DNA-PK phosphorylation sites in XRCC4 are not required for survival after radiation or for V(D)J recombination

DNA Repair (Amst). 2003 Nov 21;2(11):1239-52. doi: 10.1016/s1568-7864(03)00143-5.

Abstract

Nonhomologous end joining (NHEJ) is a major pathway for the repair of DNA double-strand breaks (DSBs) in higher eukaryotes. Several proteins, including the DNA-dependent protein kinase (DNA-PK), XRCC4 and DNA ligase IV, are required for nonhomologous end joining both in vitro and in vivo. Since XRCC4 is recruited to the DNA double-strand break with DNA-PK, and because the protein kinase activity of DNA-PK is required for its in vivo function, we reasoned that XRCC4 could be a potential physiological substrate of DNA-PK. Here, we have used mass spectrometry to map the DNA-PK phosphorylation sites in XRCC4. Two major phosphorylation sites (serines 260 and 318), as well as several minor sites were identified. All of the identified sites lie within the carboxy-terminal 100 amino acids of XRCC4. Substitution of each of these sites to alanine (in combination) reduced the ability of DNA-PK to phosphorylate XRCC4 in vitro by at least two orders of magnitude. However, XRCC4-deficient cells that were complemented with XRCC4 lacking DNA-PK phosphorylation sites were analogous to wild type XRCC4 with respect to survival after ionizing radiation and ability to repair DSBs introduced during V(D)J recombination.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alanine / metabolism
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Binding Sites
  • Cell Survival / radiation effects*
  • DNA Damage
  • DNA-Activated Protein Kinase
  • DNA-Binding Proteins / chemistry*
  • Dose-Response Relationship, Radiation
  • HeLa Cells
  • Humans
  • Nuclear Proteins
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / metabolism*
  • Radiation, Ionizing
  • Recombination, Genetic*
  • VDJ Recombinases / metabolism*

Substances

  • DNA-Binding Proteins
  • Nuclear Proteins
  • XRCC4 protein, human
  • DNA-Activated Protein Kinase
  • PRKDC protein, human
  • Protein-Serine-Threonine Kinases
  • VDJ Recombinases
  • Alanine