Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair.
Nonhomologous end-joining (NHEJ) is a major repair pathway for DNA double-strand breaks (DSBs), involving synapsis and ligation of the broken strands. We describe the use of in vivo and in vitro single-molecule methods to define the organization and interaction of NHEJ repair proteins at DSB ends. Super-resolution fluorescence microscopy allowed the precise visualization of XRCC4, XLF, and DNA ligase IV filaments adjacent to DSBs, which bridge the broken chromosome and direct rejoining. We show, by single-molecule FRET analysis of the Ku/XRCC4/XLF/DNA ligase IV NHEJ ligation complex, that end-to-end synapsis involves a dynamic positioning of the two ends relative to one another. Our observations form the basis of a new model for NHEJ that describes the mechanism whereby filament-forming proteins bridge DNA DSBs in vivo. In this scheme, the filaments at either end of the DSB interact dynamically to achieve optimal configuration and end-to-end positioning and ligation.
Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016;
Dylan A
DA
Reid
Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016;
Sarah
S
Keegan
Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016;
Alejandra
A
Leo-Macias
Department of Pathology, Biochemistry & Molecular Biology, Molecular Microbiology & Immunology, USC Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089; and.
Go
G
Watanabe
Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599.
Natasha T
NT
Strande
Department of Pathology, Biochemistry & Molecular Biology, Molecular Microbiology & Immunology, USC Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089; and.
Howard H
HH
Chang
Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016;
Betul Akgol
BA
Oksuz
Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016;
David
D
Fenyo
Department of Pathology, Biochemistry & Molecular Biology, Molecular Microbiology & Immunology, USC Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089; and.
Michael R
MR
Lieber
Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599.
Dale A
DA
Ramsden
Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016; eli.rothenberg@nyumc.org.
Eli
E
Rothenberg
D015153:Blotting, Western; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair / Q000502:physiology*; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005455:Fluorescent Antibody Technique; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular
D004268:DNA-Binding Proteins; C000607893:LIG4 protein, human; C101467:XRCC4 protein, human; D011088:DNA Ligases; D000072481:DNA Ligase ATP
DNA repair; genomic integrity; nonhomologous end-joining; single-molecule FRET; super-resolution microscopy