title: A single amino acid substitution in DNA-PKcs explains the novel phenotype of the CHO mutant, XR-C2.
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.
authors.affiliation: College of Veterinary Medicine, Department of Pathobiology and Diagnostic Investigation, Michigan State University, 350 FST, East Lansing, MI 48824, USA.
authors.forename: Timothy
authors.initials: T
authors.lastname: Woods
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Erin
authors.initials: E
authors.lastname: Convery
authors.forename: Abdellatif
authors.initials: A
authors.lastname: Errami
authors.forename: Malgorzata Z
authors.initials: MZ
authors.lastname: Zdzienicka
authors.forename: Katheryn
authors.initials: K
authors.lastname: Meek
mesh_terms: D024509:3' Flanking Region; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004260:DNA Repair; D018076:DNA, Complementary / Q000032:analysis; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D006801:Humans; D051379:Mice; D009154:Mutation; D009687:Nuclear Proteins; D010641:Phenotype; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D011836:Radiation Tolerance; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA
chemical_list: D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases