Nucleic Acids Research, 2000, Vol. 28, No. 7 1506-1513
© 2000 Oxford University Press
The C-terminal conserved domain of DNA-PKcs, missing in the SCID mouse, is required for kinase activity
MRC Cell Mutation Unit, University of Sussex, Brighton BN1 9RR, UK and 1Basel Institute for Immunology, Grenzacherstrasse 487, CH-4005 Basel, Switzerland
DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), has a phosphoinositol 3-kinase (PI 3-K) domain close to its C-terminus. Cell lines derived from the SCID mouse have been utilised as a model DNA-PKcs-defective system. The SCID mutation results in truncation of DNA-Pkcs at the extreme C-terminus leaving the PI 3-K domain intact. The mutated protein is expressed at low levels in most SCID cell lines, leaving open the question of whether the mutation abolishes kinase activity. Here, we show that a SCID cell line that expresses the mutant protein normally has dramatically impaired kinase activity. We estimate that the residual kinase activity typically present in SCID fibroblast cell lines is at least two orders of magnitude less than that found in control cells. Our results substantiate evidence that DNA-PKcs kinase activity is required for DSB rejoining and V(D)J recombination and show that the extreme C-terminal region of DNA-PKcs, present in PI 3-K-related protein kinases but absent in bona fide PI 3 lipid kinases, is required for DNA-PKcs to function as a protein kinase. We also show that expression of mutant DNA-PKcs protein confers a growth disadvantage, providing an explanation for the lack of DNA-PKcs expression in most SCID cell lines.
* To whom correspondence should be addressed. Tel: +44 1273 678482; Fax: +44 1273 678121; Email: p.a.jeggo@sussex.ac.uk
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