Autophosphorylation of the catalytic subunit of the DNA-dependent protein kinase is required for efficient end processing during DNA double-strand break repair.
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.
Ding
Qi
Q
College of Veterinary Medicine and Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, Michigan 48824, USA.
Reddy
Yeturu V R
YV
Wang
Wei
W
Woods
Timothy
T
Douglas
Pauline
P
Ramsden
Dale A
DA
Lees-Miller
Susan P
SP
Meek
Katheryn
K
D000818:Animals; D034961:Antigens, Nuclear / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D016466:CHO Cells; D020134:Catalytic Domain; D006224:Cricetinae; D004249:DNA Damage; D004265:DNA Helicases; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004260:DNA Repair; D018076:DNA, Complementary / Q000378:metabolism; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins; D009841:Oligonucleotides / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D013997:Time Factors; D014162:Transfection; D045567:VDJ Recombinases
D034961:Antigens, Nuclear; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009841:Oligonucleotides; D011994:Recombinant Proteins; D001224:Aspartic Acid; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases; D004265:DNA Helicases; C106893:XRCC5 protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen