Search Organism: All OrganismsArabidopsis thalianaBacillus subtilis 168Bos taurusCaenorhabditis elegansCanis familiarisDanio rerioDrosophila melanogasterEquus caballusEscherichia coli K12Gallus gallusHomo sapiensMacaca mulattaMus musculusNematostella vectensisOryza sativaPan troglodytesRattus norvegicusSaccharomyces cerevisiaeSchizosaccharomyces pombeStrongylocentrotus purpuratusTakifugu rubripesXenopus laevis home help / support contribute downloads mirrors about us J. Biochem. Sep (1998); 124(3):519-27 High mobility group proteins 1 and 2 can function as DNA-binding regulatory components for DNA-dependent protein kinase in vitro. Yumoto Y, Shirakawa H, Yoshida M, Suwa A, Watanabe F, Teraoka H Department of Pathological Biochemistry, Medical Research Institute, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101-0062, Japan. Abstract: The DNA-dependent protein kinase (DNA-PK) holoenzyme consists of a 470-kDa catalytic subunit (DNA-PKcs), a DNA-binding regulatory component known as Ku protein, and double-stranded DNA (dsDNA) with ends. We previously reported that the activity of DNA-PK in vitro is stimulated by non-histone chromosomal high mobility group proteins (HMG) 1 and 2 comprising two similar repeats, termed domains A and B, and an acidic C-terminal. Here we demonstrate that in vitro HMG1 and 2 can completely replace Ku protein as the DNA-binding regulatory component of DNA-PK. DNA-PKcs and Ku protein were separately purified from Raji nuclear extracts, and reconstituted into the DNA-PK holoenzyme in the presence of dsDNA. DNA-PKcs alone catalyzed DNA-dependent phosphorylation at a very low but significant level, and HMG1 and 2 markedly stimulated the phosphorylation of alpha-casein and a specific peptide substrate in a DNA-dependent manner. The HMG2-domains (A+B) polypeptide devoid of the C-terminal acidic region was more effective for DNA-PKcs stimulation than the full-length HMG2, and HMG2-domain A and -domain B polypeptides. Anti(Ku protein) antibodies inhibited the DNA-dependent phosphorylation activity of the DNA-PKcs:Ku protein complex, but not that of DNA-PKcs alone or when it was complexed with HMG1 or 2. These results demonstrate that HMG1 and 2 can function as the DNA-binding regulatory component for DNA-PKcs in vitro, and imply that a conformational change of dsDNA, which is elicited by regulatory components, is important for the stimulation of DNA-PK activity of DNA-PKcs. [PUBMED: 9722660] Copyright © 2009, Tyers Lab, The Samuel Lunenfeld Research Institute, Mount Sinai Hospital, All Rights Reserved. terms and conditions - privacy policy - Osprey Network Visualization System BioGRID: A General Repository for Interaction Datasets. Chris Stark, Bobby-Joe Breitkreutz, Teresa Reguly, Lorrie Boucher, Ashton Breitkreutz, Mike Tyers. Nucleic Acids Res. Jan 1;34:D535-9.