Cleaver J E, McDowell M, Jones C, Wood R, Karentz D
Laboratory of Radiobiology and Environmental Health, University of California, San Francisco 94143-0750.
Somat Cell Mol Genet. 1994 Jul;20(4):327-37. doi: 10.1007/BF02254721.
A series of ultraviolet (UV)-resistant cell lines have been generated from a UV-sensitive XP group A cell line homozygous for a stop codon (TGA) in the chromosome 9 XPA gene. Three lines generated by chemical mutagenesis acquired the ability to excise (6-4) photoproducts but not cyclobutane dimers from the whole genome; two lines generated by a fusion procedure with hamster cells acquired the ability to excise both (6-4) photoproducts and cyclobutane dimers from the whole genome. A central region of the hamster XPA gene was cloned and sequenced. With the use of species-specific primers in the polymerase chain reaction, we found that the hybrid cell lines do not contain a hamster XPA gene. Sequence analysis showed that all of the UV-resistant cell lines contain reversions of the human stop codon, resulting in missense mutations (glycine or leucine for arginine) or wild-type sequences. The concentration of XPA protein in revertant cell lines was about one-half that in normal cells, which would be expected from heterozygous cells; there was no evidence that the mutant proteins were less stable than the wild-type proteins. These results are consistent with the idea that the XPA protein initiates repair by binding to damaged sites with various affinities, depending on the photoproduct and the transcriptional state of the region. A concentration of XPA protein near 50% is needed before repair can proceed into nontranscribed regions of the genome. The revertant cell lines represent a class of missense mutations in the XPA gene that may have altered specificity and that can be used to understand some of the regulatory differences in repair of photoproducts in various regions of the genome.
从一个对紫外线敏感的 XP 组 A 细胞系（该细胞系在 9 号染色体 XPA 基因中因一个终止密码子（TGA）而纯合）产生了一系列抗紫外线的细胞系。通过化学诱变产生的三个细胞系获得了从整个基因组中切除（6-4）光产物但不能切除环丁烷二聚体的能力；通过与仓鼠细胞的融合程序产生的两个细胞系获得了从整个基因组中切除（6-4）光产物和环丁烷二聚体的能力。克隆并测序了仓鼠 XPA 基因的一个中心区域。在聚合酶链反应中使用物种特异性引物，我们发现杂种细胞系不包含仓鼠 XPA 基因。序列分析表明，所有抗紫外线的细胞系都含有人类终止密码子的回复突变，导致错义突变（精氨酸突变为甘氨酸或亮氨酸）或野生型序列。回复突变细胞系中 XPA 蛋白的浓度约为正常细胞中的一半，这与杂合细胞的预期相符；没有证据表明突变蛋白比野生型蛋白更不稳定。这些结果与以下观点一致，即 XPA 蛋白通过以不同亲和力结合受损位点来启动修复，这取决于光产物和该区域的转录状态。在修复能够进入基因组的非转录区域之前，需要 XPA 蛋白浓度接近 50%。回复突变细胞系代表了 XPA 基因中的一类错义突变，这些突变可能改变了特异性，可用于理解基因组不同区域光产物修复中的一些调控差异。
