Abstract
DNA double-strand break (DSB) is considered most critical among DNA damages induced by radiation. Eukaryotic cells have evolved two major mechanisms to heal DSB: non-homologous end joining (NHEJ) and homologous recombination (HR). In NHEJ, DNA-PKcs, Ku86 and Ku70 comprise a sensor for DSB and XRCC4, DNA ligase IV and XLF join two DNA ends at the final step. However, there are several important missing links: true substrate(s) of DNA-PK and assembly of NHEJ machinery in vivo. We demonstrated that XRCC4 is phophorylated after irradiation in a manner dependent on DNA-PKcs in our earlier study. We have so far identified four phosphorylation sites. Phosphorylation specific antibody were generated against each of these sites and showed that all of them were phosphorylated in living cells in response to ionizing radiation. We also mutated these phosphorylation sites into alanine and found that three of these phosphorylation sites would be important for DNA repair function, as loss of them lead to elevated radiosensitivity with deficient DNA repair capability. In addition, we established a detergent-based fractionation analysis, which enable us to detect chromatin-binding of XRCC4. Using this system, we are now trying to clarify the requirements for the recruitment of XRCC4 to DSB sites. We further isolated higher order chromatin bound complex associated with XRCC4 by immunoprecipitation and analyzed its components by Western blotting and mass spectrometry. We would also discuss possible application of the outcome of these studies in cancer diagnosis and therapeutics.
