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Molecular and Cellular Biology, May 1999, p. 3877-3884, Vol. 19, No. 5
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Requirement for the Kinase Activity of Human DNA-Dependent Protein Kinase Catalytic Subunit in DNA Strand Break Rejoining

Akihiro Kurimasa,1 Satoshi Kumano,1,2 Nikolai V. Boubnov,3 Michael D. Story,4 Chang-Shung Tung,5 Scott R. Peterson,1 and David J. Chen1,*

Life Sciences Division1 and Theoretical Biology and Biophysics,5 Los Alamos National Laboratory, Los Alamos, New Mexico 87545; Molecular and Cell Genetics, School of Life Sciences, Faculty of Medicine, Tottori University, Tottori 683, Japan2; Department of Biochemistry and Molecular Biology, St. Louis University, St. Louis, Missouri 631043; and Department of Experimental Radiotherapy, M. D. Anderson Cancer Center, University of Texas, Houston, Texas 770304

Received 5 October 1998/Returned for modification 13 November 1998/Accepted 16 February 1999

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is an enormous, 470-kDa protein serine/threonine kinase that has homology with members of the phosphatidylinositol (PI) 3-kinase superfamily. This protein contributes to the repair of DNA double-strand breaks (DSBs) by assembling broken ends of DNA molecules in combination with the DNA-binding factors Ku70 and Ku80. It may also serve as a molecular scaffold for recruiting DNA repair factors to DNA strand breaks. This study attempts to better define the role of protein kinase activity in the repair of DNA DSBs. We constructed a contiguous 14-kb human DNA-PKcs cDNA and demonstrated that it can complement the DNA DSB repair defects of two mutant cell lines known to be deficient in DNA-PKcs (M059J and V3). We then created deletion and site-directed mutations within the conserved PI 3-kinase domain of the DNA-PKcs gene to test the importance of protein kinase activity for DSB rejoining. These DNA-PKcs mutant constructs are able to express the protein but fail to complement the DNA DSB or V(D)J recombination defects of DNA-PKcs mutant cells. These results indicate that the protein kinase activity of DNA-PKcs is essential for the rejoining of DNA DSBs in mammalian cells. We have also determined a model structure for the DNA-PKcs kinase domain based on comparisons to the crystallographic structure of a cyclic AMP-dependent protein kinase. This structure gives some insight into which amino acid residues are crucial for the kinase activity in DNA-PKcs.

* Corresponding author. Mailing address: DNA Damage and Repair Group, Life Sciences Division, MS-M888, Los Alamos National Laboratory, Los Alamos, NM 87545. Phone: (505) 667-2789. Fax: (505) 665-0123. E-mail: dchen{at}telomere.lanl.gov.

Molecular and Cellular Biology, May 1999, p. 3877-3884, Vol. 19, No. 5
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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