In vitro complementation of Tdp1 deficiency indicates a stabilized enzyme-DNA adduct from tyrosyl but not glycolate lesions as a consequence of the SCAN1 mutation |
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| Authors: | Amy J. Hawkins Mark A. Subler Konstantin Akopiants Jenny L. Wiley Shirley M. Taylor Ann C. Rice Jolene J. Windle Kristoffer Valerie Lawrence F. Povirk |
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| Affiliation: | 1. Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA 23298, USA;2. Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA 23298, USA;3. Department of Radiation Oncology, Virginia Commonwealth University, Richmond, VA 23298, USA;4. Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA 23298, USA;5. Department of Neurology, Virginia Commonwealth University, Richmond, VA 23298, USA;6. The Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA;1. Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, VA, USA;2. Department of Neurology, Virginia Commonwealth University, Richmond, VA, USA;3. Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA, USA;4. Department of Pharmacotherapy and Outcomes Science, Virginia Commonwealth University, Richmond, VA, USA;5. Center for Biomarker Research and Precision Medicine, Virginia Commonwealth University, Richmond, VA, USA;1. Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA;2. Molecular Genetics Section, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA;3. Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA;1. Research Institute of the Children’s Hospital of Philadelphia, Philadelphia, PA, USA;2. Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA;3. Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA;4. Department of Bioengineering, School of Engineering and Applied Sciences, University of Pennsylvania, Philadelphia, PA, USA;5. W.F. Goodman Center for Comparative Medical Genetics, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA;1. Department of Gastroenterology, Hepatology, and Nutrition, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA;2. Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA;3. Department of Biostatistics, West Virginia University Health Sciences Center, Morgantown, West Virginia, USA;4. Department of Surgical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA;5. Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy;6. Department of Digestive Tract and General Surgery, Saitama Medical University, Saitama, Japan;7. Division of Surgical Oncology, Hereditary Cancer Registry, Hospital Sirio Libanes, Sao Paulo, Brazil;8. Department of Gastroenterology, Institut de Malalties Digestives i Metabòliques, Hospital Clinic, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Institut d''Investigacions Biomèdiques August Pi I Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain;9. Division of Internal Medicine, University of Michigan Health System, Ann Arbor, Michigan, USA;10. Section of Colon and Rectal Surgery, Washington University School of Medicine, St. Louis, Missouri, USA;11. Unit of Hereditary Digestive Tract Tumors, Fondazione IRCCS, Istituto Nazionale dei Tumori, Milan, Italy;12. Wolfson Unit for Endoscopy, St. Marks Hospital, Harrow, Middlesex, United Kingdom;13. Department of Internal Medicine, Huntsman Cancer Institute, University of Utah, Salt Lake City, Utah, USA;14. Department of Medical and Surgical Sciences, S. Orsola-Malpighi University Hospital, Bologna, Italy;15. Division of Gastroenterology, Department of Veterans Affairs Eastern Colorado Health Care System and University of Colorado School of Medicine, Denver, Colorado, USA;16. Department of Surgery, Hyogo College of Medicine, Hyogo, Japan;17. The Danish Polyposis Register, Gastrointestinal Unit, Hvidovre University Hospital, Copenhagen, Denmark;18. The Polyposis Registry, St. Mark’s Hospital, Harrow, Middlesex, United Kingdom;19. Division of Gastroenterology, Hepatology and Nutrition, Nationwide Children’s Hospital, The Ohio State University, Columbus, Ohio, USA;20. Gastroenterology and Nutrition Service, Memorial Sloan-Kettering Cancer Center, New York, New York, USA;21. Department of Surgical Gastroenterology, Aalborg Universitetshospital, Aalborg, Denmark;22. Division of Population Sciences, Division of Gastroenterology, Dana-Farber Cancer Institute, Brigham and Women''s Hospital, and Harvard Medical School, Boston, Massachusetts, USA;23. Center for Hereditary Tumors, HELIOS Klinikum Wuppertal, University Witten-Herdecke, Wuppertal, Germany |
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| Abstract: | A homozygous H493R mutation in the active site of tyrosyl-DNA phosphodiesterase (TDP1) has been implicated in hereditary spinocerebellar ataxia with axonal neuropathy (SCAN1), an autosomal recessive neurodegenerative disease. However, it is uncertain how the H493R mutation elicits the specific pathologies of SCAN1. To address this question, and to further elucidate the role of TDP1 in repair of DNA end modifications and general physiology, we generated a Tdp1 knockout mouse and carried out detailed behavioral analyses as well as characterization of repair deficiencies in extracts of embryo fibroblasts from these animals. While Tdp1?/? mice appear phenotypically normal, extracts from Tdp1?/? fibroblasts exhibited deficiencies in processing 3′-phosphotyrosyl single-strand breaks and 3′-phosphoglycolate double-strand breaks (DSBs), but not 3′-phosphoglycolate single-strand breaks. Supplementing Tdp1?/? extracts with H493R TDP1 partially restored processing of 3′-phosphotyrosyl single-strand breaks, but with evidence of persistent covalent adducts between TDP1 and DNA, consistent with a proposed intermediate-stabilization effect of the SCAN1 mutation. However, H493R TDP1 supplementation had no effect on phosphoglycolate (PG) termini on 3′ overhangs of double-strand breaks; these remained completely unprocessed. Altogether, these results suggest that for 3′-phosphoglycolate overhang lesions, the SCAN1 mutation confers loss of function, while for 3′-phosphotyrosyl lesions, the mutation uniquely stabilizes a reaction intermediate. |
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