Interaction of DNA-dependent protein kinase with DNA and with Ku: biochemical and atomic-force microscopy studies.
DNA-dependent protein kinase (DNA-PK or the scid factor) and Ku are critical for DNA end-joining in V(D)J recombination and in general non-homologous double-strand break repair. One model for the function of DNA-PK is that it forms a complex with Ku70/86, and this complex then binds to DNA ends, with Ku serving as the DNA-binding subunit. We find that DNA-PK can itself bind to linear DNA fragments ranging in size from 18 to 841 bp double-stranded (ds) DNA, as indicated by: (i) mobility shifts; (ii) crosslinking between the DNA and DNA-PK; and (iii) atomic-force microscopy. Binding of the 18 bp ds DNA to DNA-PK activates it for phosphorylation of protein targets, and this level of activation is not increased by addition of purified Ku70/86. Ku can stimulate DNA-PK activity beyond this level only when the DNA fragments are long enough for the independent binding to the DNA of both DNA-PK and Ku. Atomic-force microscopy indicates that under such conditions, the DNA-PK binds at the DNA termini, and Ku70/86 assumes a position along the ds DNA that is adjacent to the DNA-PK.
Yaneva
M
M
Department of Pathology, Washington University, St Louis, MO 63110, USA.
Kowalewski
T
T
Lieber
M R
MR
D034961:Antigens, Nuclear; D015153:Blotting, Western; D002364:Caseins / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D004265:DNA Helicases; D004260:DNA Repair; D004278:DNA, Superhelical / Q000378:metabolism; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000502:physiology; D004800:Enzymes, Immobilized; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen; D018625:Microscopy, Atomic Force; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D009690:Nucleic Acid Conformation; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014466:Ultraviolet Rays
D034961:Antigens, Nuclear; D002364:Caseins; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D004800:Enzymes, Immobilized; D009687:Nuclear Proteins; D011994:Recombinant Proteins; 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