Mechanisms of Homologous Recombination in Yeast

Columbia University Health Sciences, New York NY

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Abstract

The long-term objective of this research program is to understand the molecular mechanisms of homologous recombination in a model eukaryote, the budding yeast Saccharomyces cerevisiae. Homologous recombination (HR) plays two essential roles during the life cycle of most organisms. It is required to repair lethal lesions in DMA, such as double-strand breaks, and it is essential for the pairing and segregation of homologous chromosomes during meiosis. The importance of these functions is evidenced by increased mutagenesis, and mitotic and meiotic aneuploidy in the absence of recombination. Since many human cancer-prone syndromes are associated with increased genome instability, an understanding of the mechanisms of recombination is likely to be important in understanding these diseases. Homologous recombination initiates at single-stranded DNA (ssDNA) formed by 5'-3'resection at double-strand breaks (DSBs), or at stalled replication forks. Rad51 catalyzes synapsis and strand exchange between ssDNA bound within the Rad51 nucleoprotein filament and homologous duplex DNA, in a reaction requiring RPA, Rad52, Rad54, Rad55 and Rad57. Much progress has been made in understanding Rad51-catalyzedstrand invasion, but the identity of the resection nuclease and factors required for the post-invasion step are largely unknown. We have developed a simple genetic assay for one-ended strand invasion that requires extensive DNA synthesis to complete recombinational repair. This assay will be used to characterize the post-invasion steps of HR using physical monitoring methods and to identify the replication factors involved. The role of BIR in gross chromosomal rearrangements will also be investigated. The second aim is directed at identification of the resection nuclease. Because formation of ssDNA at break sites is considered an essential prerequisite for Rad51 -promoted strand invasion, it is surprising that none of the RAD52 group genes encodes the resection nuclease. This suggests redundancy for this step, or that the resection nuclease is essential for viability. Several genetic screens are proposed to identify genes encoding nucleases. Finally, we have shown high frequency R/AD57-independentrecombination of repeated sequences;genetic studies are proposed to identify components of the R/\D57-independent recombination pathway.