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J. Biol. Chem., Vol. 279, Issue 38, 39408-39413, September 17, 2004
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From the
Lineberger Comprehensive Cancer Center and Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, College of Veterinary Medicine and Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, Michigan 48824, and ¶Departments of Biochemistry and Molecular Biology and Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada
Repair of chromosome breaks by non-homologous end joining requires the XRCC4-ligase IV complex, Ku, and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PKcs must also retain kinase activity and undergo autophosphorylation at six closely linked sites (ABCDE sites). We describe here an end-joining assay using only purified components that reflects cellular requirements for both Ku and kinase-active DNA-PKcs and investigate the mechanistic basis for these requirements. A need for DNA-PKcs autophosphorylation is sufficient to explain the requirement for kinase activity, in part because autophosphorylation is generally required for end-joining factors to access DNA ends. However, DNA-PKcs with all six ABCDE autophosphorylation sites mutated to alanine allows access to ends through autophosphorylation of other sites, yet our in vitro end-joining assay still reflects the defectiveness of this mutant in cellular end joining. In contrast, mutation of ABCDE sites to aspartate, a phosphorylation mimic, supports high levels of end joining that is now independent of kinase activity. This is likely because DNA-PKcs with aspartate substitutions at ABCDE sites allow access to DNA ends while retaining affinity for Ku-bound ends and stabilizing recruitment of the XRCC4-ligase IV complex. Autophosphorylation at ABCDE sites thus apparently directs a rearrangement of the DNA-PK complex that ensures access to broken ends and joining steps are coupled together within a synaptic complex, making repair more accurate.
Received for publication, June 9, 2004 , and in revised form, July 15, 2004.
* This work was supported by U. S. Public Health Service Grants CA-84442 (to D. A. R.) and AI32600 and AI42938 (to K. M.) and by the Alberta Heritage Foundation for Medical Research and the Canadian Institutes for Health Research (to the laboratory of S. P. L.-M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|| To whom correspondence should be addressed: Lineberger Comprehensive Cancer Center and Dept. of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, CB-7295, Chapel Hill, NC 27599. Tel.: 919-966-9839; Fax: 919-966-3015. E-mail: dale_ramsden{at}med.unc.edu.
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