He C H, Ramotar D
Hôpital Maisonneuve-Rosemont, Centre de Recherche, Montreal, QC, Canada.
Biochem Cell Biol. 1999;77(4):375-82.
Bleomycin is an antitumor drug that kills cells by introducing lesions in DNA. Thus, normal cells exposed to bleomycin must rely on efficient DNA repair mechanisms to survive. In the yeast Saccharomyces cerevisiae, the transcriptional activator Imp2 is required to fend off the toxic effects of bleomycin. However, it remains unclear whether Imp2 controls the expression of a protein that either repairs bleomycin-induced DNA lesions, or detoxifies the drug, and or both. To gain further insight into the mechanisms by which yeast cells mount a response towards bleomycin, we began to sequentially characterize the genetic defect in a collection of bleomycin-sensitive mutants that were previously isolated by mini-Tn3 transposon mutagenesis. A rescue plasmid designed to integrate at the site of the mini-Tn3 insertion was used to identify the defective gene in one of the mutant strains, HCY53, which was not allelic to IMP2. We showed that in strain HCY53, the mini-Tn3 was inserted at the distal end of an essential gene RPB7, which encodes one of the two subunits, Rpb4-Rbp7, that forms a subcomplex with RNA polymerase II. Since rpb7 null mutants are nonviable, it would appear that the rpb7::mini-Tn3 allele produces a protein that retains partial biological function thus permitting cell viability, but which is unable to provide bleomycin resistance to strain HCY53. The defective phenotype of strain HCY53 could be corrected by a plasmid bearing the entire RPB7 gene. Two dimensional gel analysis revealed that the expression of several proteins were diminished or absent in the rpb7::mini-Tn3 mutant when challenged with bleomycin. These results are in accord with our previous report that bleomycin resistance in yeast is controlled at the transcriptional level.
博来霉素是一种抗肿瘤药物，它通过在DNA中引入损伤来杀死细胞。因此，暴露于博来霉素的正常细胞必须依靠有效的DNA修复机制才能存活。在酿酒酵母中，转录激活因子Imp2是抵御博来霉素毒性作用所必需的。然而，目前尚不清楚Imp2是否控制一种能够修复博来霉素诱导的DNA损伤或使药物解毒，或两者兼有的蛋白质的表达。为了进一步深入了解酵母细胞对博来霉素产生反应的机制，我们开始对一组先前通过mini-Tn3转座子诱变分离的博来霉素敏感突变体中的遗传缺陷进行序列分析。一个设计用于整合到mini-Tn3插入位点的拯救质粒被用来鉴定其中一个突变菌株HCY53中的缺陷基因，该菌株与IMP2非等位。我们发现，在菌株HCY53中，mini-Tn3插入到一个必需基因RPB7的远端，该基因编码与RNA聚合酶II形成亚复合物之一的两个亚基Rpb4-Rbp7中的一个。由于rpb7缺失突变体无法存活，似乎rpb7::mini-Tn3等位基因产生的蛋白质保留了部分生物学功能，从而允许细胞存活，但无法为菌株HCY53提供博来霉素抗性。携带完整RPB7基因的质粒可以纠正菌株HCY53的缺陷表型。二维凝胶分析显示，当用博来霉素处理时，rpb7::mini-Tn3突变体中几种蛋白质的表达减少或缺失。这些结果与我们之前关于酵母中博来霉素抗性在转录水平上受到控制的报道一致。
