A large number of DNA lesions occur within a cell each day jeopardizing the integrity of the genome, of which DNA double strand breaks (DSBs) are the most toxic lesions. Unrepaired or misrepaired DSBs can result in senescence, inducted apoptosis, or chromosomal aberrations, including translocations and deletions. These chromosomal aberrations can lead to genomic instability and tumorigenesis. To counter DSBs, organisms have evolved a complex mechanism of DNA damage response that includes recognition of the broken DNA molecule, cellular signaling including modulation of the cell cycle via checkpoints, and ultimately the repair of the DNA lesion. Two prominent pathways mediate the repair of DSBs in mammalian cells: homologous recombination (HR) and non-homologous end-joining (NHEJ). Although much work has been performed to identify and characterize the factors of each DSB repair pathway, important questions regarding cross-talk amongst pathways remain unresolved. These include elucidating factors that initially bind to the DSB ends and stabilize them, whether these factors influence recruitment of downstream factors and whether these factors modulate pathway choice/switching between NHEJ and HR for the repair of DSBs. In this study, we will take a novel ?Ku-centric? view of DSB repair pathway choice in mammalian systems, which challenges the paradigm established in yeast models. We hypothesize that Ku binds to DSBs in all cell cycle phases to protect DNA ends, and that phosphorylation-mediated dissociation of Ku from DSBs is one of the key mechanisms responsible for modulating pathway choice between NHEJ and HR in S phase. Furthermore, we postulate that dysregulation of this process will result in genomic instability and increased susceptibility to tumorigenesis and most importantly, will provide an insight into the etiology of spontaneous tumors. To test this hypothesis, we propose the following specific aims: 1) To determine the kinase(s) responsible for Ku70 phosphorylation and the mechanism mediating Ku dissociation from DSBs in response to DNA double-strand breaks; 2) To test the hypothesis that Ku phosphorylation is essential for homologous recombination repair (HR), DNA damage response (DDR), and maintenance of genome stability in human cells; and 3) To test the hypothesis that Ku phosphorylation-dead mutations cause genome instability and lead to tumorigenicity in mouse model. Finally, since a number of human genetic diseases and initiation of carcinogenesis are directly associated with impaired or misrepaired DNA lesions and mutations in DSB repair genes, we believe basic mechanistic insights into DSB repair mechanisms and regulation of DSB repair pathway choice could lead to the identification of possible drug targets, which would ultimately translate into clinical benefits.
The importance of DNA double-strand break (DSB) repair is supported by the observations that a number of human genetic diseases and initiation of carcinogenesis are directly associated with mutations in DSB repair genes and defective repair of DNA lesions. To cope with DSBs, cells have evolved multiple repair pathways for which a cell must properly which pathway to employ for each specific DSB. We predict that inappropriate DSB repair pathway choice will lead to sporadic tumor formation, which is supported by the discovery of a number of mutations in factors that likely play a role in DSB repair pathway choice correlate with sporadic tumorigenesis. The goal of this study is to understand one of the major unresolved questions in the field of DNA repair: what molecular events underlie the choice between the different pathways for repair of DSBs and if this process is dysregulated does it result in genomic instability and carcinogenesis? We believe basic mechanistic insights into DSB repair mechanisms and regulation of DSB repair pathway choice could lead to the identification of possible drug targets, which would ultimately translate into new opportunities for cancer treatment.
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