Differential phosphorylation of DNA-PKcs regulates the interplay between end-processing and end-ligation during nonhomologous end-joining

Mol Cell. 2015 Apr 2;58(1):172-85. doi: 10.1016/j.molcel.2015.02.024. Epub 2015 Mar 26.


Nonhomologous end-joining (NHEJ) is a major DNA double-strand break repair pathway that is conserved in eukaryotes. In vertebrates, NHEJ further acquires end-processing capacities (e.g., hairpin opening) in addition to direct end-ligation. The catalytic subunit of DNA-PK (DNA-PKcs) is a vertebrate-specific NHEJ factor that can be autophosphorylated or transphosphorylated by ATM kinase. Using a mouse model expressing a kinase-dead (KD) DNA-PKcs protein, we show that ATM-mediated transphosphorylation of DNA-PKcs regulates end-processing at the level of Artemis recruitment, while strict autophosphorylation of DNA-PKcs is necessary to relieve the physical blockage on end-ligation imposed by the DNA-PKcs protein itself. Accordingly, DNA-PKcs(KD/KD) mice and cells show severe end-ligation defects and p53- and Ku-dependent embryonic lethality, but open hairpin-sealed ends normally in the presence of ATM kinase activity. Together, our findings identify DNA-PKcs as the molecular switch that coordinates end-processing and end-ligation at the DNA ends through differential phosphorylations.

Publication types

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

MeSH terms

  • Animals
  • Antigens, Nuclear / genetics
  • Antigens, Nuclear / metabolism
  • Ataxia Telangiectasia Mutated Proteins / genetics
  • Ataxia Telangiectasia Mutated Proteins / metabolism
  • B-Lymphocytes / cytology
  • B-Lymphocytes / metabolism*
  • Cell Line
  • DNA Breaks, Double-Stranded
  • DNA End-Joining Repair / genetics*
  • DNA-Activated Protein Kinase / genetics*
  • DNA-Activated Protein Kinase / metabolism
  • DNA-Binding Proteins / genetics*
  • DNA-Binding Proteins / metabolism
  • Endonucleases / genetics*
  • Endonucleases / metabolism
  • Female
  • Gene Expression Regulation
  • Ku Autoantigen
  • Male
  • Mice
  • Mice, Transgenic
  • Nuclear Proteins / genetics*
  • Nuclear Proteins / metabolism
  • Phosphorylation
  • Signal Transduction
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism


  • Antigens, Nuclear
  • DNA-Binding Proteins
  • Nuclear Proteins
  • Tumor Suppressor Protein p53
  • Ataxia Telangiectasia Mutated Proteins
  • Atm protein, mouse
  • DNA-Activated Protein Kinase
  • Prkdc protein, mouse
  • Endonucleases
  • Dclre1c protein, mouse
  • Xrcc6 protein, mouse
  • Ku Autoantigen