Woods Timothy, Wang Wei, Convery Erin, Errami Abdellatif, Zdzienicka Malgorzata Z, Meek Katheryn
College of Veterinary Medicine, Department of Pathobiology and Diagnostic Investigation, Michigan State University, 350 FST, East Lansing, MI 48824, USA.
Nucleic Acids Res. 2002 Dec 1;30(23):5120-8. doi: 10.1093/nar/gkf625.
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.
我们最近描述了一种属于XRCC7互补组（XR - C2）的CHO DSBR突变体，它具有有趣的表型：对辐射敏感，但在VDJ重组中只有适度缺陷。该细胞系仅表达略低水平的DNA - PKcs，但DNA - PK活性检测不到。对来自XR - C2的DNA - PKcs转录本进行的有限序列分析揭示了一个点突变，该突变导致从C末端起六个残基处的甘氨酸被谷氨酸取代。为了确定这个单一取代是否导致了XR - C2细胞中的表型，我们将该突变引入到一个DNA - PKcs表达载体中。虽然转染这个表达载体显著恢复了DNA - PKcs缺陷细胞中的VDJ重组缺陷，但辐射抗性并未恢复。因此，在DNA - PKcs缺陷细胞中表达这种突变形式的DNA - PKcs基本上重现了在XR - C2中观察到的表型，并且我们得出结论，这个单一氨基酸取代是导致在XR - C2中观察到的非同源末端连接缺陷的原因。
