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.
Smider
V
V
Departments of Medicine and Biochemistry, Stanford University Medical Center, Stanford, California 94305, USA.
Rathmell
W K
WK
Brown
G
G
Lewis
S
S
Chu
G
G
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