Homologous recombination is an important DNA repair function that helps to ensure the integrity of the genome prior to cellular division. Mitotic recombination typically involves identical sequences on sister chromatids, but the presence of large amounts of repetitive DNA in eukaryotic genomes affords the opportunity for other types of interactions to occur. Interactions between dispersed repeated sequences can result in a variety of deleterious genome rearrangements, and such rearrangements are causative factors in a number of human diseases. Cells thus have evolved a surveillance mechanism that enforces strict sequence identity requirements during recombination, and this mechanism primarily involves antirecombination activity of the mismatch repair machinery.
Aims 1 and 2 of this proposal are to further examine the regulation of recombination between non-identical sequences in the yeast Saccharomyces cerevisiae.
In Aim 1, genetic approaches will be used to elucidate the mechanism of anti-recombination in both wild-type and recombination-compromised strains, with a focus on the roles of the Sgs1 helicase and PCNA in this process.
In Aim 2, the impact of sequence divergence on the resolution of recombination events will be examined, as the resolution step determines whether or not the event will impact genome structure. Both a transformation assay and an HO-based chromosomal assay will be used to assess the effect of sequence divergence on gene conversion versus crossover events in wild-type and appropriate mutant strains. In addition to the effect of primary DNA sequence on recombination, recombination also can be influenced by the co-occurrence of other DNA metabolic processes such as transcription and DNA replication.
Aim 3 of the proposal will continue studies of transcription-stimulated recombination, and these studies will be expanded to examine the molecular basis of transcription-stimulated mutation. A doxycycline (Dox)-regulated system will be developed that will allow the transcription rate of a Iys2 frameshift allele, which can serve as either a recombination substrate or as a mutational target, to be varied over a very broad range. The Dox system will be used to (1) determine the relationship between the level of transcription and recombination/mutation rates, (2) to examine the relevance of the direction of replication fork movement to transcription-stimulated recombination, and (3) to genetically investigate the molecular origin of a novel type of transcription-associated mutation, a -2 frameshift mutation.
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