Binding of inositol phosphate to DNA-PK and stimulation of double-strand break repair

Cell. 2000 Sep 15;102(6):721-9. doi: 10.1016/s0092-8674(00)00061-1.

Abstract

In mammalian cells, double-strand breaks in DNA can be repaired by nonhomologous end-joining (NHEJ), a process dependent upon Ku70/80, DNA-PKcs, XRCC4, and DNA ligase IV. Starting with HeLa cell-free extracts, which promote NHEJ in a reaction dependent upon all of these proteins, we have purified a novel factor that stimulates DNA end-joining in vitro. Using a combination of phosphorus NMR, mass spectroscopy, and strong anion exchange chromatography, we identify this factor as inositol hexakisphosphate (IP6). Purified IP6 is bound by DNA-PK and specifically stimulates DNA-PK-dependent end-joining in vitro. The involvement of inositol phosphate in DNA-PK-dependent NHEJ is of particular interest since the catalytic domain of DNA-PKcs is similar to that found in the phosphatidylinositol 3 (PI 3)-kinase family.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Fractionation
  • Cell-Free System
  • DNA / metabolism*
  • DNA Repair / physiology*
  • DNA-Activated Protein Kinase
  • DNA-Binding Proteins / analysis
  • DNA-Binding Proteins / isolation & purification
  • DNA-Binding Proteins / metabolism
  • Genetic Complementation Test
  • HeLa Cells
  • Humans
  • Magnetic Resonance Spectroscopy
  • Mass Spectrometry
  • Nuclear Proteins
  • Phytic Acid / analysis
  • Phytic Acid / isolation & purification
  • Phytic Acid / metabolism*
  • Protein-Serine-Threonine Kinases / metabolism*
  • Tritium

Substances

  • DNA-Binding Proteins
  • Nuclear Proteins
  • Tritium
  • Phytic Acid
  • DNA
  • DNA-Activated Protein Kinase
  • PRKDC protein, human
  • Protein-Serine-Threonine Kinases