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. 1998 Apr 15;26(8):1965–1973. doi: 10.1093/nar/26.8.1965

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

A Priestley 1, H J Beamish 1, D Gell 1, A G Amatucci 1, M C Muhlmann-Diaz 1, B K Singleton 1, G C Smith 1, T Blunt 1, L C Schalkwyk 1, J S Bedford 1, S P Jackson 1, P A Jeggo 1, G E Taccioli 1
PMCID: PMC147487  PMID: 9518490

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.

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Selected References

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