Ionizing radiation and V(D)J recombination generate DNA double-strand breaks (DSBs), which are repaired by non-homologous end-joining (NHEJ). Proteins required for NHEJ include DNA-dependent protein kinase (DNA-PK), which consists of a DNA binding subunit (Ku) and a catalytic subunit (DNA-PKcs). It is not yet known how DNA-PK and other factors bring the broken ends of the DNA together, process the ends for joining, and regulate the reaction by phosphorylation. The long-term objective is to elucidate each step in NHEJ, which will lead to a better understanding of cancer, immunity, and aging.
The specific aims are:1.To define the mechanism for synapsis of DNA ends during NHEJ. Preliminary studies show that DNA-PKcs brings DNA ends together in a synaptic complex at low salt. Proposed studies will study the effect of DNAPKcs autophosphorylation on DNA synapsis, determine the factors required for synapsis to occur at physiological salt, and determine the number of DNA-PKcs molecules in the synaptic complex.2. To define how the DNA ends are processed for joining during NHEJ. A cell-free system for NHEJ detected efficient end-processing in wild type extracts and deficient end-processing in extracts from Werner's Syndrome, a disease of premature aging and cancer, and Nijmegan Breakage Syndrome, a disease of radiation sensitivity and cancer. Experiments will define the roles of the associated proteins, Wm and Nbs1. End-processing included templated nucleotide addition and increased with addition of dNTPs. Experiments will determine if DNA polymerase(s) are involved. The cell free system will be used to characterize the joining of hairpin ends, which occurs during V(D)J recombination, identify the endonuclease that opens the hairpin ends, and determine how hairpin opening is regulated by DNA-PKcs.3. To define how NHEJ is regulated by protein phosphorylation. To determine the role of DNA-PKcs kinase activity in NHEJ, DNA-PKcs-mutant extracts will be supplemented with the active kinase or wortmannin-inactivated kinase and assayed for processing and joining of DNA ends. Experiments will determine whether phosphatases play a role in NHEJ, and identify physiologically relevant targets of DNA-PKcs. ? ?
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