Kobayashi T, Takeuchi S, Saijo M, Nakatsu Y, Morioka H, Otsuka E, Wakasugi M, Nikaido O, Tanaka K
Institute for Molecular and Cellular Biology, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
Nucleic Acids Res. 1998 Oct 15;26(20):4662-8. doi: 10.1093/nar/26.20.4662.
To analyze the function of the xeroderma pigmentosum group A (XPA) protein in strand-specific DNA repair, we examined repair of UV-induced cyclobutane pyrimidine dimer (CPD) in transcribed and non-transcribed strands of the dihydrofolate reductase gene of xeroderma pigmentosum group A (XP-A) cell line (XP12ROSV) which was transfected with various types of mutant XPA cDNA. The transfectant overexpressing mutant XPA with a defect in the interaction with either ERCC1, replication protein A (RPA), or general transcription factor TFIIH, showed more or less decreased repair of CPD in each strand in parallel, while in the transfectant overexpressing R207G (Arg207to Gly) mutant XPA derived from XP129, a UV-resistant XP12ROSV revertant, the rate of CPD repair was almost normal in each strand. We also examined the dose responses of the XPA protein on CPD repair in each strand by the modulation of the expression levels of wild-type or R207G mutant XPA using an inducible expression system, LacSwitchtrade mark promoter. There were good correlations between the rate of CPD repair in each strand and the amount of XPA protein produced in these Lac cells. Our results indicate that the XPA protein is equally important for the CPD repair in both transcribed and non-transcribed strands and that the R207G mutation found in XP129 may not be responsible for a selective defect in CPD repair in the non-transcribed strand in XP129.
为了分析着色性干皮病A组（XPA）蛋白在链特异性DNA修复中的功能，我们检测了转染了各种类型突变XPA cDNA的着色性干皮病A组（XP - A）细胞系（XP12ROSV）二氢叶酸还原酶基因转录链和非转录链中紫外线诱导的环丁烷嘧啶二聚体（CPD）的修复情况。过表达与ERCC1、复制蛋白A（RPA）或通用转录因子TFIIH相互作用存在缺陷的突变XPA的转染细胞，各链中CPD的修复或多或少同时出现下降，而在过表达源自紫外线抗性XP12ROSV回复株XP129的R207G（精氨酸207突变为甘氨酸）突变XPA的转染细胞中，各链中CPD的修复率几乎正常。我们还通过使用诱导表达系统LacSwitch™启动子调节野生型或R207G突变XPA的表达水平，检测了XPA蛋白对各链中CPD修复的剂量反应。这些Lac细胞中各链CPD的修复率与产生的XPA蛋白量之间存在良好的相关性。我们的结果表明，XPA蛋白对于转录链和非转录链中的CPD修复同样重要，并且在XP129中发现的R207G突变可能与XP129非转录链中CPD修复的选择性缺陷无关。
