Abstract
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.
Publication types
- Research Support, U.S. Gov't, P.H.S.
MeSH terms
- Antigens, Nuclear*
- Blotting, Western
- Caseins / metabolism
- DNA / chemistry
- DNA / metabolism*
- DNA / ultrastructure
- DNA Helicases*
- DNA Repair
- DNA, Superhelical / metabolism
- DNA-Activated Protein Kinase
- DNA-Binding Proteins / isolation & purification
- DNA-Binding Proteins / metabolism*
- DNA-Binding Proteins / ultrastructure
- Electrophoresis, Polyacrylamide Gel
- Enzyme Activation / physiology
- Enzymes, Immobilized
- HeLa Cells
- Humans
- Ku Autoantigen
- Microscopy, Atomic Force
- Nuclear Proteins / isolation & purification
- Nuclear Proteins / metabolism*
- Nuclear Proteins / ultrastructure
- Nucleic Acid Conformation
- Phosphorylation
- Protein Serine-Threonine Kinases / isolation & purification
- Protein Serine-Threonine Kinases / metabolism*
- Protein Serine-Threonine Kinases / ultrastructure
- Recombinant Proteins / genetics
- Recombinant Proteins / metabolism
- Ultraviolet Rays
Substances
- Antigens, Nuclear
- Caseins
- DNA, Superhelical
- DNA-Binding Proteins
- Enzymes, Immobilized
- Nuclear Proteins
- Recombinant Proteins
- DNA
- DNA-Activated Protein Kinase
- PRKDC protein, human
- Protein Serine-Threonine Kinases
- DNA Helicases
- XRCC5 protein, human
- Xrcc6 protein, human
- Ku Autoantigen