Abstract
Analysis of the dynamics of the factors involved in DNA damage response is indispensable for comprehensive understandings of the radiation effects, especially the initial process of recognizing DNA double strand breaks on living organisms. However, previous studies have been performed mostly in fixed cells, which could not allow spatiotemporal analysis. We have established a live cell imaging system, by which the dynamic process of the recruitment of DNA damage checkpoint factors can be visualized. The experimental system uses the EGFP-tagged 53BP1 and MDC1 genes, and fluorescence microscope equipped with time-lapse digital imaging module. Upon irradiation, 53BP1 and MDC1 proteins were recruited to the sites of DNA double strand breaks, and they formed discrete foci. We confirmed the growth of the 53BP1 and MDC1 foci that were observed in fixed cells. In addition, we found that some foci emerged at the sites where previously showed no foci. The delayed foci induction has never been described in the previous literature, suggesting that illegitimate repair causes secondary changes in the higher-order chromatin structure.
