Abstract
DNA double-strand breaks (DSBs) are the major DNA lesions induced by ionizing radiation. Unrepired and/or misrepaired DSBs cause genome instability and leads to the onset of cancer. DNA-dependent protein kinase complex (DNA-PK), consisting of Ku70 and Ku80 heterodimer and DNA-PK catalytic subunit (DNA-PKCS), is the key component of the non-homologous end-joining pathway of DNA DSB repair. To elucidate the mechanisms by which DNA-PK complex is recruited to DSBs in vivo and the temporal relationship with other DNA repair factors, we utilize a UV microbeam laser system to generate DSBs and live cell imaging approach to analyze protein dynamics at the laser induced DNA damage sites in live cells. Combination with genetics analysis and live cell imaging techniques, we are able to determine the temporal relationship among Ku, DNA-PKcs, XRCC4 and XLF at DSB sites upon laser damage. Further more, the use of photobleaching techniques (FRAP) has allowed us to determine that phosphorylation status of DNA-PKCS influence the stability of its binding to DNA ends. This allowed us to determine that disabling the kinase activity and phosphorylation status of DNA-PKcs leads to a more rigid binding of DNA-PKCS to DNA ends, which may interfere with effective ligation.
