DNA-PKcs and ATM co-regulate DNA double-strand break repair |
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| Authors: | Meena Shrivastav Cheryl A Miller Leyma P De Haro Stephen T Durant Benjamin PC Chen David J Chen Jac A Nickoloff |
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| Institution: | 1. Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87131, United States;2. Division of Molecular Radiation Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, United States;1. Department of Orthopaedic Surgery, West China Hospital, Sichuan University, NO. 37 Guoxuexiang Rd, Wuhou District, Chengdu 610041, China;2. Jining NO. 1 People''s Hospital, NO. 6 Jiankang Rd, Central District, Jining City, Shandong Province 272002, China;1. Department of Restorative Dentistry, University of Washington School of Dentistry, Seattle, WA 98195-7456, USA;2. Savannah River National Laboratory, Savannah River Nuclear Solutions, LLC, Aiken, SC 29808, USA;1. Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan;2. Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan;3. Department of Medical Laboratory Science and Biotechnology, College of Health Sciences, Kaohsiung Medical University, Kaohsiung 80708, Taiwan;4. Department of Electrical Engineering, College of Engineering, National Sun Yat-Sen University, Kaohsiung 80424, Taiwan;1. Education and Research Support Center, Graduate School of Medicine, Gunma University, Maebashi, Japan;2. Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Brighton BN19 RQ, UK;3. Radiation Biology and DNA Repair, Darmstadt University of Technology, 64287 Darmstadt, Germany;1. The Howard Hughes Medical Institute and Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA;2. Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA;3. Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA;4. Radiation Biology and DNA Repair, Darmstadt University of Technology, 64287 Darmstadt, Germany |
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| Abstract: | DNA double-strand breaks (DSBs) are repaired by nonhomologous end-joining (NHEJ) and homologous recombination (HR). The NHEJ/HR decision is under complex regulation and involves DNA-dependent protein kinase (DNA-PKcs). HR is elevated in DNA-PKcs null cells, but suppressed by DNA-PKcs kinase inhibitors, suggesting that kinase-inactive DNA-PKcs (DNA-PKcs-KR) would suppress HR. Here we use a direct repeat assay to monitor HR repair of DSBs induced by I-SceI nuclease. Surprisingly, DSB-induced HR in DNA-PKcs-KR cells was 2- to 3-fold above the elevated HR level of DNA-PKcs null cells, and ～4- to 7-fold above cells expressing wild-type DNA-PKcs. The hyperrecombination in DNA-PKcs-KR cells compared to DNA-PKcs null cells was also apparent as increased resistance to DNA crosslinks induced by mitomycin C. ATM phosphorylates many HR proteins, and ATM is expressed at a low level in cells lacking DNA-PKcs, but restored to wild-type level in cells expressing DNA-PKcs-KR. Several clusters of phosphorylation sites in DNA-PKcs, including the T2609 cluster, which is phosphorylated by DNA-PKcs and ATM, regulate access of repair factors to broken ends. Our results indicate that ATM-dependent phosphorylation of DNA-PKcs-KR contributes to the hyperrecombination phenotype. Interestingly, DNA-PKcs null cells showed more persistent ionizing radiation-induced RAD51 foci (but lower HR levels) compared to DNA-PKcs-KR cells, consistent with HR completion requiring RAD51 turnover. ATM may promote RAD51 turnover, suggesting a second (not mutually exclusive) mechanism by which restored ATM contributes to hyperrecombination in DNA-PKcs-KR cells. We propose a model in which DNA-PKcs and ATM coordinately regulate DSB repair by NHEJ and HR. |
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