Real-time analysis of DNA damage response in living cell: novel methods, experimental significance and future perspective

While influence of ionizing radiation on cell has been analyzed by its sensitivity and mutation frequency, molecular mechanisms of the repair process of damaged DNA has been characterized mainly by in vitro analysis using synthesized DNA and purified proteins or cellular extracts. However, even in the most extensively characterized nucleotide excision repair in vitro repair activity attains only a few percentages of that obtained within cell, suggesting that there are effective but still uncovered mechanisms for the repair of DNA damage within cell. Therefore, to understand cellular mechanisms of DNA damage response, its molecular analysis in living cell is absolutely required. To come up with this demand, we have developed various local irradiation systems and analyzed real-time damage response of various proteins and protein complexes in living cell. Real-time visualization of cellular response to DNA damage is especially useful to identify novel proteins and mechanisms. Our recent findings of DNA strand breaks response in human cells will be presented. [References] Hong, Z. et al. Recruitment of mismatch repair proteins to the site of DNA damage in human cells. J. Cell Sci. in press., Hong, Z. et al. A polycomb group protein, PHF1, is involved in the response to DNA double-strand breaks in human cell. Nucleic Acids Res. 36, 2939-2947, 2008. Prasad, R. et al. HMGB1 is a co-factor in mammalian base excision repair. Mol. Cell, 27, 829-841, 2007, Kanno, S. et al. A novel human AP endonuclease with conserved zinc-finger-like motifs involved in DNA strand break responses, EMBO J. 26, 2094-2103, 2007, Hashiguchi, K. et al. Recruitment of DNA repair synthesis machinery to sites of DNA damage/repair in living cells. Nucleic Acids Res. 35, 2913-2923, 2007.