C-NHEJ without indels is robust and requires synergistic function of distinct XLF domains

Nat Commun. 2018 Jun 27;9(1):2484. doi: 10.1038/s41467-018-04867-5.


To investigate the fidelity of canonical non-homologous end joining (C-NHEJ), we developed an assay to detect EJ between distal ends of two Cas9-induced chromosomal breaks that are joined without causing insertion/deletion mutations (indels). Here we find that such EJ requires several core C-NHEJ factors, including XLF. Using variants of this assay, we find that C-NHEJ is required for EJ events that use 1-2, but not ≥3, nucleotides of terminal microhomology. We also investigated XLF residues required for EJ without indels, finding that one of two binding domains is essential (L115 or C-terminal lysines that bind XRCC4 and KU/DNA, respectively), and that disruption of one of these domains sensitizes XLF to mutations that affect its dimer interface, which we examined with molecular dynamic simulations. Thus, C-NHEJ, including synergistic function of distinct XLF domains, is required for EJ of chromosomal breaks without indels.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Cell Line
  • Chromosome Breakage*
  • DNA End-Joining Repair*
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Fibroblasts
  • INDEL Mutation
  • Ku Autoantigen / chemistry
  • Ku Autoantigen / genetics
  • Ku Autoantigen / metabolism
  • Mice
  • Molecular Dynamics Simulation
  • Mouse Embryonic Stem Cells
  • Protein Binding / genetics
  • Protein Domains / genetics*
  • Protein Multimerization


  • DNA-Binding Proteins
  • XLF protein, mouse
  • XRCC4 protein, mouse
  • Xrcc6 protein, mouse
  • Ku Autoantigen