title: Molecular and biochemical characterisation of DNA-dependent protein kinase-defective rodent mutant irs-20.
abstract: 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.
authors.affiliation: MRC Cell Mutation Unit, University of Sussex, Brighton BN1 9RR, UK.
authors.forename: A
authors.initials: A
authors.lastname: Priestley
authors.forename: H J
authors.initials: HJ
authors.lastname: Beamish
authors.forename: D
authors.initials: D
authors.lastname: Gell
authors.forename: A G
authors.initials: AG
authors.lastname: Amatucci
authors.forename: M C
authors.initials: MC
authors.lastname: Muhlmann-Diaz
authors.forename: B K
authors.initials: BK
authors.lastname: Singleton
authors.forename: G C
authors.initials: GC
authors.lastname: Smith
authors.forename: T
authors.initials: T
authors.lastname: Blunt
authors.forename: L C
authors.initials: LC
authors.lastname: Schalkwyk
authors.forename: J S
authors.initials: JS
authors.lastname: Bedford
authors.forename: S P
authors.initials: SP
authors.lastname: Jackson
authors.forename: P A
authors.initials: PA
authors.lastname: Jeggo
authors.forename: G E
authors.initials: GE
authors.lastname: Taccioli
mesh_terms: 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
chemical_list: 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