Variation in efficiency of DNA mismatch repair at different sites in the yeast genome

Abstract

Evolutionary studies have suggested that mutation rates vary significantly at different positions in the eukaryotic genome. The mechanism that is responsible for this context-dependence of mutation rates is not understood. We demonstrate experimentally that frameshift mutation rates in yeast microsatellites depend on the genomic context and that this variation primarily reflects the context-dependence of the efficiency of DNA mismatch repair. We measured the stability of a 16.5-repeat polyGT tract by using a reporter gene (URA3-GT) in which the microsatellite was inserted in-frame into the yeast URA3 gene. We constructed 10 isogenic yeast strains with the reporter gene at different locations in the genome. Rates of frameshift mutations that abolished the correct reading frame of this gene were determined by fluctuation analysis. A 16-fold difference was found among these strains. We made mismatch-repair-deficient (msh2) derivatives of six of the strains. Mutation rates were elevated for all of these strains, but the differences in rates among the strains were substantially reduced. The simplest interpretation of this result is that the efficiency of DNA mismatch repair varies in different regions of the genome, perhaps reflecting some aspect of chromosome structure.

Fig. 1.

Construction of yeast strains with the URA3-GT reporter gene in different chromosome contexts. The plasmid pMBW1 (9) contains a frameshift reporter gene (URA3-GT). This fusion gene has small segments derived from the yeast LYS2 and HIS4 genes, and most of the sequence of the yeast URA3 gene. There is an in-frame insertion (33 bp) of polyGT sequences near the beginning of the gene, and transcription of the gene is controlled by the galactose-inducible GAL1,10 promoter. The DNA fragments used for transformation were generated by PCR amplification of pMBW1 using primers that had homology to the sequences flanking the URA3-GT gene at their 3′ ends (≈20 bp) and homology to chromosomal DNA sequences at their 5′ ends. These fragments were transformed into the haploid yeast strain LS48 (deleted for URA3), and Ura⁺ transformants were selected in galactose-containing medium.

Fig. 2.

Context-dependent rates of microsatellite instability in wild-type (MMR⁺) and msh2 mutant (MMR^-) yeast strains. White bars show the rates derived from MMR⁺ cells, and gray bars show the rates for the isogenic MMR^- strain; 6 of the 10 strains had an isogenic msh2 derivative. (Absence of a gray bar indicates that an msh2 derivative was not analyzed.) Error bars indicate the 95% confidence limits on the mutation rates.

Fig. 3.

Alterations in the length of a microsatellite by DNA polymerase slippage. Replication of a poly GT/CA tract that is seven repeats in length is shown. A slippage event in which the displaced repeat is on the primer strand, if uncorrected, will result in a tract that is eight repeats in length in the polyGT strand. Alternatively, a displaced repeat on the template strand results in six-repeat poly GT strand.