Molecular and biochemical characterisation of DNA-dependent protein kinase-defective rodent mutant irs-20.
The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) is a member of a sub-family of phosphatidylinositol (PI) 3-kinases termed PIK-related kinases. A distinguishing feature of this sub-family is the presence of a conserved C-terminal region downstream of a PI 3-kinase domain. Mutants defective in DNA-PKcs are sensitive to ionising radiation and are unable to carry out V(D)J recombination. Irs-20 is a DNA-PKcs-defective cell line with milder gamma-ray sensitivity than two previously characterised mutants, V-3 and mouse scid cells. Here we show that the DNA-PKcs protein from irs-20 cells can bind to DNA but is unable to function as a protein kinase. To verify the defect in irs-20 cells and provide insight into the function and expression of DNA-PKcs in double-strand break repair and V(D)J recombination we introduced YACs encoding human and mouse DNA-PKcs into defective mutants and achieved complementation of the defective phenotypes. Furthermore, in irs-20 we identified a mutation in DNA-PKcs that causes substitution of a lysine for a glutamic acid in the fourth residue from the C-terminus. This represents a strong candidate for the inactivating mutation and provides supportive evidence that the extreme C-terminal motif is important for protein kinase activity.
Priestley
A
A
MRC Cell Mutation Unit, University of Sussex, Brighton BN1 9RR, UK.
Beamish
H J
HJ
Gell
D
D
Amatucci
A G
AG
Muhlmann-Diaz
M C
MC
Singleton
B K
BK
Smith
G C
GC
Blunt
T
T
Schalkwyk
L C
LC
Bedford
J S
JS
Jackson
S P
SP
Jeggo
P A
PA
Taccioli
G E
GE
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002470:Cell Survival / Q000528:radiation effects*; D018244:Chromosomes, Artificial, Yeast; D006224:Cricetinae; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D004307:Dose-Response Relationship, Radiation; D005720:Gamma Rays; D015723:Gene Library; D006736:Horses; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D009687:Nuclear Proteins; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency* / Q000378:metabolism*; D014162:Transfection; D045567:VDJ Recombinases
D004247:DNA; D004254:DNA Nucleotidyltransferases; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D045567:VDJ Recombinases; C497263:PRKDC protein, human