A noncatalytic function of the ligation complex during nonhomologous end joining

J Cell Biol. 2013 Jan 21;200(2):173-86. doi: 10.1083/jcb.201203128.

Abstract

Nonhomologous end joining is the primary deoxyribonucleic acid (DNA) double-strand break repair pathway in multicellular eukaryotes. To initiate repair, Ku binds DNA ends and recruits the DNA-dependent protein kinase (DNA-PK) catalytic subunit (DNA-PKcs) forming the holoenzyme. Early end synapsis is associated with kinase autophosphorylation. The XRCC4 (X4)-DNA Ligase IV (LIG4) complex (X4LIG4) executes the final ligation promoted by Cernunnos (Cer)-X4-like factor (XLF). In this paper, using a cell-free system that recapitulates end synapsis and DNA-PKcs autophosphorylation, we found a defect in both activities in human cell extracts lacking LIG4. LIG4 also stimulated the DNA-PKcs autophosphorylation in a reconstitution assay with purified components. We additionally uncovered a kinase autophosphorylation defect in LIG4-defective cells that was corrected by ectopic expression of catalytically dead LIG4. Finally, our data support a contribution of Cer-XLF to this unexpected early role of the ligation complex in end joining. We propose that productive end joining occurs by early formation of a supramolecular entity containing both DNA-PK and X4LIG4-Cer-XLF complexes on DNA ends.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell-Free System / metabolism
  • Cells, Cultured
  • DNA Damage
  • DNA End-Joining Repair / physiology*
  • DNA Helicases / metabolism
  • DNA Helicases / physiology
  • DNA Ligase ATP
  • DNA Ligases / metabolism
  • DNA Ligases / physiology*
  • DNA Repair Enzymes / metabolism
  • DNA Repair Enzymes / physiology
  • DNA-Activated Protein Kinase / metabolism
  • DNA-Binding Proteins / metabolism
  • DNA-Binding Proteins / physiology*
  • Genomic Instability
  • Holoenzymes
  • Humans
  • Ku Autoantigen
  • Phosphorylation

Substances

  • DNA-Binding Proteins
  • Holoenzymes
  • LIG4 protein, human
  • NHEJ1 protein, human
  • XRCC4 protein, human
  • DNA-Activated Protein Kinase
  • DNA Helicases
  • XRCC5 protein, human
  • Ku Autoantigen
  • DNA Ligases
  • DNA Repair Enzymes
  • DNA Ligase ATP