title: Two-Stage Synapsis of DNA Ends during Non-homologous End Joining.
abstract: Repair of DNA double-strand breaks (DSBs) is essential for genomic stability. The most common DSB repair mechanism in human cells, non-homologous end joining (NHEJ), rejoins broken DNA ends by direct ligation. It remains unclear how components of the NHEJ machinery assemble a synaptic complex that bridges DNA ends. Here, we use single-molecule imaging in a vertebrate cell-free extract to show that synapsis of DNA ends occurs in at least two stages that are controlled by different NHEJ factors. DNA ends are initially tethered in a long-range complex whose formation requires the Ku70/80 heterodimer and the DNA-dependent protein kinase catalytic subunit. The ends are then closely aligned, which requires XLF, a non-catalytic function of XRCC4-LIG4, and DNA-PK activity. These results reveal a structural transition in the synaptic complex that governs alignment of DNA ends. Our approach provides a means of studying physiological DNA DSB repair at single-molecule resolution.
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Thomas G W
authors.initials: TG
authors.lastname: Graham
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA. Electronic address: johannes_walter@hms.harvard.edu.
authors.forename: Johannes C
authors.initials: JC
authors.lastname: Walter
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA. Electronic address: joseph_loparo@hms.harvard.edu.
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Loparo
mesh_terms: D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism; D002474:Cell-Free System; D023902:Chromosome Pairing / Q000235:genetics*; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair / Q000235:genetics*; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics*; D045643:DNA Repair Enzymes / Q000378:metabolism; D051747:DNA-Activated Protein Kinase / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D000072200:Ku Autoantigen; D057054:Molecular Imaging; D011485:Protein Binding
chemical_list: D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; C000607893:LIG4 protein, human; C508551:NHEJ1 protein, human; C101467:XRCC4 protein, human; D051747:DNA-Activated Protein Kinase; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; D011088:DNA Ligases; D045643:DNA Repair Enzymes; D000072481:DNA Ligase ATP