Homologous recombination occurs at high frequency during meiosis and plays a critical role in promoting reductional segregation of chromosomes. Meiotic recombination is initiated by the programmed induction of DNA double strand breaks (DSBs) and involves a mechanism related to recombinational repair of DNA damage in mitotic cells. Defects in meiotic recombination cause chromosome non-disjunction and loss, both of which lead to birth defects and spontaneous abortion. Failure of recombinational repair in mitosis is implicated in the etiology of breast cancer and other malignancies. The central step of recombination is homologous strand invasion, a process promoted by proteins called recombinases. In order to promote strand invasion, recombinases must first form filaments on regions of single stranded DNA. This assembly process is regulated by recombinase accessory factors. During meiosis in the budding yeast S. cerevisiae, strand invasion is promoted by two structural and functional homologs of the E. coli recombinase RecA. These two are Rad51, which also promotes recombinational repair in mitosis, and Dmc1, which is meiosis-specific. The two recombinases, while capable of acting alone, often cooperate at sites of recombination. The proposed experiments seek to elucidate the mechanisms underlying recombinase cooperation before and during the strand invasion stage and to determine how those mechanisms regulate the outcome of recombination.
The specific aims of the proposal include: 1. Biochemical characterization of interactions between Dmc1, the single strand DNA binding protein RPA, and several proteins known to be accessory factors for Rad51-mediated strand invasion.
This aim will test the hypothesis that assembly of Dmc1 is regulated by a manner distinct from that regulating assembly of Rad51. 2. To examine the genetic requirements for specific association of Rad51 and Dmc1 at sites of recombination in living cells. 3. To test the hypothesis that Rad51 and Dmc1 assemble on opposite DNA ends created by a DSB and do so via a concerted mechanism. 4. To determine if meiotic functions constrain the strand invasion activity of a DNA end created by a DNA double strand break relative to its partner end. 5. To use a novel genomics approach to identify proteins that interact with Dmc1. ? ?
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