Title: Live cell imaging and in situ analysis of DNA damage responses in mammalian cells.

Professor David Chen
Radiation Biology, UTSW

DNA double-strand breaks (DSBs) are the major DNA lesions induced by ionizing radiation. Unrepaired 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. Our result clearly demonstrated that DNA-PKcs and Ku proteins are localized to DSBs sites within second after laser exposure. Combination with genetics analysis and live cell imaging techniques, we are able to confirm the temporal relationship between DNA-PKcs and Ku proteins 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 and hence affect the efficiency of DSB repair. Our live cell imaging and protein dynamics studies provide new approaches to reveal the fundamental functions of DNA repair proteins in vivo.

UT Arlington Physics