Constitutively active Artemis nuclease recognizes structures containing single-stranded DNA configurations |
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| Institution: | 1. Department of Pathology, Keck School of Medicine of University of Southern California, Los Angeles, CA, 90089, USA;2. Norris Comprehensive Cancer Center, Keck School of Medicine of University of Southern California, Los Angeles, CA, 90089, USA;3. Department of Biological Sciences, Molecular and Computational Biology Section, University of Southern California, Los Angeles, CA, 90089, USA;1. Institute for Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Laboratory Center, Erling Skjalgssons Gate 1, 7491 Trondheim, Norway;2. The NNF Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark;3. Howard Hughes Medical Institute, Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Department of Genetics, Harvard Medical School, Boston, MA 02115, United States;4. St. Olavs Hospital, Trondheim University Hospital, Clinic of Medicine, Postboks 3250 Sluppen, 7006 Trondheim, Norway;1. Programa de Pós-Graduação em Ciências Fisiológicas, Universidade Federal do Rio Grande-FURG, Av. Itália km 8, 96203-900 Rio Grande, Rio Grande do Sul, Brazil;2. Instituto de Ciências Biológicas, Universidade Federal do Rio Grande-FURG, Av. Itália km 8, 96203-900 Rio Grande, Rio Grande do Sul, Brazil;1. Department of Plant Pathology and Microbiology, Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University, Rehovot, 76100, Israel;2. Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden;3. de Botton Institute for Protein Profiling, the Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science, Rehovot, 76100, Israel;1. Department of Pharmacology and Toxicology and Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, United States;2. Centre for Genome Engineering, University of Calgary, Calgary, AB, Canada;3. Department of Oncology, Cross Cancer Institute and University of Alberta, Edmonton, AB, Canada;1. USC Norris Comprehensive Cancer Ctr, Departments of Pathology, of Biochemistry & Molecular Biology, of Molecular Microbiology & Immunology, University of Southern California Keck School of Medicine, and the Molecular and Computational Biology Section of the Department of Biological Sciences, 1441 Eastlake Ave., Rm. 5428, Los Angeles, CA, 90089-9176, United States;2. Department of Urology, USC Norris Comprehensive Cancer Ctr, Los Angeles, CA, 90089 United States |
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| Abstract: | The Artemis nuclease recognizes and endonucleolytically cleaves at single-stranded to double-stranded DNA (ss/dsDNA) boundaries. It is also a key enzyme in the non-homologous end joining (NHEJ) DNA double-strand break repair pathway. Previously, a truncated form, Artemis-413, was developed that is constitutively active both in vitro and in vivo. Here, we use this constitutively active form of Artemis to detect DNA structures with ss/dsDNA boundaries that arise under topological stress. Topoisomerases prevent abnormal levels of torsional stress through modulation of positive and negative supercoiling. We show that overexpression of Artemis-413 in yeast cells carrying genetic mutations that ablate topoisomerase activity have an increased frequency of DNA double-strand breaks (DSBs). Based on the biochemical activity of Artemis, this suggests an increase in ss/dsDNA-containing structures upon increased torsional stress, with DSBs arising due to Artemis cutting at these ss/dsDNA structures. Camptothecin targets topoisomerase IB (Top1), and cells treated with camptothecin show increased DSBs. We find that expression of Artemis-413 in camptothecin-treated cells leads to a reduction in DSBs, the opposite of what we find with topoisomerase genetic mutations. This contrast between outcomes not only confirms that topoisomerase mutation and topoisomerase poisoning have distinct effects on cells, but also demonstrates the usefulness of Artemis-413 to study changes in DNA structure. |
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| Keywords: | DNA repair Recombination Topoisomerases DNA double-strand breaks Artemis |
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