title: Different DNA End Configurations Dictate Which NHEJ Components Are Most Important for Joining Efficiency.
abstract: The nonhomologous DNA end-joining (NHEJ) pathway is a key mechanism for repairing dsDNA breaks that occur often in eukaryotic cells. In the simplest model, these breaks are first recognized by Ku, which then interacts with other NHEJ proteins to improve their affinity at DNA ends. These include DNA-PKcsand Artemis for trimming the DNA ends; DNA polymerase μ and λ to add nucleotides; and the DNA ligase IV complex to ligate the ends with the additional factors, XRCC4 (X-ray repair cross-complementing protein 4), XLF (XRCC4-like factor/Cernunos), and PAXX (paralog of XRCC4 and XLF). In vivo studies have demonstrated the degrees of importance of these NHEJ proteins in the mechanism of repair of dsDNA breaks, but interpretations can be confounded by other cellular processes. In vitro studies with NHEJ proteins have been performed to evaluate the nucleolytic resection, polymerization, and ligation steps, but a complete system has been elusive. Here we have developed a NHEJ reconstitution system that includes the nuclease, polymerase, and ligase components to evaluate relative NHEJ efficiency and analyze ligated junctional sequences for various types of DNA ends, including blunt, 5' overhangs, and 3' overhangs. We find that different dsDNA end structures have differential dependence on these enzymatic components. The dependence of some end joining on only Ku and XRCC4·DNA ligase IV allows us to formulate a physical model that incorporates nuclease and polymerase components as needed.
authors.affiliation: From the Departments of Pathology, Biochemistry & Molecular Biology, and Molecular Microbiology & Immunology and the Section of Molecular & Computational Biology, Department of Biological Sciences, Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA, 90033 and.
authors.forename: Howard H Y
authors.initials: HHY
authors.lastname: Chang
authors.affiliation: From the Departments of Pathology, Biochemistry & Molecular Biology, and Molecular Microbiology & Immunology and the Section of Molecular & Computational Biology, Department of Biological Sciences, Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA, 90033 and.
authors.forename: Go
authors.initials: G
authors.lastname: Watanabe
authors.affiliation: From the Departments of Pathology, Biochemistry & Molecular Biology, and Molecular Microbiology & Immunology and the Section of Molecular & Computational Biology, Department of Biological Sciences, Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA, 90033 and.
authors.forename: Christina A
authors.initials: CA
authors.lastname: Gerodimos
authors.affiliation: the Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge CB2 1QN, United Kingdom.
authors.forename: Takashi
authors.initials: T
authors.lastname: Ochi
authors.affiliation: the Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge CB2 1QN, United Kingdom.
authors.forename: Tom L
authors.initials: TL
authors.lastname: Blundell
authors.affiliation: the Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge CB2 1QN, United Kingdom.
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Jackson
authors.affiliation: From the Departments of Pathology, Biochemistry & Molecular Biology, and Molecular Microbiology & Immunology and the Section of Molecular & Computational Biology, Department of Biological Sciences, Norris Comprehensive Cancer Center, University of Southern California Keck School of Medicine, Los Angeles, CA, 90033 and. Electronic address: lieber@usc.edu.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Lieber
mesh_terms: D000818:Animals; D002460:Cell Line; D059766:DNA End-Joining Repair; D000072481:DNA Ligase ATP / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D045643:DNA Repair Enzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D000072200:Ku Autoantigen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera
chemical_list: D004268:DNA-Binding Proteins; C508551:NHEJ1 protein, human; C101467:XRCC4 protein, human; C106893:XRCC5 protein, human; D000072200:Ku Autoantigen; D045643:DNA Repair Enzymes; D000072481:DNA Ligase ATP
keywords: DNA damage; DNA recombination; DNA repair; chromosomes; double-strand DNA break; enzyme; ligase; nuclease