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Molecular and biochemical characterisation of DNA-dependent protein kinase-defective rodent mutant irs-20

Citation

Priestley, A and Beamish, HJ and Gell, D and Amatucci, AG and Muhlmann-Diaz, MC and Singleton, BK and Smith, GC and Blunt, T and Schalkwyk, LC and Bedford, JS and Jackson, SP and Jeggo, PA and Taccioli, GE, Molecular and biochemical characterisation of DNA-dependent protein kinase-defective rodent mutant irs-20, Nucleic Acids Research, 26, (8) pp. 1965-1973. ISSN 0305-1048 (1998) [Refereed Article]

DOI: doi:10.1093/nar/26.8.1965

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 γ-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.

Item Details

Item Type:Refereed Article
Research Division:Biological Sciences
Research Group:Biochemistry and Cell Biology
Research Field:Enzymes
Objective Division:Expanding Knowledge
Objective Group:Expanding Knowledge
Objective Field:Expanding Knowledge in the Biological Sciences
Author:Gell, D (Dr David Gell)
ID Code:64590
Year Published:1998
Web of Science® Times Cited:68
Deposited By:Menzies Institute for Medical Research
Deposited On:2010-08-12
Last Modified:2011-08-10
Downloads:0

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