Homologous recombination is a ubiquitous DNA metabolic process. Besides its contribution to evolution, recombination constitutes an important DNA repair pathway and appears to be required for accurate chromosome metabolism during meiosis. Impaired or aberrant recombination can lead to genomic instability, typically found in tumor cells, and to human disorders like in Bloom s or Werner s syndrome or ataxia telangiectasia. Recombination is a major pathway for the repair of DNA double-strand breaks which can be caused by cellular processes, certain anti-cancer drugs, or ionizing radiation. The common use of ionizing radiation in medical diagnosis and cancer therapy requires an understanding of the cellular responses to ionizing radiation-induced DNA damage. Practical applications of recombination, such as somatic gene therapy and transgenesis, will also benefit from a better understanding of the recombination mechanisms. The long term goal of this proposal is to elucidate the molecular mechanisms of recombinational DNA repair in eukaryotes. The focus is on the function of the Rad54 protein in the model system Saccharomyces cerevisiae. Mutations in RAD54 lead to extreme sensitivity to ionizing radiation. Based on the severity of the rad54 mutant phenotypes, Rad54p is one of the most important proteins in recombinational repair together with the Rad51 and Rad52 proteins.
The specific aims are: (1) The cellular functions of Rad54p will be established by genetic analysis. This will define a novel role of Rad54p in chromosome stability and mutation avoidance. (2) The DNA binding properties of Rad54p and the significance of its dsDNA-specific ATPase activity will be determined by biochemical analysis. These experiments test the molecular models for Rad54p and are a prerequisite to understand its exact role in recombinational repair. (3) The structural basis and the biological significance of the Rad54p:Rad51p will be established by in vivo and in vitro analysis. This will provide critical tests for the molecular models and important insights into the role of Rad54p in the formation, function and/or disassembly of the Rad51:DNA filament during the central step of homology search and strand exchange in recombination. (4) In vitro and in vivo approaches will be integrated to identify and characterize proteins and their genes that interact with Rad54p. This will help to understand the role of Rad54p as well as identify and characterize novel DNA repair proteins.
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