Synapsis of DNA ends by DNA-dependent protein kinase.
The catalytic subunit of DNA-dependent protein kinase (DNA-PK(CS)) is required for a non-homologous end-joining pathway that repairs DNA double-strand breaks produced by ionizing radiation or V(D)J recombination; however, its role in this pathway has remained obscure. Using a neutravidin pull-down assay, we found that DNA-PK(CS) mediates formation of a synaptic complex containing two DNA molecules. Furthermore, kinase activity was cooperative with respect to DNA concentration, suggesting that activation of the kinase occurs only after DNA synapsis. Electron microscopy revealed complexes of two DNA ends brought together by two DNA-PK(CS) molecules. Our results suggest that DNA-PK(CS) brings DNA ends together and then undergoes activation of its kinase, presumably to regulate subsequent steps for processing and ligation of the ends.
DeFazio
Lisa G
LG
Departments of Medicine and Biochemistry, Stanford University, Stanford, CA 94305, USA.
Stansel
Rachel M
RM
Griffith
Jack D
JD
Chu
Gilbert
G
D034961:Antigens, Nuclear; D023902:Chromosome Pairing / Q000502:physiology*; D004247:DNA / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D004249:DNA Damage; D004265:DNA Helicases; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen; D008957:Models, Genetic; D009687:Nuclear Proteins / Q000378:metabolism; D009690:Nucleic Acid Conformation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D009687:Nuclear 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