Double-stranded DNA breaks (DSBs) are the primary genotoxic lesion of ionizing radiation (IR), and DSB induction appears to determine the efficacy of IR and other DMA metabolism-based anti-tumor drugs as cancer therapeutic agents.! Homologous recombination (HR) is an important DSB repair pathway, which is essential for cellular radiation resistance and genome stability. Defects in HR lead to genomic instability and have been implicated in the etiology of cancer. The long-term goal of this proposal is to elucidate the mechanism of HR with a present focus on post-synapsis and resolution, stages that are particularly poorly understood in eukaryotes. Analysis of recombination reactions reconstituted from purified proteins and in vivo experiments are combined to determine the mechanism of the dsDNA-specific ATPase Rad54 in Rad51-mediated recombination; to determine the mechanism of the mutual stimulation of the Rad51 and Rad54 proteins; and to determine the function of the Mus81-Mms4 DMA structure-specific endonuclease, which was first identified in my laboratory through its interaction with Rad54 protein. The results will provide novel mechanistic insights for Rad51, Rad54, and Mus81-Mms4, which are critical for HR in eukaryotes.
The specific aims are: (1) Determine the biochemical function of Rad54 during recombination. Rad54 remodels Rad51-dsDNA complexes. We will test if Rad54 modulates access of DMA polymerases to the 3'- OH end of the invading strand in the important transition from DMA strand invasion to repair synthesis. (2) Determine the in vivo function of Rad54. Analysis of in vivo pairing intermediates in recombinational DSB repair will be used to distinguish between the 3'-OH access model and competing models. (3) Determine the mechanism of the mutual stimulation of the Rad51 and Rad54 proteins. Using separation-of-function mutants, we will establish the mechanism of the mutual stimulation of the Rad51 and Rad54 proteins and provide a rigorous test if the Rad51-Rad54 interaction is of biological significance. (4) Identify the biochemical and cellular function of the Mus81-Mms4 endonuclease. The pathways leading to resolution of junction structures in recombination are still unclear. Biochemical and in vivo experiments will determine the substrate-specificity and function of the Mus81-Mms4 endonuclease in HR.
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