Binding of inositol phosphate to DNA-PK and stimulation of double-strand break repair.
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.
Hanakahi
L A
LA
Imperial Cancer Research Fund, South Mimms, Hertfordshire, United Kingdom.
Bartlet-Jones
M
M
Chappell
C
C
Pappin
D
D
West
S C
SC
D002458:Cell Fractionation; D002474:Cell-Free System; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair / Q000502:physiology*; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D005816:Genetic Complementation Test; D006367:HeLa Cells; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D009687:Nuclear Proteins; D010833:Phytic Acid / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D014316:Tritium
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D014316:Tritium; D010833:Phytic Acid; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases