Different frameshift mutation spectra in non-repetitive DNA of MutSalpha- and MutLalpha-deficient fission yeast cells

A frameshift reversion assay has been established for Schizosaccharomyces pombe, which allows detection of deletions and insertions of nucleotides in a non-repetitive DNA sequence. Compared to wild type, frameshift mutation rates were increased in the mismatch repair (MMR) mutants msh2, msh6, mlh1, and pms1, but not in a swi4 strain (defective in the Msh3 homologue). Rates were also elevated in the DNA nuclease-deficient strains rad2 (defective in the FEN-1 homologue) and exo1. In MutSalpha-deficient strains, msh2 and msh6, most of the reversions were 1bp deletions. In contrast, mlh1 and pms1 mutants, defective in MutLalpha, accumulated significantly more 2bp insertions, preferentially of the type CG to (CG)(2). Such duplications were less frequent in double mutants additionally defective in msh2, msh6, rad2, or exo1. Thus, accumulation of (CG)(2) in MutLalpha-deficient strains depends on the presence of MutSalpha, Rad2 and Exo1.     

Spontaneous frameshift mutations in Saccharomyces cerevisiae: accumulation during DNA replication and removal by proofreading and mismatch repair activities

The msh6 mismatch repair gene of Schizosaccharomyces pombe was cloned, sequenced, and inactivated. Strains bearing all combinations of inactivated msh6, msh2, and swi4 (the S. pombe MSH3 ortholog) alleles were tested for their defects in mitotic and meiotic mismatch repair. Mitotic mutation rates were similarly increased in msh6 and msh2 mutants, both for reversion of a base-base substitution as well as of an insertion of one nucleotide in a mononucleotide run. Tetrad analysis and intragenic two-factor crosses revealed that meiotic mismatch repair was affected in msh6 to the same extent as in msh2 background. In contrast, loss of Swi4 likely did not cause a defect in mismatch repair, but rather resulted in reduced recombination frequency. Consistently, a mutated swi4 caused a two- to threefold reduction of recombinants in intergenic crosses, while msh2 and msh6 mutants were not significantly different from wild type. In summary, our study showed that Msh6 plays the same important role as Msh2 in the major mismatch repair pathway of S. pombe, while Swi4 rather functions in recombination.     

Multiple factors insulate Msh2-Msh6 mismatch repair activity from defects in Msh2 domain I

DNA mismatch repair (MMR) is a highly conserved mutation avoidance mechanism that corrects DNA polymerase misincorporation errors. In initial steps in MMR, Msh2-Msh6 binds mispairs and small insertion/deletion loops, and Msh2-Msh3 binds larger insertion/deletion loops. The msh2Δ1 mutation, which deletes the conserved DNA-binding domain I of Msh2, does not dramatically affect Msh2-Msh6-dependent repair. In contrast, msh2Δ1 mutants show strong defects in Msh2-Msh3 functions. Interestingly, several mutations identified in patients with hereditary non-polyposis colorectal cancer map to domain I of Msh2; none have been found in MSH3. To understand the role of Msh2 domain I in MMR, we examined the consequences of combining the msh2Δ1 mutation with mutations in two distinct regions of MSH6 and those that increase cellular mutational load (pol3-01 and rad27). These experiments reveal msh2Δ1-specific phenotypes in Msh2-Msh6 repair, with significant effects on mutation rates. In vitro assays demonstrate that msh2Δ1-Msh6 DNA binding is less specific for DNA mismatches and produces an altered footprint on a mismatch DNA substrate. Together, these results provide evidence that, in vivo, multiple factors insulate MMR from defects in domain I of Msh2 and provide insights into how mutations in Msh2 domain I may cause hereditary non-polyposis colorectal cancer.     

Stabilization of Microsatellite Sequences by Variant Repeats in the Yeast Saccharomyces Cerevisiae

We examined the effect of a single variant repeat on the stability of a 51-base pair (bp) microsatellite (poly GT). We found that the insertion stabilizes the microsatellite about fivefold in wild-type strains. The stabilizing effect of the variant base was also observed in strains with mutations in the DNA mismatch repair genes pms1, msh2 and msh3, indicating that this effect does not require a functional DNA mismatch repair system. Most of the microsatellite alterations in the pms1, msh2 and msh3 strains were additions or deletions of single GT repeats, but about half of the alterations in the wild-type and msh6 strains were large (>8 bp) deletions or additions.     

Expansions and contractions in 36-bp minisatellites by gene conversion in yeast

The instability of simple tandem repeats, such as human minisatellite loci, has been suggested to arise by gene conversions. In Saccharomyces cerevisiae, a double-strand break (DSB) was created by the HO endonuclease so that DNA polymerases associated with gap repair must traverse an artificial minisatellite of perfect 36-bp repeats or a yeast Y' minisatellite containing diverged 36-bp repeats. Gene conversions are frequently accompanied by changes in repeat number when the template contains perfect repeats. When the ends of the DSB have nonhomologous tails of 47 and 70 nucleotides that must be removed before repair DNA synthesis can begin, 16% of gene conversions had rearrangements, most of which were contractions, almost always in the recipient locus. When efficient removal of nonhomologous tails was prevented in rad1 and msh2 strains, repair was reduced 10-fold, but among survivors there was a 10-fold reduction in contractions. Half the remaining events were expansions. A similar decrease in the contraction rate was observed when the template was modified so that DSB ends were homologous to the template; and here, too, half of the remaining rearrangements were expansions. In this case, efficient repair does not require RAD1 and MSH2, consistent with our previous observations. In addition, without nonhomologous DSB ends, msh2 and rad1 mutations did not affect the frequency or the distribution of rearrangements. We conclude that the presence of nonhomologous ends alters the mechanism of DSB repair, likely through early recruitment of repair proteins including Msh2p and Rad1p, resulting in more frequent contractions of repeated sequences.     

Saccharomyces cerevisiae pol30 (Proliferating Cell Nuclear Antigen) Mutations Impair Replication Fidelity and Mismatch Repair

To understand the role of POL30 in mutation suppression, 11 Saccharomyces cerevisiae pol30 mutator mutants were characterized. These mutants were grouped based on their mutagenic defects. Many pol30 mutants harbor multiple mutagenic defects and were placed in more than one group. Group A mutations (pol30-52, -104, -108, and -126) caused defects in mismatch repair (MMR). These mutants exhibited mutation rates and spectra reminiscent of MMR-defective mutants and were defective in an in vivo MMR assay. The mutation rates of group A mutants were enhanced by a msh2 or a msh6 mutation, indicating that MMR deficiency is not the only mutagenic defect present. Group B mutants (pol30-45, -103, -105, -126, and -114) exhibited increased accumulation of either deletions alone or a combination of deletions and duplications (4 to 60 bp). All deletion and duplication breakpoints were flanked by 3 to 7 bp of imperfect direct repeats. Genetic analysis of one representative group B mutant, pol30-126, suggested polymerase slippage as the likely mutagenic mechanism. Group C mutants (pol30-100, -103, -105, -108, and -114) accumulated base substitutions and exhibited synergistic increases in mutation rate when combined with msh6 mutations, suggesting increased DNA polymerase misincorporation as a mutagenic defect. The synthetic lethality between a group A mutant, pol30-104, and rad52 was almost completely suppressed by the inactivation of MSH2. Moreover, pol30-104 caused a hyperrecombination phenotype that was partially suppressed by a msh2 mutation. These results suggest that pol30-104 strains accumulate DNA breaks in a MSH2-dependent manner.     

EXO1 and MSH6 are high-copy suppressors of conditional mutations in the MSH2 mismatch repair gene of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, Msh2p, a central component in mismatch repair, forms a heterodimer with Msh3p to repair small insertion/deletion mismatches and with Msh6p to repair base pair mismatches and single-nucleotide insertion/deletion mismatches. In haploids, a msh2Delta mutation is synthetically lethal with pol3-01, a mutation in the Poldelta proofreading exonuclease. Six conditional alleles of msh2 were identified as those that conferred viability in pol3-01 strains at 26 degrees but not at 35 degrees. DNA sequencing revealed that mutations in several of the msh2(ts) alleles are located in regions with previously unidentified functions. The conditional inviability of two mutants, msh2-L560S pol3-01 and msh2-L910P pol3-01, was suppressed by overexpression of EXO1 and MSH6, respectively. Partial suppression was also observed for the temperature-sensitive mutator phenotype exhibited by msh2-L560S and msh2-L910P strains in the lys2-Bgl reversion assay. High-copy plasmids bearing mutations in the conserved EXO1 nuclease domain were unable to suppress msh2-L560S pol3-01 conditional lethality. These results, in combination with a genetic analysis of msh6Delta pol3-01 and msh3Delta pol3-01 strains, suggest that the activity of the Msh2p-Msh6p heterodimer is important for viability in the presence of the pol3-01 mutation and that Exo1p plays a catalytic role in Msh2p-mediated mismatch repair.     

Role of the DNA repair nucleases Rad13, Rad2 and Uve1 of Schizosaccharomyces pombe in mismatch correction.

Repair of mismatched DNA occurs mainly by the long-patch mismatch repair (MMR) pathway, requiring Msh2 and Pms1. In Schizosaccharomyces pombe mismatches can be repaired by a short-patch repair system, containing nucleotide excision repair (NER) factors. We studied mismatch correction efficiency in cells with inactivated DNA repair nucleases Rad13, Rad2 or Uve1 in MMR proficient and deficient background. Rad13 incises 3' of damaged DNA during NER. Rad2 has a function in the Uve1-dependent repair of DNA damages and in replication. Loss of Rad13 caused a strong reduction of short-patch processing of mismatches formed during meiotic recombination. Mitotic mutation rates were increased, but not to the same extent as in the NER mutant swi10, which is defective in 5' incision. The difference might be caused by an additional role of Rad13 in base excision repair or due to partial redundancy with other 3' endonucleases. Meiotic mismatch repair was not or only slightly affected in rad2 and uve1 mutants. In addition, inactivation of uve1 caused only weak effects on mutation avoidance. Mutation rates were elevated when rad2 was mutated, but not further increased in swi10 rad2 and rad13 rad2 double mutants, indicating an epistatic relationship. However, the mutation spectra of rad2 were different from that of swi10 and rad13. Thus, the function of Rad2 in mutation avoidance is rather independent of NER. rad13, swi10 and rad2, but not uve1 mutants were sensitive to the DNA-damaging agent methyl methane sulphonate. Cell survival was further reduced in the double mutants swi10 rad2, rad13 rad2 and, surprisingly, swi10 rad13. These data confirm that NER and Rad2 act in distinct damage repair pathways and further indicate that the function of Rad13 in repair of alkylated bases is partially independent of NER.     

Sequence composition and context effects on the generation and repair of frameshift intermediates in mononucleotide runs in Saccharomyces cerevisiae

DNA polymerase slippage occurs frequently in tracts of a tandemly repeated nucleotide, and such slippage events can be genetically detected as frameshift mutations. In long mononucleotide runs, most frameshift intermediates are repaired by the postreplicative mismatch repair (MMR) machinery, rather than by the exonucleolytic proofreading activity of DNA polymerase. Although mononucleotide runs are hotspots for polymerase slippage events, it is not known whether the composition of a run and the surrounding context affect the frequency of slippage or the efficiency of MMR. To address these issues, 10-nucleotide (10N) runs were inserted into the yeast LYS2 gene to create +1 frameshift alleles. Slippage events within these runs were detected as Lys(+) revertants. 10G or 10C runs were found to be more unstable than 10A or 10T runs, but neither the frequency of polymerase slippage nor the overall efficiency of MMR was greatly influenced by sequence context. Although complete elimination of MMR activity (msh2 mutants) affected all runs similarly, analyses of reversion rates in msh3 and msh6 mutants revealed distinct specificities of the yeast Msh2p-Msh3p and Msh2p-Msh6p mismatch binding complexes in the repair of frameshift intermediates in different sequence contexts.     

Msh2 separation of function mutations confer defects in the initiation steps of mismatch repair

In eukaryotes the MSH2-MSH3 and MSH2-MSH6 heterodimers initiate mismatch repair (MMR) by recognizing and binding to DNA mismatches. The MLH1-PMS1 heterodimer then interacts with the MSH proteins at or near the mismatch site and is thought to act as a mediator to recruit downstream repair proteins. Here we analyzed five msh2 mutants that are functional in removing 3' non-homologous tails during double-strand break repair but are completely defective in MMR. Because non-homologous tail removal does not require MSH6, MLH1, or PMS1 functions, a characterization of the msh2 separation of function alleles should provide insights into early steps in MMR. Using the Taq MutS crystal structure as a model, three of the msh2 mutations, msh2-S561P, msh2-K564E, msh2-G566D, were found to map to a domain in MutS involved in stabilizing mismatch binding. Gel mobility shift and DNase I footprinting assays showed that two of these mutations conferred strong defects on MSH2-MSH6 mismatch binding. The other two mutations, msh2-S656P and msh2-R730W, mapped to the ATPase domain. DNase I footprinting, ATP hydrolysis, ATP binding, and MLH1-PMS1 interaction assays indicated that the msh2-S656P mutation caused defects in ATP-dependent dissociation of MSH2-MSH6 from mismatch DNA and in interactions between MSH2-MSH6 and MLH1-PMS1. In contrast, the msh2-R730W mutation disrupted MSH2-MSH6 ATPase activity but did not strongly affect ATP binding or interactions with MLH1-PMS1. These results support a model in which MMR can be dissected into discrete steps: stable mismatch binding and sensing, MLH1-PMS1 recruitment, and recycling of MMR components.     

ATP binding and hydrolysis by Saccharomyces cerevisiae Msh2–Msh3 are differentially modulated by mismatch and double-strand break repair DNA substrates

In Saccharomyces cerevisiae, Msh2–Msh3-mediated mismatch repair (MMR) recognizes and targets insertion/deletion loops for repair. Msh2–Msh3 is also required for 3′ non-homologous tail removal (3′NHTR) in double-strand break repair. In both pathways, Msh2–Msh3 binds double-strand/single-strand junctions and initiates repair in an ATP-dependent manner. However, we recently demonstrated that the two pathways have distinct requirements with respect to Msh2–Msh3 activities. We identified a set of aromatic residues in the nucleotide binding pocket (FLY motif) of Msh3 that, when mutated, disrupted MMR, but left 3′NHTR largely intact. One of these mutations, msh3Y942A, was predicted to disrupt the nucleotide sandwich and allow altered positioning of ATP within the pocket. To develop a mechanistic understanding of the differential requirements for ATP binding and/or hydrolysis in the two pathways, we characterized Msh2–Msh3 and Msh2–msh3Y942A ATP binding and hydrolysis activities in the presence of MMR and 3′NHTR DNA substrates. We observed distinct, substrate-dependent ATP hydrolysis and nucleotide turnover by Msh2–Msh3, indicating that the MMR and 3′NHTR DNA substrates differentially modify the ATP binding/hydrolysis activities of Msh2–Msh3. Msh2–msh3Y942A retained the ability to bind DNA and ATP but exhibited altered ATP hydrolysis and nucleotide turnover. We propose that both ATP and structure-specific repair substrates cooperate to direct Msh2–Msh3-mediated repair and suggest an explanation for the msh3Y942A separation-of-function phenotype.     

Binding and repair of mismatched DNA mediated by Rhp14, the fission yeast homologue of human XPA.

Rhp14 of Schizosaccharomyces pombe is homologous to human XPA and Saccharomyces cerevisiae Rad14, which act in nucleotide excision repair of DNA damages induced by ultraviolet light and chemical agents. Cells with disrupted rhp14 were highly sensitive to ultraviolet light, and epistasis analysis with swi10 (nucleotide excision repair) and rad2 (Uve1-dependent ultraviolet light damage repair pathway) revealed that Rhp14 is an important component of nucleotide excision repair for ultraviolet light-induced damages. Moreover, defective rhp14 caused instability of a GT repeat, similar to swi10 and synergistically with msh2 and exo1. Recombinant Rhp14 with an N-terminal hexahistidine tag was purified from Escherichia coli. Complementation studies with a rhp14 mutant demonstrated that the tagged Rhp14 is functional in repair of ultraviolet radiation-induced damages and in mitotic mutation avoidance. In bandshift assays, Rhp14 showed a preference to substrates with mismatched and unpaired nucleotides. Similarly, XPA bound more efficiently to C/C, A/C, and T/C mismatches than to homoduplex DNA. Our data show that mismatches and loops in DNA are substrates of nucleotide excision repair. Rhp14 is likely part of the recognition complex but alone is not sufficient for the high discrimination of nucleotide excision repair for modified DNA.     

Frameshift intermediates in homopolymer runs are removed efficiently by yeast mismatch repair proteins.

A change in the number of base pairs within a coding sequence can result in a frameshift mutation, which almost invariably eliminates the function of the encoded protein. A frameshift reversion assay with Saccharomyces cerevisiae that can be used to examine the types of insertions and deletions that are generated during DNA replication, as well as the editing functions that remove such replication errors, has been developed. Reversion spectra have been obtained in a wild-type strain and in strains defective for defined components of the postreplicative mismatch repair system (msh2, msh3, msh6, msh3 msh6, pms1, and mih1 mutants). Comparison of the spectra reveals that yeast mismatch repair proteins preferentially remove frameshift intermediates that arise in homopolymer tracts and indicates that some of the proteins have distinct substrate or context specificities.     

Distinct roles for the Saccharomyces cerevisiae mismatch repair proteins in heteroduplex rejection, mismatch repair and nonhomologous tail removal

The Saccharomyces cerevisiae mismatch repair (MMR) protein MSH6 and the SGS1 helicase were recently shown to play similarly important roles in preventing recombination between divergent DNA sequences in a single-strand annealing (SSA) assay. In contrast, MMR factors such as Mlh1p, Pms1p, and Exo1p were shown to not be required or to play only minimal roles. In this study we tested mutations that disrupt Sgs1p helicase activity, Msh2p-Msh6p mismatch recognition, and ATP binding and hydrolysis activities for their effect on preventing recombination between divergent DNA sequences (heteroduplex rejection) during SSA. The results support a model in which the Msh proteins act with Sgs1p to unwind DNA recombination intermediates containing mismatches. Importantly, msh2 mutants that displayed separation-of-function phenotypes with respect to nonhomologous tail removal during SSA and heteroduplex rejection were characterized. These studies suggest that nonhomologous tail removal is a separate function of Msh proteins that is likely to involve a distinct DNA binding activity. The involvement of Sgs1p in heteroduplex rejection but not nonhomologous tail removal further illustrates that subsets of MMR proteins collaborate with factors in different DNA repair pathways to maintain genome stability.     

CTG repeat instability and size variation timing in DNA repair-deficient mice

Type 1 myotonic dystrophy is caused by the expansion of an unstable CTG repeat in the DMPK gene. We have investigated the molecular mechanisms underlying the CTG repeat instability by crossing transgenic mice carrying >300 unstable CTG repeats in their human chromatin environment with mice knockout for genes involved in various DNA repair pathways: Msh2 (mismatch repair), Rad52 and Rad54 (homologous recombination) and DNA-PKcs (non-homologous end-joining). Genes of the non-homologous end-joining and homologous recombination pathways did not seem to affect repeat instability. Only lack of Rad52 led to a slight decrease in expansion range. Unexpectedly, the absence of Msh2 did not result in stabilization of the CTG repeats in our model. Instead, it shifted the instability towards contractions rather than expansions, both in tissues and through generations. Furthermore, we carefully analyzed repeat transmissions with different Msh2 genotypes to determine the timing of intergenerational instability. We found that instability over generations depends not only on parental germinal instability, but also on a second event taking place after fertilization.     

Separation-of-Function Mutations in Saccharomyces cerevisiae MSH2 That Confer Mismatch Repair Defects but Do Not Affect Nonhomologous-Tail Removal during Recombination

Yeast Msh2p forms complexes with Msh3p and Msh6p to repair DNA mispairs that arise during DNA replication. In addition to their role in mismatch repair (MMR), the MSH2 and MSH3 gene products are required to remove 3' nonhomologous DNA tails during genetic recombination. The mismatch repair genes MSH6, MLH1, and PMS1, whose products interact with Msh2p, are not required in this process. We have identified mutations in MSH2 that do not disrupt genetic recombination but confer a strong defect in mismatch repair. Twenty-four msh2 mutations that conferred a dominant negative phenotype for mismatch repair were isolated. A subset of these mutations mapped to residues in Msh2p that were analogous to mutations identified in human nonpolyposis colorectal cancer msh2 kindreds. Approximately half of the these MMR-defective mutations retained wild-type or nearly wild-type activity for the removal of nonhomologous DNA tails during genetic recombination. The identification of mutations in MSH2 that disrupt mismatch repair without affecting recombination provides a first step in dissecting the Msh-effector protein complexes that are thought to play different roles during DNA repair and genetic recombination.     

Microsatellite instability in yeast: dependence on repeat unit size and DNA mismatch repair genes.

We examined the stability of microsatellites of different repeat unit lengths in Saccharomyces cerevisiae strains deficient in DNA mismatch repair. The msh2 and msh3 mutations destabilized microsatellites with repeat units of 1, 2, 4, 5, and 8 bp; a poly(G) tract of 18 bp was destabilized several thousand-fold by the msh2 mutation and about 100-fold by msh3. The msh6 mutations destabilized microsatellites with repeat units of 1 and 2 bp but had no effect on microsatellites with larger repeats. These results argue that coding sequences containing repetitive DNA tracts will be preferred target sites for mutations in human tumors with mismatch repair defects. We find that the DNA mismatch repair genes destabilize microsatellites with repeat units from 1 to 13 bp but have no effect on the stability of minisatellites with repeat units of 16 or 20 bp. Our data also suggest that displaced loops on the nascent strand, resulting from DNA polymerase slippage, are repaired differently than loops on the template strand.     

Isolation and characterization of new proliferating cell nuclear antigen (POL30) mutator mutants that are defective in DNA mismatch repair

A number of studies have suggested a role for proliferating cell nuclear antigen (PCNA) in DNA mismatch repair (MMR). However, the majority of mutations in the POL30 gene encoding PCNA that cause MMR defects also cause replication and other repair defects that contribute to the increased mutation rate caused by these mutations. Here, 20 new pol30 mutants were identified and screened for MMR and other defects, resulting in the identification of two mutations, pol30-201 and pol30-204, that appear to cause MMR defects but little if any other defects. The pol30-204 mutation altered an amino acid (C81R) in the monomer-monomer interface region and resulted in a partial general MMR defect and a defect in MSH2-MSH6 binding in vitro. The pol30-201 mutation altered an amino acid (C22Y) located on the surface of the PCNA trimer that slides over the DNA but did not cause a defect in MSH2-MSH6 binding in vitro. The pol30-201 mutation caused an intermediate mutator phenotype. However, the pol30-201 mutation caused almost a complete defect in the repair of AC and GT mispairs and only a small defect in the repair of a "+T" insertion, an effect similar to that caused by an msh6Delta mutation, indicating that pol30-201 primarily effects MSH6-dependent MMR. The chromosomal double mutant msh3-FF>AA msh6-FF>AA eliminating the conserved FF residues of the PCNA interacting motif of these proteins caused a small (<10%) defect in MMR but showed synergistic interactions with mutations in POL30, indicating that the FF>AA substitution may not eliminate PCNA interactions in vivo. These results indicate that the interaction between PCNA and MMR proteins is more complex than was previously appreciated.     

Pms2 Suppresses Large Expansions of the (GAA·TTC)n Sequence in Neuronal Tissues

Expanded trinucleotide repeat sequences are the cause of several inherited neurodegenerative diseases. Disease pathogenesis is correlated with several features of somatic instability of these sequences, including further large expansions in postmitotic tissues. The presence of somatic expansions in postmitotic tissues is consistent with DNA repair being a major determinant of somatic instability. Indeed, proteins in the mismatch repair (MMR) pathway are required for instability of the expanded (CAG·CTG)_n sequence, likely via recognition of intrastrand hairpins by MutSβ. It is not clear if or how MMR would affect instability of disease-causing expanded trinucleotide repeat sequences that adopt secondary structures other than hairpins, such as the triplex/R-loop forming (GAA·TTC)_n sequence that causes Friedreich ataxia. We analyzed somatic instability in transgenic mice that carry an expanded (GAA·TTC)_n sequence in the context of the human FXN locus and lack the individual MMR proteins Msh2, Msh6 or Pms2. The absence of Msh2 or Msh6 resulted in a dramatic reduction in somatic mutations, indicating that mammalian MMR promotes instability of the (GAA·TTC)_n sequence via MutSα. The absence of Pms2 resulted in increased accumulation of large expansions in the nervous system (cerebellum, cerebrum, and dorsal root ganglia) but not in non-neuronal tissues (heart and kidney), without affecting the prevalence of contractions. Pms2 suppressed large expansions specifically in tissues showing MutSα-dependent somatic instability, suggesting that they may act on the same lesion or structure associated with the expanded (GAA·TTC)_n sequence. We conclude that Pms2 specifically suppresses large expansions of a pathogenic trinucleotide repeat sequence in neuronal tissues, possibly acting independently of the canonical MMR pathway.     

Saccharomyces cerevisiae Msh2-Msh3 acts in repair of base-base mispairs

DNA mismatch repair is thought to act through two subpathways involving the recognition of base-base and insertion/deletion mispairs by the Msh2-Msh6 heterodimer and the recognition of insertion/deletion mispairs by the Msh2-Msh3 heterodimer. Here, through genetic and biochemical approaches, we describe a previously unidentified role of the Msh2-Msh3 heterodimer in the recognition of base-base mispairs and the suppression of homology-mediated duplication and deletion mutations. Saccharomyces cerevisiae msh3 mutants did not show an increase in the rate of base substitution mutations by the CAN1 forward mutation assay compared to the rate for the wild type but did show an altered spectrum of base substitution mutations, including an increased accumulation of base pair changes from GC to CG and from AT to TA; msh3 mutants also accumulated homology-mediated duplication and deletion mutations. The mutation spectrum of mlh3 mutants paralleled that of msh3 mutants, suggesting that the Mlh1-Mlh3 heterodimer may also play a role in the repair of base-base mispairs and in the suppression of homology-mediated duplication and deletion mutations. Mispair binding analysis with purified Msh2-Msh3 and DNA substrates derived from CAN1 sequences found to be mutated in vivo demonstrated that Msh2-Msh3 exhibited robust binding to specific base-base mispairs that was consistent with functional mispair binding.