Microsatellite Instability in Yeast: Dependence on Repeat Unit Size and DNA Mismatch Repair Genes

Abstract

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.Eukaryotic genomes often contain regions of DNA (called microsatellites or minisatellites) in which a single base or a small number of bases is tandemly repeated. In this paper, repetitive tracts with repeats of 1 to 13 bp will be considered microsatellites and tracts with repeats of more than 16 bp will be considered minisatellites. Both microsatellites and minisatellites are unstable, frequently undergoing deletions and additions (10,13,19). In vitro replication experiments demonstrate that DNA polymerase frameshift errors occur in repetitive sequences (17), and most of the available in vivo data suggest that alterations in microsatellite length reflect DNA polymerase slippage events (19,27). This mechanism predicts a transient dissociation of the template and the nascent strand during replication of the microsatellite (28). Due to the repetitive nature of the tract, the two DNA strands can reassociate out of register, leaving one or more unpaired repeats on either the template or nascent strand (see Fig. 1). If the distortion caused by these unpaired bases is not removed from the newly synthesized strand by the DNA mismatch repair system, the result will be a loss (if the unpaired bases are on the template strand) or a gain (if the unpaired bases are on the nascent strand) of one or more repeats. As expected from this model, mutations in the genes required for DNA mismatch repair greatly increase the rate of instability of repetitive DNA sequences in Escherichia coli, the yeast Saccharomyces cerevisiae, and human cells (19,22,27).In E. coli, two of the proteins involved in DNA mismatch repair are MutS (involved in recognition of the DNA mismatch) and MutL (involved in interactions between MutS and other proteins) (22). Homologs of these proteins have been identified in yeast and mammals. In yeast, the effects of mutations in the mutL homologs MLH1 and PMS1 and in the mutS homologs MSH2, MSH3, and MSH6 on the stability of a 33-bp poly(GT) repeat have been examined previously (14,26,27). Mutations in MLH1, P...

Saccharomyces cerevisiae MSH2–MSH3 and MSH2–MSH6 Complexes Display Distinct Requirements for DNA Binding Domain I in Mismatch Recognition

“…2,17 This specificity is best demonstrated in the microsatellite instability assay, where msh3Δ strains displayed rates of microsatellite instability for 4, 5, and 8 nt repeat tracts (9 -60 fold above wild-type) comparable to that seen in msh2Δ strains (9 -60 fold; in all cases msh3Δ vs. msh2Δ, p > 0.65); the msh6 mutation, however, conferred little or no increased rate of instability (1.0 to 1.4 fold above wild-type; in all cases msh6Δ vs. wild-type, p > 0.24) for these tract lengths (Table 3). When the repeat tracts were decreased to 1-2 nt, msh3Δ showed a greater increase in instability (53 to 180 fold higher than wild-type) compared to msh6Δ (2.1 to 20 fold; msh3Δ vs. msh6Δ, p < 0.001), but neither strain displayed defects as large as those observed in msh2Δ (360 to 4800; msh2Δ vs. msh3Δ and msh2Δ vs. msh6Δ, p < 0.001) or msh3Δ msh6Δ strains (400 to 3100 fold).…”

“…17 The msh2Δ1 (deletion of amino acids 2-133) and msh3Δ1 (Δ149-306) alleles contain complete deletions of Domain I (Figure 1). We employed a sensitive microsatellite instability assay developed by the Petes laboratory in which DNA sequences of various repeat units were inserted in frame into the coding region of URA3.…”

“…These results also illustrated the overlapping specificities for MSH2-MSH3 and MSH2-MSH6 for small (1-2 nt) loop mispairs. 17,18 We measured rates of microsatellite instability in strains bearing the msh2Δ1 allele and other mismatch repair mutations. As shown in Table 3 and Figure 2, the effect of the msh2Δ1 mutation on microsatellite instability was similar to that seen for msh3Δ, providing evidence that only the MSH2-MSH3 repair pathway was compromised in msh2Δ1strains.…”

Escherichia coli DNA polymerase IV contributes to spontaneous mutagenesis at coding sequences but not microsatellite alleles

“…There are many reports in the literature showing that the sequence composition and length of the repeat allele plays a large role in the mutagenesis of microsatellites [9,11,37,38]. We compared various aspects of the mutational differences detected among the three microsatellite alleles.…”

“…Microsatellite sequences are often hotspots for mutation, and an increase in mutagenesis is observed as the length of the repeated sequence increases [9,10]. The predominant mutation in mono-and dinucleotide microsatellite sequences are single-unit insertions or deletions [9,[11][12][13]. One proposed mechanism to explain instability in these microsatellites is the polymerase slippage model [14,15], also referred to as slipped strand mispairing [16].…”