Abstract
Recent advance in biological imaging has revealed that cell nucleus carries a dynamic higher order architecture showing the close association with nuclear functions, transcription, replication and DNA repair. However, little is known about the dynamic organization of higher order nuclear architectures including chromatin and non-chromatin nuclear domains after induction of DNA damage. To examine the dynamic organization of higher order nuclear architecture for DNA repair, we applied local irradiation of cell nuclei using a focused UV-laser (laser-UV-microirradiation). First, we analyzed the dynamics of DNA repair proteins in fixed human fibroblast cells by immunofluorescence staining after laser-UV-microirradiation. g-H2AX foci were detected within 3 min., and accumulations of MRE11 were observed 5 - 10 min. after irradiation, respectively. RAD51 showed accumulations from 5-10 min. after irradiation, and the number of cells carrying RAD51 accumulations increased until 20 - 30 min. after irradiation. PML showed colocalization with RAD51 accumulation 12 hours after laser-UV-microirradiation. These results suggest that DNA repair associated nuclear domains are formed at sites of DNA damage by the time dependent manner. Fluorescence Recovery after Photobleaching (FRAP) technique has make it possible to examine the dynamics of specific proteins in living cells. Now we are studying the dynamics of chromatin after laser-UVA-microirradiation using FRAP analysis of GFP tagged-histones in living cells. Dynamic organization of higher order nuclear architecture for DNA repair will be discussed.
