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

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


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Western
  • Cell Line, Tumor
  • DNA Breaks, Double-Stranded*
  • DNA End-Joining Repair / physiology*
  • DNA Ligase ATP
  • DNA Ligases / metabolism*
  • DNA-Binding Proteins / metabolism*
  • Fluorescence Resonance Energy Transfer
  • Fluorescent Antibody Technique
  • Humans
  • Kinetics
  • Models, Molecular*


  • DNA-Binding Proteins
  • LIG4 protein, human
  • XRCC4 protein, human
  • DNA Ligases
  • DNA Ligase ATP