Nucleic Acids Research, 2002, Vol. 30, No. 23 5120-5128
© 2002 Oxford University Press
A single amino acid substitution in DNA-PKcs explains the novel phenotype of the CHO mutant, XR-C2
College of Veterinary Medicine, Department of Pathobiology and Diagnostic Investigation, Michigan State University, 350 FST, East Lansing, MI 48824, USA, 1 Department of Radiation Genetics and Chemical Mutagenesis, Leiden University, Medical Center, Leiden, The Netherlands and 2 Department of Molecular Cell Genetics, The Ludwil Rydygier University of Medical Sciences, Bydgoszcz, Poland
*To whom correspondence should be addressed. Tel: +1 517 432 9505; Fax: +1 517 353 9004; Email: kmeek{at}msu.edu
We recently described a CHO DSBR mutant belonging to the XRCC7 complementation group (XR-C2) that has the interesting phenotype of being radiosensitive, but having only a modest defect in VDJ recombination. This cell line expresses only slightly reduced levels of DNA-PKcs but has undetectable DNA-PK activity. Limited sequence analyses of DNA-PKcs transcripts from XR-C2 revealed a point mutation that results in an amino acid substitution of glutamic acid for glycine six residues from the C-terminus. To determine whether this single substitution was responsible for the phenotype in XR-C2 cells, we introduced the mutation into a DNA-PKcs expression vector. Whereas transfection of this expression vector significantly restores the VDJ recombination deficits in DNA-PKcs-deficient cells, radioresistance is not restored. Thus, expression of this mutant form of DNA-PKcs in DNA-PKcs- deficient cells substantially recapitulates the phenotype observed in XR-C2, and we conclude that this single amino acid substitution is responsible for the non-homologous end joining deficits observed in XR-C2.
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