Failure of hairpin-ended and nicked DNA To activate DNA-dependent protein kinase: implications for V(D)J recombination.
V(D)J recombination is initiated by a coordinated cleavage reaction that nicks DNA at two sites and then forms a hairpin coding end and blunt signal end at each site. Following cleavage, the DNA ends are joined by a process that is incompletely understood but nevertheless depends on DNA-dependent protein kinase (DNA-PK), which consists of Ku and a 460-kDa catalytic subunit (DNA-PKCS or p460). Ku directs DNA-PKCS to DNA ends to efficiently activate the kinase. In vivo, the mouse SCID mutation in DNA-PKCS disrupts joining of the hairpin coding ends but spares joining of the open signal ends. To better understand the mechanism of V(D)J recombination, we measured the activation of DNA-PK by the three DNA structures formed during the cleavage reaction: open ends, DNA nicks, and hairpin ends. Although open DNA ends strongly activated DNA-PK, nicked DNA substrates and hairpin-ended DNA did not. Therefore, even though efficient processing of hairpin coding ends requires DNA-PKCS, this may occur by activation of the kinase bound to the cogenerated open signal end rather than to the hairpin end itself.
Departments of Medicine and Biochemistry, Stanford University Medical Center, Stanford, California 94305, USA.
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Smider
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Rathmell
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Brown
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Lewis
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Chu
D000818:Animals; D004247:DNA / Q000378:metabolism*; D004254:DNA Nucleotidyltransferases / Q000235:genetics*; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D051379:Mice; D016513:Mice, SCID; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D011995:Recombination, Genetic / Q000235:genetics*; D045567:VDJ Recombinases
D004268:DNA-Binding Proteins; D004247:DNA; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases