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Proc Natl Acad Sci U S A. 2015 May 19;112(20):E2575-84. doi: 10.1073/pnas.1420115112. Epub 2015 May 4.

Organization and dynamics of the nonhomologous end-joining machinery during DNA double-strand break repair.

Author information

1
Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016;
2
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.
3
Department of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599.
4
Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016; eli.rothenberg@nyumc.org.

Abstract

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.

KEYWORDS:

DNA repair; genomic integrity; nonhomologous end-joining; single-molecule FRET; super-resolution microscopy

PMID:
25941401
PMCID:
PMC4443322
DOI:
10.1073/pnas.1420115112
[Indexed for MEDLINE]
Free PMC Article

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