During the last funding period several groups identified three genes/proteins that are required for dsb repair; this pathway is required not only for joining dsbs induced by agents like ionizing radiation, but also for the dsb intermediated that are required for the rearrangement of immunoglobulin genes, i.e., V(D)J recombination. These three proteins are Ku70, Ku80 and DNAPKcs (460 kDa). The ADNA-dependent protein kinase (DNA-PK) comprises a complex between these three proteins, but the underlying molecular mechanisms by which the complex participates in DNA rejoining pathways is not known, and the precise role of the kinase activity of this complex is not known. All of the proposed studies are aimed at addressing these unknowns. Previous studies demonstrate that DNA-PK assembles on the ends of DNA strands, in vitro, but this has not yet been shown to occur in vivo. If this complex is present at dsbs in vivo, it is likely to be a key participant in the first steps of dsbs repair. As well as being involved in damage recognition and signal transduction, the kinase complex may play a more direct role in repair at the break site; e.g., tethering broken ends together; modification of chromatin structure; nucleation of repair complex assembly. Based on the current evidenc the principal investigator proposes that DNA-PK functions by binding to the ends of broken DNA and mediates the subsequent steps of DNA rejoining through the phosphorylation of proteins that colocalize with the complex on the DNA ends, including the Ku complex. The objective of the proposed experiments is to test these ideas using unique reagents and experimental approaches that the principal investigator accumulated during the last funding period.
The specific aims are: (1) To use Atomic Force Microscopy (AFM) to test the hypothesis that DNA-PK binds to and tethers the ends of two broken DNA molecules; naked DNA or nucleosome associated DNA will be used as substrate. (2) To determine the protein domains critical for Ku70 and Ku80 heterodimerization and DNA-PKcs interactions with the Ku70/80 complex; classical deletion analysis followed by expression in mouse cell lines knocked out for Ku80 or Ku70 (3) To test the hypothesis that the kinase activity of DNA-PKcs and the phosphorylation of Ku70 and Ku80 are essential in mediating dsb rejoining and V(D)J recombination; mutant versions of the three proteins will be generated that are likely to be deficient in kinase activity and their in vivo properties examined. The properties of the DNA-PK complex have been well studied in vitro, but the principal investigator proposes to take these into in vivo studies. To do this they will express various mutant versions of the three proteins in cells with defined genetic backgrounds that lack the Ku70, the Ku80 or the DNA-PKcs functions.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Chemical Pathology Study Section (CPA)
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Pelroy, Richard
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Lawrence Berkeley National Laboratory
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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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