DNA-PK phosphorylation of RPA32 Ser4/Ser8 regulates replication stress checkpoint activation,fork restart,homologous recombination and mitotic catastrophe |
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| Affiliation: | 1. Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, CO, United States;2. Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM, United States;3. Department of Oral Biology, University of Nebraska Medical Center, Omaha, NE, United States;4. Eppley Cancer Center, University of Nebraska Medical Center, Omaha, NE, United States;1. Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), 41092 Sevilla, Spain;2. Departamento de Genética, Universidad de Sevilla, 41080, Sevilla, Spain;1. Institute for Cancer Genetics, Department of Pathology and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA;2. Pathobiology Graduate Program, Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA;3. Division of Pediatric Oncology, Hematology and Stem Cell Transplantation, Department of Pediatrics, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA;4. University of Pittsburg, Hillman Cancer Center Research Pavilion Suite 2.6, 5117 Centre Avenue, Pittsburgh, PA 15213-1863, USA;5. Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109, USA;1. Department of Biochemistry & Molecular Pharmacology, New York University School of Medicine, New York, NY, 10016, USA;2. Molecular Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA;1. Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA;2. Department of Oral Biology, University of Nebraska Medical Center, Omaha, NE 68583, USA;3. School of Interdisciplinary Informatics, College of Information Science and Technology, University of Nebraska at Omaha, Omaha, NE 68182, USA;4. Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland;5. Institute for Molecular Immunology, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), 81377 München, Germany;1. Department of Genetics, Harvard Medical School; Division of Genetics, Brigham and Women’s Hospital; Howard Hughes Medical Institute, Boston, MA 02115, USA;2. Department of Radiation Oncology, Massachusetts General Hospital, Boston, MA 02114, USA;3. Department of Medicine and Cancer Research Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA;4. Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA |
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| Abstract: | Genotoxins and other factors cause replication stress that activate the DNA damage response (DDR), comprising checkpoint and repair systems. The DDR suppresses cancer by promoting genome stability, and it regulates tumor resistance to chemo- and radiotherapy. Three members of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, ATM, ATR, and DNA-PK, are important DDR proteins. A key PIKK target is replication protein A (RPA), which binds single-stranded DNA and functions in DNA replication, DNA repair, and checkpoint signaling. An early response to replication stress is ATR activation, which occurs when RPA accumulates on ssDNA. Activated ATR phosphorylates many targets, including the RPA32 subunit of RPA, leading to Chk1 activation and replication arrest. DNA-PK also phosphorylates RPA32 in response to replication stress, and we demonstrate that cells with DNA-PK defects, or lacking RPA32 Ser4/Ser8 targeted by DNA-PK, confer similar phenotypes, including defective replication checkpoint arrest, hyper-recombination, premature replication fork restart, failure to block late origin firing, and increased mitotic catastrophe. We present evidence that hyper-recombination in these mutants is ATM-dependent, but the other defects are ATM-independent. These results indicate that DNA-PK and ATR signaling through RPA32 plays a critical role in promoting genome stability and cell survival in response to replication stress. |
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| Keywords: | DNA repair Replication stress Checkpoint regulation Homologous recombination |
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