Live cell analysis of cellular responses to various types of DNA damage induced by reactive oxygen

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Using local irradiation of UVA laser light through microscopic lens, we have developed methods to produce various types of oxidative DNA damage within human nucleus and analyzed real time response of proteins to the damage site. We identified a number of novel proteins involved in the repair of base damage, single-strand breaks and double-strand breaks. Furthermore, we applied proteome analysis by LC/MS/MS spectrometer and identified protein complexes necessary for the repair of oxidative DNA damage within human cell. DNA damage causes genome instability and cell death, but many of the cellular responses to DNA damage still remain elusive. We found, for example. a human protein, PALF (PNK and APTX-like FHA protein), with an FHA (forkhead-associated) domain and novel zinc-finger-like CYR (cysteine-tyrosine-arginine) motifs that are involved in responses to DNA damage. We found that the CYR motif is widely distributed among DNA repair proteins of higher eukaryotes, and that PALF, as well as a Drosophila protein with tandem CYR motifs, has endo- and exonuclease activities against abasic site and other types of base damage. PALF accumulates rapidly at single-strand breaks in a poly(ADP-ribose) polymerase 1 (PARP1)-dependent manner in human cells. Indeed, PALF interacts directly with PARP1 and is required for its activation and for cellular resistance to methyl-methane sulfonate. PALF also interacts directly with KU86, LIGASEIV and phosphorylated XRCC4 proteins and possesses endo/exonuclease activity at protruding DNA ends. Various treatments that produce double-strand breaks induce formation of PALF foci, which fully coincide with gammaH2AX foci. Thus, PALF and the CYR motif may play important roles in DNA repair of higher eukaryotes.