Abstract
DNA damage causes genome instability and cell death, but many of the cellular responses to DNA damage remain elusive. We apply in situ analysis of DNA damage responses by using laser micro irradiation of living human cells. We found a human protein, PALF, with a FHA (forkhead-associated) domain and novel zinc-finger-like CYR (Cystein-Tyrosine-Arginine) motifs that are involved in responses to DNA damage. The CYR motif is widely distributed amongst DNA repair proteins of higher eukaryotes, and that PALF, as well as a Drosophila protein with tandem CYR motifs, have AP-endonuclease and exonuclease activities. 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 γH2AX foci. Furthermore, we report another approach to screen and identify damage accumulating proteins by laser-micro-irradiation as well.
