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

• Published in: Nucleic acids research 2007-06, Vol.35 (12), p.3869-3878
• Main Authors: Povirk, Lawrence F, Zhou, Rui-Zhe, Ramsden, Dale A, Lees-Miller, Susan P, Valerie, Kristoffer
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Ataxia Telangiectasia Mutated Proteins; Catalysis; Cell Cycle Proteins - metabolism; Cell Extracts; Cell Line, Tumor; DNA Ligase ATP; DNA Ligases - metabolism; DNA-Activated Protein Kinase - chemistry; DNA-Activated Protein Kinase - genetics; DNA-Activated Protein Kinase - metabolism; DNA-Binding Proteins - metabolism; HeLa Cells; Humans; Nuclear Proteins - chemistry; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Nucleic Acid Enzymes; Phosphorylation; Protein-Serine-Threonine Kinases - metabolism; Serine - metabolism; Threonine - metabolism; Tumor Suppressor Proteins - metabolism
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkm339

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 [rightward arrow] A or S/T [rightward arrow] 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 [rightward arrow] D-substituted than with the S/T [rightward arrow] 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.