EXCEED THE SPACE PROVIDED. The repair of DNA double strand breaks (DSBs), induced by ionizing radiation (IR) or generated during the process of V(D)J recombination, is of paramount importance to the cell, as misrepair of DSBs can lead to cell death or promote tumorigenesis. The DNA-dependent protein kinase (DNA-PK) is the key component of the non-homologous end-joining (NHEJ) pathway of DNA DSB repair in mammalian cells. We have previously shown that the kinase activity of the DNA-PK catalytic subunit (DNA-PKcs) is absolutely essential for the repair of DSBs by NHEJ. However, nothing is known about the in vivotargets of DNA-PKcs (whether itself and/or other factors), and about the mechanisms underlying the requirement of DNA-PKcs kinase activity for NHEJ. We propose, in this grant, to define the phosphorylation events mediated by DNA-PK and to understand the mechanisms by which these modifications modulate NHEJ. Towards this goal we propose to 1) Identify relevant phosphorylation sites on DNA-PKcs, demonstrate in vivo phosphorylation at these sites, and understand the biological consequences of abrogation of phosphorylation; 2) Examine the localization of phospho-DNA-PKcs with respect to DSBs, determine the mechanism by which phosphorylation might modulate DNA-PK function, and understand how DNA-PKcs phosphorylation is coordinated with other early events at a break; 3) Identify phosphorylation sites and the function of phosphorylation of two known targets of DNA-PK, XRCC4 and WRN, and identify novel targets of DNA-PKcs kinase activity. We have recently made a breakthrough, by identifying one of the sites on DNA-PKcs that it is phosphorylated in vivo in response to DNA damage and by demonstrating that abrogation of phosphorylation leads to radiation sensitivity. We have also identified putative phosphorylation sites on XRCC4 and WRN, and are pursuing a novel method to identify new targets of DNA-PKcs. The work proposed in this grant should provide us with a mechanistic basis for the requirement of the kinase activity of DNA-PKcs in NHEJ. PERFORMANCE SITE ========================================Section End===========================================

National Institute of Health (NIH)
National Cancer Institute (NCI)
Method to Extend Research in Time (MERIT) Award (R37)
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Chemical Pathology Study Section (CPA)
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Pelroy, Richard
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University of Texas Sw Medical Center Dallas
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Mori, Eiichiro; Davis, Anthony J; Hasegawa, Masatoshi et al. (2016) Lysines 3241 and 3260 of DNA-PKcs are important for genomic stability and radioresistance. Biochem Biophys Res Commun 477:235-40
Davis, Anthony J; Lee, Kyung-Jong; Chen, David J (2013) The N-terminal region of the DNA-dependent protein kinase catalytic subunit is required for its DNA double-stranded break-mediated activation. J Biol Chem 288:7037-46
Wang, Hailong; Shi, Linda Z; Wong, Catherine C L et al. (2013) The interaction of CtIP and Nbs1 connects CDK and ATM to regulate HR-mediated double-strand break repair. PLoS Genet 9:e1003277
Chung, Young Min; Park, See-Hyoung; Tsai, Wen-Bin et al. (2012) FOXO3 signalling links ATM to the p53 apoptotic pathway following DNA damage. Nat Commun 3:1000
Sun, Jingxin; Lee, Kyung-Jong; Davis, Anthony J et al. (2012) Human Ku70/80 protein blocks exonuclease 1-mediated DNA resection in the presence of human Mre11 or Mre11/Rad50 protein complex. J Biol Chem 287:4936-45
Shao, Zhengping; Davis, Anthony J; Fattah, Kazi R et al. (2012) Persistently bound Ku at DNA ends attenuates DNA end resection and homologous recombination. DNA Repair (Amst) 11:310-6
Pankotai, Tibor; Bonhomme, Celine; Chen, David et al. (2012) DNAPKcs-dependent arrest of RNA polymerase II transcription in the presence of DNA breaks. Nat Struct Mol Biol 19:276-82
Levy-Barda, Adva; Lerenthal, Yaniv; Davis, Anthony J et al. (2011) Involvement of the nuclear proteasome activator PA28? in the cellular response to DNA double-strand breaks. Cell Cycle 10:4300-10
Lee, Kyung-Jong; Lin, Yu-Fen; Chou, Han-Yi et al. (2011) Involvement of DNA-dependent protein kinase in normal cell cycle progression through mitosis. J Biol Chem 286:12796-802
Kozlov, Sergei V; Graham, Mark E; Jakob, Burkhard et al. (2011) Autophosphorylation and ATM activation: additional sites add to the complexity. J Biol Chem 286:9107-19

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