Abstract
DNA double strand break (DSBs) are major threats to the genomic integrity of cells. Eukaryotic cells have multiple pathways to repair DSBs, such as recombinational repair and end-joining. Several proteins involved in DNA repair, especially recombinational repair, have been shown to form higher order nuclear structures, radiation induced repair foci (RIRF), after induction of DSBs. However, little is known about how RIRF are formed after induction of DSBs. Recent advance in bio-imaging techniques make it possible to study the dynamics of nuclear structures formed by specific proteins in living cells.
RAD51, a eukaryotic RecA homologue, plays a central role in homologous recombinational repair of DSBs in yeast and is conserved from yeast to human. RAD51 showed punctuate nuclear localization in human cells, called RAD51 foci, typically during the S phase. To examine the dynamics of RAD51 after induction of DNA damage, we applied local irradiation of cell nuclei with a focused UV-laser (laser-UV-microirradiation). By use of laser-UVA microirradiation the localization of RAD51 at damaged sites containing DSBs could be demonstrated. The accumulation of RAD51 at microirradiated sites was visible 5 - 10 minutes after irradiation, and the number of cells with RAD51 accumulations increases until a plateau is reached 20 - 30 minutes after irradiation. We also studied the dynamics of RAD51 at microirradiated sites by combing lase-UV-maicroirradiation with living cell observation system. In this workshop, we would like to introduce our recent findings obtained by this new system and discuss about the dynamic organization of DNA repair system.
