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Independent and sequential recruitment of NHEJ and HR factors to DNA damage sites in mammalian cells

¹ Department of Biological Chemistry, School of Medicine
² Beckman Laser Institute, Department of Surgery, Laser Microbeam and Medical Program, University of California, Irvine, Irvine, CA 92697
³ Department of Electrical Engineering and Bioscience, School of Science and Engineering, Waseda University, Tokyo 169-8555, Japan
⁴ Molecular Radiation Biology Division, Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, TX 75390
⁵ Cancer Research UK Institute for Cancer Studies, University of Birmingham, Edgbaston, Birmingham B15 2TT, England, UK

Correspondence to Kyoko Yokomori: kyokomor{at}uci.edu

Abstract

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.

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.

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