Modulation of DNA-dependent protein kinase activity in chlorambucil-treated cells |
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| Institution: | 1. Department of Microbiology and Immunology, University of Texas Medical Branch at Galveston, Galveston, TX 77555, USA;2. Department of Human Biological Chemistry and Genetics, Sealy Center for Molecular Sciences, University of Texas Medical Branch at Galveston, Galveston, TX 77555, USA;1. Department of Biological Chemistry and Pharmacology, Columbus, OH 43210, USA;2. Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA;3. Ohio State Biochemistry Program, Columbus, OH 43210, USA;1. European Molecular Biology Laboratory, Grenoble Outstation, Univ. Grenoble Alpes-EMBL-CNRS, 71 avenue des Martyrs, 38042 France;2. Unit for Virus Host-Cell Interactions, Univ. Grenoble Alpes-EMBL-CNRS, 71 avenue des Martyrs, 38042 France;3. Department of Oncological Sciences, Icahn School of Medicine at Sinai, One Gustave L. Levy Place, NY 10029, USA;4. iRTSV, CEA-Grenoble, 17, rue des Martyrs, 38054 Grenoble Cedex 9, France;1. Microbiology and Physiological Systems Department, University of Massachusetts Medical School, University of Massachusetts, Worcester, MA 01655, USA;2. Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Ridgefield, CT 06877, USA;1. Council for Scientific and Industrial Research (CSIR)-Institute of Genomics and Integrative Biology, New Delhi, India;2. Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India;3. Department of Dermatology, PGIMER Dr. Ram Manohar Lohia Hospital, New Delhi, India;4. Department of Dermatology, Kalinga Institute of Medical Sciences, Kalinga Institute of Industrial Technology, Bhubaneswar, Odisha;5. Department of Dermatology, Dr. D.Y. Patil Medical College, Hospital & Research Centre, Dr. D.Y. Patil Vidyapeeth, Pune, India;6. Department of Bioscience and Bioengineering, Indian Institute of Technology, Jodhpur, Rajasthan, India;7. Translational Medicine Unit, Biomedical Research Centre, University of Salford, Manchester, United Kingdom;8. National Institute of Immunology, New Delhi, India;1. University of California Riverside, Department of Biomedical Sciences, Riverside, CA 92521, USA;2. University of Sheffield, Department of Biomedical Science, Sheffield S10 2TN, UK |
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| Abstract: | DNA-dependent protein kinase (DNA-PK) is activated in a two-step process whereby the Ku heterodimer first binds to the DNA double-strand breaks (dsbs) and then the DNA-PK catalytic subunit (cs) is recruited to form a repair complex. Oxidative stress is simultaneously generated along with DNA damage by ionizing radiation or chemotherapeutic agents whose impact on the DNA-PK activity has not previously been investigated. Here we show that the DNA damage-induced kinase activity of DNA-PK was modulated by oxidative stress, which was induced along with DNA dsbs in chlorambucil (Cbl)-exposed cells. Pretreatment with the antioxidants, 2(3)-t-butyl-4-hydroxyanisole or N-acetyl-l-cysteine enhanced the amount of DNA-PKcs phosphorylated at threonine 2609 (DNA-PKpThr2609) at the DNA dsbs and DNA-PK activity. Conversely, oxidative stress induced by l-buthionine (SR)-sulfoximine or glucose oxidase decreased the DNA-PK activity in Cbl-exposed cells. In addition, DNA-PKpThr2609 was poorly detectable at the site of DNA dsbs, as shown by colocalization to DNA-end-binding pH2AX or p53BP1. There was no change in the protein levels of DNA-PKcs, Ku70, or Ku86. Data from these studies provide the first evidence that oxidative stress effects posttranslational modification and assembly of DNA-PK complex at DNA dsbs, and thereby repair of DNA dsbs. |
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