DNA nucleotide excision-repair synthesis is dependent of pertubations of deoxynucleoside triphosphate pool size |
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| Institution: | 1. Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL, USA;2. Howard Hughes Medical Institute and Children’s Medical Center Research Institute, UT Southwestern Medical Center, Dallas, TX, USA;1. Center for Cancer Research, National Cancer Institute (NCI), National Institutes of Health (NIH), Bethesda, MD 20892, USA;2. Center for Molecular Medicine, National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), Bethesda, MD 20892, USA;3. NIH-Georgetown University Graduate Partnership Program, Georgetown University Medical School, Washington, DC 20057, USA;4. Clinical Investigator Development Program, NCI, NIH, Bethesda, MD 20892, USA;5. Experimental Transplantation and Immunology Branch, NCI, NIH, Bethesda, MD 20892, USA;6. Laboratory of Molecular Immunology and the Immunology Center, NHLBI, NIH, Bethesda, MD 20892, USA;7. Hematology Branch, NHLBI, NIH, Bethesda, MD 20892, USA;8. Medical Scientist Training Program, The Ohio State University College of Medicine, Columbus, OH 43210, USA;9. Thoracic and GI Oncology Branch, NCI, NIH, Bethesda, MD 20892, USA;10. Sidra Medical and Research Center, Doha, Qatar;1. Laboratory of Angiogenesis and Vascular Metabolism, VIB Center for Cancer Biology, VIB, Leuven 3000, Belgium;2. State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou 510060, Guangdong, P.R. China;3. Laboratory of Angiogenesis and Vascular Metabolism, VIB-KU Leuven Center for Cancer Biology, Department of Oncology, KU Leuven, Leuven 3000, Belgium;4. The Koch Institute for Integrative Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA;5. Department of Developmental Biology and Cancer Research, The Hebrew University, Jerusalem 91120, Israel;6. Laboratory for Molecular Membrane Neuroscience, RIKEN Brain Science Institute, Wako City, Saimata 351-0198, Japan;7. Department of Bioscience and Biotechnology, Kyushu University, Fukuoka 812-8581, Japan;8. Dana-Farber Cancer Institute, Boston, MA 02115, USA;1. Department of Molecular Medicine, School of Advanced Medical Science, Tabriz University of Medical Sciences, Tabriz, Iran;2. Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran;3. Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran;4. Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran;5. Department of Medical Biotechnology, School of Advanced Medical Science, Tabriz University of Medical Sciences, Tabriz, Iran;6. Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada;1. Department of Biochemistry, Cancer Metabolism and Epigenetic Unit, Faculty of Science, Cancer and Mutagenesis Unit, King Fahd Center for Medical Research, King Abdulaziz University, Jeddah, Saudi Arabia;2. Molecular Oncology Laboratories, Department of Oncology, University of Oxford, Weatherall Institute of Molecular Medicine, Oxford OX3 9DS, UK |
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| Abstract: | We have investigated the effects of fluctuations in deoxynucleoside triphosphate (dNTP) pool size on DNA repair and, conversely, the effect of DNA repair on dNTP pool size. In confluent normal human skin fibroblasts, dNTP pool size was quantitated by the formation of 3H]TTP from 3H]thymidine; DNA repair was examined by repair replication in cultures irradiated with UV light. As defined by HPLC analysis, the 3H]TTP pool was formed within 30 min of the addition of 3H]thymidine and remained relatively constant for the next 6 h. Addition of 2–10 mM hydroxyurea (HU) caused a gradual 2–4-fold increase in the 3H]TTP pool as HU inhibited DNA synthesis but not TTP production. No difference was seen between the 3H]TTP pool size in cells exposed to 20 M/m2 and unrradiated controls, although DNA-repair synthesis was readily quantitated in the former. This result was observed even though the repair replication protocol caused an 8–10-fold reduction in the size of the 3H]TTP pool relative to the initial studies. In the UV excision-repair studies the precense of hydroxyurea did not alter the specific activity of 3H] thymidine 5'-monophospahte incorporated into parental DNA due to repaier replication. These results suggest that fluctuations in the deoxynucleoside triphosphate pools do not limit the extent of excision-repair sythesis in human cells and demonstrate that DNA nucleotide excision-repair synthesis does not significantly diminish the size of the 3H]TTP pool. |
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