| DNA double-strand breaks arise when cells are exposed to ionizing radiation or chemotherapeutic agents. DSBs are also intermediates during V(D)J and class switch recombination, processes that generate diversity in the immune repertoire by recombining different antibody gene segments. Independent of the source, DS:Bs in mammalian cells can be repaired by the process of nonhomologous end joining (NHEJ). During NHEJ, broken DNA ends are recognized, processed, and ligated in a nontemplated manner. The DNA-dependent protein kinase (DNA-PK) is a protein complex required for NHEJ. DNA-PK is composed of the Ku heterodimer, which recognizes the break and recruits the catalytic subunit, DNA-PKcs, to the DNA ends. Once bound to an end, DNA-PKcs is stimulated for its kinase activity. In order to understand the biochemistry of DNA-PKcs, we measured DNA binding and kinase activation under conditions where DNA-PKcs is active in the absence of Ku. We found that DNA-PKcs most avidly binds and is activated by DNA with both double- and single-stranded components. This finding is consistent with predictions from an electron crystallography reconstruction of DNA-PKcs. We used a biochemical pull-down assay and electron microscopy to show that two DNA-PKcs molecules mediate the synapsis of two DNA ends. Kinetic experiments suggested that DNA-PKcs kinase is not efficiently activated until after formation of the synaptic complex. We propose a model in which DNA-PKcs binds DNA ends and brings them together, and then single-stranded overhangs at the breaks interact with opposing DNA-PKcs proteins to stabilize the synaptic complex. DNA-PKcs then becomes an active kinase and may regulate the activities of other NHEJ proteins. Finally, we showed that inorganic polyphosphate, an abundant but enigmatic molecule in mammalian cells, inhibits DNA-PKcs kinase activity and NHEJ in a cell-free system. |
