Damage recognition by repair/checkpoint factors is the critical first step of the DNA damage response. DNA double strand breaks (DSBs) activate checkpoint signaling and are repaired by nonhomologous end-joining (NHEJ) and homologous recombination (HR) pathways. However, in vivo kinetics of the individual factor responses and the mechanism of pathway choice are not well understood. We report cell cycle and time course analyses of checkpoint activation by ataxia-telangiectasia mutated and damage site recruitment of the repair factors in response to laser-induced DSBs. We found that MRN acts as a DNA damage marker, continuously localizing at unrepaired damage sites. Damage recognition by NHEJ factors precedes that of HR factors. HR factor recruitment is not influenced by NHEJ factor assembly and occurs throughout interphase. Damage site retention of NHEJ factors is transient, whereas HR factors persist at unrepaired lesions, revealing unique roles of the two pathways in mammalian cells.
Independent and sequential recruitment of NHEJ and HR factors to DNA damage sites in mammalian cells
Jong-Soo Kim's present address is Neuroscience Research Institute, Gachon Medical School, Namdong-gu, Incheon 405-760, Korea.
Abbreviations used in this paper: ad, after damage; A-T, ataxia-telangiectasia; A-TLD, A-T-like disorder; A-TM, A-T mutated; A-TR, A-T and Rad3 related; DNA–PKcs, DNA-dependent protein kinase catalytic subunit; DSB, DNA double strand break; HR, homologous recombination; IR, ionizing radiation; IRIF, IR-induced foci; Nd:YAG, neodymium/yttrium-aluminum garnet; NHEJ, nonhomologous end joining; MRN, Mre11–Rad50–Nbs1; PARP-1, poly(ADP-ribose) polymerase 1; PIKK, PI3 kinase-related protein kinase; RPA, replication protein A; ssDNA, single-stranded DNA.
Jong-Soo Kim, Tatiana B. Krasieva, Hitoshi Kurumizaka, David J. Chen, A. Malcolm R. Taylor, Kyoko Yokomori; Independent and sequential recruitment of NHEJ and HR factors to DNA damage sites in mammalian cells . J Cell Biol 1 August 2005; 170 (3): 341–347. doi: https://doi.org/10.1083/jcb.200411083
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