Binding of Inositol Phosphate to DNA-PK and Stimulation of Double-Strand Break Repair

• Published in: Cell Vol. 102; no. 6; pp. 721 - 729
• Main Authors: Hanakahi, Les A, Bartlet-Jones, Michael, Chappell, Claire, Pappin, Darryl, West, Stephen C
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.09.2000
• Subjects: Cell Fractionation; Cell-Free System; DNA - metabolism; DNA ligase IV; DNA Repair - physiology; DNA-Activated Protein Kinase; DNA-Binding Proteins - analysis; DNA-Binding Proteins - isolation & purification; DNA-Binding Proteins - metabolism; DNA-dependent protein kinase; Genetic Complementation Test; HeLa Cells; Humans; inositol hexaphosphate; Magnetic Resonance Spectroscopy; Mass Spectrometry; Nuclear Proteins; Phytic Acid - analysis; Phytic Acid - isolation & purification; Phytic Acid - metabolism; Protein-Serine-Threonine Kinases - metabolism; Tritium
• ISSN: 0092-8674; 1097-4172
• DOI: 10.1016/S0092-8674(00)00061-1

In mammalian cells, double-strand breaks in DNA can be repaired by n on h omologous e nd -j oinin g (NHEJ), a process dependent upon Ku70/80, DNA-PK cs, 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 (IP 6). Purified IP 6 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-PK cs is similar to that found in the phosphatidylinositol 3 (PI 3)-kinase family.