A single amino acid substitution in DNA-PKcs explains the novel phenotype of the CHO mutant, XR-C2

Nucleic Acids Res. 2002 Dec 1;30(23):5120-8. doi: 10.1093/nar/gkf625.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3' Flanking Region
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Animals
  • Base Sequence
  • CHO Cells*
  • Cell Line
  • Cricetinae
  • DNA Repair*
  • DNA, Complementary / analysis
  • DNA-Activated Protein Kinase
  • DNA-Binding Proteins*
  • Humans
  • Mice
  • Mutation
  • Nuclear Proteins
  • Phenotype
  • Protein Serine-Threonine Kinases / genetics*
  • Protein Serine-Threonine Kinases / metabolism
  • Radiation Tolerance
  • Recombination, Genetic
  • Sequence Analysis, DNA

Substances

  • DNA, Complementary
  • DNA-Binding Proteins
  • Nuclear Proteins
  • DNA-Activated Protein Kinase
  • PRKDC protein, human
  • Protein Serine-Threonine Kinases