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.
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.
Povirk
Lawrence F
LF
Department of Pharmacology and Toxicology, Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, USA. LPOVIRK@vcu.edu
Zhou
Rui-Zhe
RZ
Ramsden
Dale A
DA
Lees-Miller
Susan P
SP
Valerie
Kristoffer
K
D019943:Amino Acid Substitution; D064007:Ataxia Telangiectasia Mutated Proteins; D002384:Catalysis; D018797:Cell Cycle Proteins / Q000378:metabolism; D002457:Cell Extracts; D045744:Cell Line, Tumor; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism; D051747:DNA-Activated Protein Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000378:metabolism
D018797:Cell Cycle Proteins; D002457:Cell Extracts; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D025521:Tumor Suppressor Proteins; D013912:Threonine; D012694:Serine; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D011088:DNA Ligases; D000072481:DNA Ligase ATP