Nucleic Acids Research Advance Access originally published online on May 25, 2007
Nucleic Acids Research 2007 35(12):3869-3878; doi:10.1093/nar/gkm339
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Nucleic Acids Research, 2007, Vol. 35, No. 12 3869-3878
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Phosphorylation in the serine/threonine 2609–2647 cluster promotes but is not essential for DNA-dependent protein kinase-mediated nonhomologous end joining in human whole-cell extracts
1Department of Pharmacology and Toxicology and 4Department of Radiation Oncology, Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, 2Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599 and 3Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada T2N 4N1
*To whom correspondence should be addressed. Tel: 804 828 9640; Fax: 804 827 0635; Email: LPOVIRK{at}vcu.edu
Received March 19, 2007. Revised April 19, 2007. Accepted April 21, 2007.
Previous work suggested that phosphorylation of DNA-PKcs at several serine/threonine (S/T) residues at positions 2609–2647 promotes DNA-PK-dependent end joining. In an attempt to clarify the role of such phosphorylation, end joining was examined in extracts of DNA-PKcs-deficient M059J cells. Joining of ends requiring gap filling prior to ligation was completely dependent on complementation of these extracts with exogenous DNA-PKcs. DNA-PKcs with either S/T 
A or S/T D substitutions at all six sites in the 2609–2647 cluster also supported end joining, but with markedly lower efficiency than wild-type protein. The residual end joining was greater with the S/T D-substituted than with the S/T A-substituted protein. A specific inhibitor of the kinase activity of DNA-PK, KU57788, completely blocked end joining promoted by wild type as well as both mutant forms of DNA-PK, while inhibition of ATM kinase did not. The fidelity of end joining was not affected by the mutant DNA-PKcs alleles or the inhibitors. Overall, the results support a role for autophosphorylation of the 2609–2647 cluster in promoting end joining and controlling the accessibility of DNA ends, but suggest that DNA-PK-mediated phosphorylation at other sites, on either DNA-PKcs or other proteins, is at least as important as the 2609–2647 cluster in regulating end joining.
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