DNA-dependent protein kinase is activated by nicks and larger single-stranded gaps.
DNA-PK is a DNA-activated serine/threonine protein kinase capable of phosphorylating a number of nuclear DNA-binding proteins. Purified human DNA-PK has two subunits, a 350-kDa polypeptide, Prkdc, which binds ATP and is presumed to contain the catalytic site, and the Ku autoantigen which mediates DNA binding and activation. Previous studies have shown that DNA-PK is activated in vitro by linear double-stranded DNA fragments; however, the Ku subunit binds a broader range of DNA structures. Here we show that EBP-80, a protein originally isolated as a transcription factor for a retroviral long terminal repeat element and subsequently found to be similar to if not identical with Ku, also mediates kinase activation. The EBP-80-Prkdc complex is activated by nanomolar concentrations of DNA constructs containing single-to-double strand transitions, including a closed stem-loop structure and single strand gaps of 0 (nick), 6, and 30 nucleotides. Kinase activation parallels the ability of EBP-80 to bind these and other constructs. Our results extend the range of DNA configurations known to activate DNA-PK and are consistent with the participation of this enzyme complex in several nuclear functions.
Morozov
V E
VE
Laboratory of Biochemistry, National Cancer Institute, Bethesda, Maryland 20892.
Falzon
M
M
Anderson
C W
CW
Kuff
E L
EL
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D034961:Antigens, Nuclear; D001324:Autoantigens / Q000378:metabolism; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004265:DNA Helicases; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D000072200:Ku Autoantigen; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D034961:Antigens, Nuclear; D001324:Autoantigens; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D010455:Peptides; D000255:Adenosine Triphosphate; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen