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

Priestley, A and Beamish, HJ and Gell, D and Amatucci, AG and Muhlmann-Diaz, MC and Singleton, BK and Smith, GCM and Blunt, T and Schalkwyk, LC and Bedford, JS and Jackson, SP and Jeggo, PA and Taccioli, GE (1998) 'Molecular and biochemical characterisation of DNA-dependent protein kinase-defective rodent mutant irs-20.' Nucleic Acids Research, 26 (8). 1965 - 1973. ISSN 0305-1048

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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 Type: Article
Subjects: Q Science > QH Natural history > QH426 Genetics
Divisions: Faculty of Science and Health > Biological Sciences, School of
Depositing User: Jim Jamieson
Date Deposited: 23 Oct 2014 12:14
Last Modified: 17 Aug 2017 17:46
URI: http://repository.essex.ac.uk/id/eprint/11096

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