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Molecular and Cellular Biology, November 1998, p. 6853-6858, Vol. 18, No. 11
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Failure of Hairpin-Ended and Nicked DNA To Activate DNA-Dependent Protein Kinase: Implications for V(D)J Recombination

Vaughn Smider,1 W. Kimryn Rathmell,1 Greg Brown,2 Susanna Lewis,2 and Gilbert Chu1,*

Departments of Medicine and Biochemistry, Stanford University Medical Center, Stanford, California 94305,1 and Department of Immunology, University of Toronto, and Division of Immunology/Cancer, Hospital for Sick Children, Toronto, Ontario, Canada2

Received 1 April 1998/Returned for modification 9 June 1998/Accepted 12 August 1998

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.

* Corresponding author. Mailing address: M211, Division of Oncology, Stanford University Medical Center, Stanford, CA 94305-5115. Phone: (650) 725-6442. Fax: (650) 725-1420. E-mail: chu{at}cmgm.stanford.edu.

Molecular and Cellular Biology, November 1998, p. 6853-6858, Vol. 18, No. 11
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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