Mutations in the MSH3 gene preferentially lead to deletions within tracts of simple repetitive DNA in Saccharomyces cerevisiae.

Abstract

Eukaryotic genomes contain tracts of DNA in which a single base or a small number of bases are repeated (microsatellites). Mutations (2,3,6), whereas the msh3 mutation has only a 2-fold effect on the mutation rate (5); reversion rates of a frameshift mutation are elevated about 1000-fold by MSH2, MLH1, or PMS1 and about 10-fold by MSH3. Homologues to the yeast MSH3 gene exist in mice (7) and humans (8).Alterations in the length of simple repetitive DNA tracts in yeast are likely to reflect DNA polymerase slippage events (9, 10). In wild-type strains, the displaced repeats resulting from DNA polymerase slippage (Fig. 1) are often corrected by excising the mismatched region from the newly synthesized strand and repairing the gap by using information derived from the template strand. Although repeats are displaced on only one strand in each slippage event, it should be noted that both strands reflect a distortion of the helix. As expected from this model, yeast strains with pmsl, msh2, or mlhl mutations have greatly increased levels of instability in microsatellite sequences (9).Mutations in human homologs of MSH2, MLH1, and PMS1 lead to a variety of hereditary tumors (11)(12)(13)(14)(15) (9) and contained the assay plasmids pSH31 (MS85) or pSH91 (MS96). MS128 was derived from AMY125 by insertion of an msh2 deletion allele [constructed by using the plasmid pll-2-Tn1OLUK7-7 (6) obtained from E. Alani, Cornell University, Ithaca, NY], transformation with the assay plasmid pSH91, and insertion of a LEU2 gene by using CV9. MS111 was constructed by transforming AMY125 with CV9 (MS71), transforming MS71 with pEN33 (strain GCY140; contains deletion mutation of MSH3), and transforming GCY140 with pSH91. MS125 was constructed by transforming GCY178 with pSH91; GCY178 was made by doing a two-step transplacement of GCY141 (an msh3 mutant strain isogenic with GCY140) with the plasmid p3O6M2RIA to introduce the msh2 mutation. MS110 and MS111 are isogenic, as are MS124 and MS125, except for the assay plasmid. MS94 was constructed by transforming AMY125 with the plasmid pII-2-Tn1OLUK7-7 to introduce the msh2 mutation, followed by transformation with the assay plasmid pSH31.Assays of Microsatellite Instability. Two plasmids, pSH31 (22) and pSH91 (9), were used to measure microsatellite instability. Since the methods used to monitor tract instability have been published (22), the description of these methods is abbreviated. The plasmid pSH31 contained an out-of-frame 29-bp poly(GT) tract within the coding sequence of 13-galactosidase (Fig. 2a). Strains containing this plasmid formed white colonies on plates containing 5-bromo-4-chloro-3-indolyl f-D-galactoside (X-Gal); strains that had an alteration in the poly(GT) tract length that restored the correct reading frame of the 13-galactosidase gene, however, formed blue colonies (as confirmed by DNA sequence analysis). To determine a rate of instability, we determined the frequency of blue colonies in 15-20 cultures. If any of the cultures had no blue Abbreviations: X-Gal, ...

Biochemistry and genetics of eukaryotic mismatch repair.

“…Furthermore, the frequency of insertion events approximately equaled the frequency of deletion events when insertionldeletion mispairs were repaired after transformation of wild-type S. cerevisiae cells, whereas there was a strong disparity in favor of deletion events among the fraction of insertionldeletion mispairs that appeared to be repaired in a pmsl mutant (Bishop and Kolodner 1986;Bishop et al 1987). A similar shift toward deletion events in mismatch repair mutants was seen when alterations in GT tract length resulting from replication errors were analyzed (Strand et al 1993(Strand et al , 1995. One of several possible explanations for these data is that there could be a separate, less-efficient repair pathway that selectively repairs insertionldeletion mispairs by deletion.…”

“…Unlike either msh2 or msh6 mutants, msh3 mutants have little if any mutator phenotype in forward mutation and frameshift reversion assays and show increased dinucleotide repeat instability, albeit at lower levels, than that seen in msh2 mutants (New et al 1993;Alani et al 1994;Strand et al 1995;Marsischky et al 1996;). Strikingly, when double mutant strains were analyzed, msh3 and msh6 mutations showed a large synergistic effect on the rate of accumulation of mutations in frameshift reversion assays that measure the formation and repair of single-base insertion1 deletion mispairs (Marsischky et al 1996).…”

Oligonucleotide transformation of yeast reveals mismatch repair complexes to be differentially active on DNA replication strands

“…There are several observations in the literature, however, that have never been satisfactorily explained, and we suggest that these could reflect a strand asymmetry of MMR. Assays for dinucleotide repeat slippage, for example, found that 87% of events were due to a gain of repeat units in msh6 strains, whereas 85% of events were due to a loss in msh3 strains (41,42). Similarly, an examination of loss or gain of bases in mononucleotide repeats found a complex pattern that depended not only on the genetic background but also on whether there were Cs or Gs on the coding strand (43).…”

The Rate and Spectrum of Microsatellite Mutation in Caenorhabditis elegans and Daphnia pulex

“…Despite the central significance of microsatellite mutations to issues of genomic instability, forensic testing, and population genetic analyses, the rate of origin and spectrum of effects of such mutations are still poorly understood, with many estimates being derived from reporter constructs in yeast cultures (e.g., Henderson and Petes 1992;Strand et al 1995;Wierdl et al 1996;Sia et al 1997). Our long-term series of mutationaccumulation lines of C. elegans and D. pulex provide a useful platform for a more direct evaluation of the properties of microsatellite mutations in two key model organisms.…”

“…This apparent reduction in the ability to repair a common form of DNA instability may be a consequence of the compromised ability of natural selection to maintain optimal features of genomic architecture in lineages of multicellular organisms with reduced effective population sizes (Lynch 2006(Lynch , 2007. For example, because mismatch repair (MMR) plays a central role in the elimination of post-replication slippage errors, specifically in microsatellite loci (Strand et al 1995;Wierdl et al 1996Wierdl et al , 1997Eshleman and Markowitz 1996;Boyer et al 2002;Yamadaet al 2002), a decline in the efficiency of MMR might be responsible in part for increased mutation rates in multicellular species.…”