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Molecular and Cellular Biology, August 2003, p. 5836-5848, Vol. 23, No. 16
0270-7306/03/$08.00+0     DOI: 10.1128/MCB.23.16.5836-5848.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Autophosphorylation of the Catalytic Subunit of the DNA-Dependent Protein Kinase Is Required for Efficient End Processing during DNA Double-Strand Break Repair

Qi Ding,1 Yeturu V. R. Reddy,2 Wei Wang,1 Timothy Woods,1 Pauline Douglas,3 Dale A. Ramsden,2,4 Susan P. Lees-Miller,3 and Katheryn Meek1*

College of Veterinary Medicine and Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, Michigan 48824,1 Departments of Biochemistry and Molecular Biology and Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4,3 Lineberger Comprehensive Cancer Center,2 Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina4

Received 17 October 2002/ Returned for modification 22 November 2002/ Accepted 14 May 2003

The DNA-dependent protein kinase (DNA-PK) plays an essential role in nonhomologous DNA end joining (NHEJ) by initially recognizing and binding to DNA breaks. We have shown that in vitro, purified DNA-PK undergoes autophosphorylation, resulting in loss of activity and disassembly of the kinase complex. Thus, we have suggested that autophosphorylation of the DNA-PK catalytic subunit (DNA-PKcs) may be critical for subsequent steps in DNA repair. Recently, we defined seven autophosphorylation sites within DNA-PKcs. Six of these are tightly clustered within 38 residues of the 4,127-residue protein. Here, we show that while phosphorylation at any single site within the major cluster is not critical for DNA-PK's function in vivo, mutation of several sites abolishes the ability of DNA-PK to function in NHEJ. This is not due to general defects in DNA-PK activity, as studies of the mutant protein indicate that its kinase activity and ability to form a complex with DNA-bound Ku remain largely unchanged. However, analysis of rare coding joints and ends demonstrates that nucleolytic end processing is dramatically reduced in joints mediated by the mutant DNA-PKcs. We therefore suggest that autophosphorylation within the major cluster mediates a conformational change in the DNA-PK complex that is critical for DNA end processing. However, autophosphorylation at these sites may not be sufficient for kinase disassembly.

* Corresponding author. Mailing address: Michigan State University, 350 FST, East Lansing, MI 48824. Phone: (517) 432-9505. Fax: (517) 353-9004. E-mail: kmeek{at}msu.edu.

Molecular and Cellular Biology, August 2003, p. 5836-5848, Vol. 23, No. 16
0022-538X/03/$08.00+0     DOI: 10.1128/MCB.23.16.5836-5848.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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