ABSTRACT
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-PKCSdisrupts 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.
ACKNOWLEDGMENTS
We thank O. Hammarsten, B. J. Hwang, D. Brutlag, and J. Danska for helpful conversations and P. Mayerfeld and J. Short for loaning the TD-700 fluorometer.
This research was supported by grant DAMD 17-94-J-4350 from the U.S. Army Medical Research and Materiel Command to G.C. and grant MT-13219 from the Medical Research Council of Canada to S.L., who is a Research Scientist of the National Cancer Institute of Canada supported with funds from the Canadian Cancer Society. V.S. is supported by the Life and Health Insurance Medical Research Fund sponsored by the American United Life Insurance Company. W.K.R. is supported by the Medical Scientist Training Program.