Mismatch Repair Acts As a Barrier to Homeologous Recombination in \u3cem\u3eSaccharomyces cerevisiae\u3c/em\u3e: A Dissertation

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 SPT15function 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 pms1deletion 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 from each strain to determine the percentage of products that fell into a given class (crossovers or gene conversions). Each percentage was then multiplied by the overall rate to arrive at a rate of recombination for individual events. Typically 90-100% of homologous and homeologous recombinant products could be accounted for, either as crossovers or gene conversions. Recombination for all classes of products was inhibited when divergent sequences were present, indicating that homeology blocks formation of both crossovers and gene conversions. Sequence analysis of a limited number of homeologous recombinants indicated that transfer of DNA occurred in continuous stretches and that endpoints fell within regions of 3-11 base pairs of perfect homology. Mutations in the mismatch repair genes MSH2 or MSH3that elevate the overall rate of homeologous recombination produced similar rate increases in formation of each recombinant class. This suggests that mismatch correction proteins block multiple types of homeologous recombination events. Taken together, these results support the hypothesis that homeologous and homologous recombination occur by the same (or similar) pathways, with mismatch repair superimposed as an extra level of control over the fidelity of the process. I also investigated whether this homeologous recombination system would be useful in a genetic screen to identify novel genes or new alleles of genes known to increase exchanges between diverged DNA sequences. Hyperhomeologous recombination mutants were isolated following ethylmethane sulfonate mutagenesis of yeast that harbored the spt15 homeologous duplication. Preliminary characterization of these mutants demonstrated that some of these isolates yielded phenotypes that were consistent with mutations in mismatch repair genes verifying the utility of this method to identify such mutants. To improve the use of this system for a mutant screen, I developed a second generation homeologous duplication using URA3. These new starter strains are expected to be important for efficient isolation and characterization of hyperhomeologous recombination mutants