Open AccessMolecular and biochemical characterisation of DNA-dependent protein kinase-defective rodent mutant irs-20
Author(s) -
Anne Priestley,
Heather Beamish,
David A. Gell,
Aldo G Amatucci,
María C. Mühlmann-Díaz,
Belinda K. Singleton,
Graeme C.M. Smith,
Tracy Blunt,
Leonard C. Schalkwyk,
Joel S. Bedford,
Stephen Jackson,
Penny A. Jeggo,
Guillermo E. Taccioli
Publication year - 1998
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/26.8.1965
Subject(s) - dna pkcs , biology , microbiology and biotechnology , mutant , kinase , complementation , protein subunit , protein kinase a , dna repair , dna , biochemistry , gene
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.