1992). The plasmid pNKl206 was obtained from NANCY KLECKNER (HUISMAN and KLECKNER 1987). The TnlOLLK construct was made as follows. Yep1 3 DNA (BROACH, STRATHERN and HICKS 1979) was digested with BglII and the 2.6-kb fragment harboring the LEU2 gene was isolated. This fragment was then inserted into the BamHI site located between the lacZ and RunR sequences of TnlOLK of pNK1206 to yield pTNlOLLK (Lac Leu Kan). The orientation of the BglII fragment in the BamHI site has not been determined. In order to transform yeast and replace the URA3 marker of the TnlOLUK insertion by recombination with TNIOLLK containing a LEU2 marker, pTn I O LLK was digested with BclI and NruI and the DNA used directly in LiCl transformation (ITO et al. 1983). BclI and NruI cleave pTNlOLLK at sites in the lacZ and kanR sequences, respectively. Transposon mutagenesis: Plasmids pI-A5 and pII-2 (REENAN and KOLODNER 1992) were transformed into NK5830/pNK629 (HUISMAN and KLECKNER 1987) selecting for ampicillin (PI-A5 and pII-2) and tetracycline (pNK629) resistance and then mutagenized with TnlOLUK by infection with phage X 1224 following a method similar to HUISMAN and KLECKNER (1987). The resulting pools of mutagenized plasmid DNA were used to transform NK80 17 (HUISMAN and KLECKNER 1987) and plasmid DNA was isolated from individual transformants (HOLMES and QuIGLEY 198 1). An individual mutant plasmid DNA was isolated from each pool to assure independence of insertions. Insertions into the desired fragments were then identified by restriction mapping. These insertion mutations were then introduced into their homologous location in the yeast genome using the one step transplacement method (ROTHSTEIN 1991). The allele number for each mutation obtained is listed in Figure 1. Polymerase chain reaction (PCR) techniques: Primers used in PCR were the sequencing primers as described in REENAN and KOLODNER (1992). For PCR analysis of the presence of insertion mutations, DNA was isolated from individual transformants and PCR was performed as described in REENAN and KOLODNER (1992) except that 5 pmol of each primer and 1 pg of yeast DNA were used as substrate in each 5O-pl reaction. The primers used are listed in the legend to Figure 2. Growth protocols for MSH2IMSH2 viability experiments: Minimal vegetative growth regimen: Two wild-type or msh2::TnlOLUK haploids were mated and single colonies (23 mm) were isolated on rich medium (YPD). These diploid colonies were used to inoculate 5 ml of presporulation medium (YPAc) at low cell density and growth was allowed to proceed to saturation. The culture was then washed with sporulation medium and then incubated for 24 hr in sporulation medium. Zero growth regimen: Haploid strains were patched onto rich medium (YPD) directly from frozen stocks and allowed to grow overnight. Haploids of opposite mating-type were suspended in liquid YPD, mixed and plated back onto a YPD plate. The mating was allowed to proceed for 4 hr on rich medium and then the mating mixture was transferred directly to sporulation medium, allowing no vegetative growth. Sporulation was allowed to proceed for 24 hr. Determination of mutation and recombination rates: Mutation rates were determined by a fluctuation test and two or three independent experiments were performed for each strain tested (LEA and COULSEN 1949). Strains to be tested were plated for single colonies at 30" on YPD plates. Eleven single colonies (>3 mm) were excised from the plate and resuspended in sterile water. Appropriate dilutions were then plated to determine the number of viable cells and canavanine resistant cells per culture and these data were analyzed by the method of LEA and COUJSEN (1 949). Using this method, ro = M(1.24 + In M) where ro is the median number of canavanine-resistant colony-forming units per culture among the 11 platings and M is the average number of canavanine-resistant mutations per culture. M was solved by interpolation and then used to calculate the mutation or recombination rate, r = M/N where N is the final average number of viable cells per plating. Meiotic recombination was measured by determining the frequency of His+ cells present before and after sporulation of individual cultures of cells. Strains were grown to an ODGoo f 0.5 in YPD and then washed with presporulation medium (YPAc) twice. These cells were resuspended at low density in YPAc (OD600 of 0.0025) and growth was continued until an ODfioo f 1 .O was reached. The cells were then washed twice in sporulation medium and resuspended in sporulation medium. These cells were the 0 time point and were sonically disrupted and plated on plates lacking histidine and minimal complete plates to determine the frequency of recombinants. The remaining cells were allowed to sporulate for 20 hr and analyzed as described above. The frequency of His+ cells before and after induction of meiosis is given.