The long-term objective of the proposed research is to understand the cellular mechanisms for the repair of DNA double strand breaks (DSBs) in eukaryotes. The repair of DSBs by homologous recombination is essential to maintain genome integrity and for the accurate segregation of chromosomes during meiosis. Defects in DNA repair pathways are associated with several cancer-prone syndromes in humans. Recent studies implicate the human breast cancer genes, BRCAI and BRCA2, in the homologous recombination pathway through their association with Rad51 and phenotypic characterization of cell lines with hypomorphic alleles. Therefore, understanding the mechanisms of DSB repair will be important for understanding these human pathologies. In eukaryotes, components of the recombinational repair pathway are highly conserved, and the Rad51 protein plays a key role in DSB repair by catalyzing synapsis and strand exchange between homologous duplexes. However, Rad51 requires a number of other proteins, including replication factor A (RPA), Rad52, RadS4, Rad55 and Rad57, to accomplish strand exchange. The major goal of this proposal is to further characterize the role of Rad51 and two of the accessory proteins, Rad55 and Rad57, in this process. We have isolated a novel class of rad51 alleles that partially suppress the DNA repair defects of rad55 and rad57 strains. These will be subject to genetic and biochemical analyses to understand the mechanism for suppression. To directly determine the role of Rad55 and Rad57 in stimulation of Rad5 1-catalyzed strand exchange, both proteins will be purified and characterized in vitro for DNA binding, ATPase and strand exchange activity in the presence or absence of Rad5 1 and RPA. Gene replacement using double-stranded linear fragments occurs with high efficiency in rad51 mutants. We propose to investigate the genetic control of gene targeting, role of transcription of the target locus, and mechanisms using a variety of targeting vectors. The use of gene replacement for targeted gene disruption has application for gene therapy, thus an understanding of the mechanism is of fundamental importance.
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