Mismatch Repair Acts As a Barrier to Homeologous Recombination in Saccharomyces cerevisiae : A Dissertation

Abstract

Homeologous recombination refers to genetic exchanges between DNA partners containing similar but not identical DNA sequences. Heteroduplex intermediates in such exchanges are expected to contain multiple DNA mismatches at positions of sequence divergence and hence are potential targets for mismatch correction. Thus recombination of this type is of particular interest in understanding the role of DNA mismatch correction on recombination fidelity. Previous studies that examined this question in prokaryotic systems suggested that mismatch repair acts as a barrier to recombination between diverged sequences. The central hypothesis of this thesis is that mismatch correction acts as a barrier to homeologous recombination in yeast. The objectives of these studies was to elucidate the role of mismatch correction in homeologous recombination as a means of dissecting its mechanism in eukaryotic organisms. To examine homeologous genetic events in yeast, I developed an in vivo assay system to evaluate recombination between diverged DNA sequences. A homeologous gene pair consisting of Saccharomyces cerevisiae SPT15 and its Schizosaccharomyces pombe homolog were present as a direct repeat on chromosome V, with the exogenous S. pombe sequences inserted either upstream or downstream of the endogenous S. cerevisiae gene . Each gene carried a different inactivating mutation, rendering this starting strain Spt15 - . Recombinants that regenerated SPT15 function were identified by genetic selection and the rates of recombination in different backgrounds were compared. The homeologous mitotic recombination assay was utilized to test the role of S. cerevisiae mismatch repair genes PMS1, MSH2 and MSH3 on recombination fidelity. In strains wild type for mismatch repair, homeologous recombination was reduced 150-180 fold relative to homologous controls, indicating that multiply mispaired sequences act in cis as part of an inhibitory mechanism. In the upstream orientation of the homeologous gene pair, msh2 or msh3 mutations resulted in 17-fold and 9.6-fold elevations in recombination and the msh2 msh3 double mutant exhibited an 43-fold increase, implying that each MSH gene can function independently in trans to prevent homeologous recombination. Homologous recombination was not significantly affected by the msh mutations. In the other orientation, only msh2 strains were elevated (12-fold) for homeologous recombination. A mutation in MSH3 did not affect the rate of recombination in this orientation. Surprisingly, a pms1 deletion mutant did not exhibit elevated homeologous recombination in either case. Next, I investigated whether mismatch correction acts as a specific or general obstacle to homeologous recombination by blocking one or many exchange pathways. To answer this question, I performed structural analysis on numerous recombinant products…