Guzder S N, Qiu H, Sommers C H, Sung P, Prakash L, Prakash S
Department of Biology, University of Rochester, New York 14642.
Nature. 1994 Jan 6;367(6458):91-4. doi: 10.1038/367091a0.
The RAD3 gene of Saccharomyces cerevisiae is required for excision repair of ultraviolet-damaged DNA and is essential for cell viability. The RAD3-encoded protein shares a high degree of homology with the human ERCC2(XPD) gene product. Mutations in XPD, besides causing the cancer-prone syndrome xeroderma pigmentosum, can also result in Cockayne's syndrome and trichothiodystrophy. To investigate the role of RAD3 in viability, we examined here the effect of a recessive, temperature-sensitive (ts) conditional lethal mutation of the gene on transcription by RNA polymerase II. Upon transfer to the restrictive temperature, the rad3-ts mutant rapidly ceases growth and poly(A)+ RNA synthesis is inhibited drastically. Messenger RNA levels of all the genes examined, HIS3, TRP3, STE2, MET19, RAD23, CDC7, CDC9 and ACT1, decline rapidly upon loss of RAD3 activity. The synthesis of heat-shock-inducible HSP26 mRNA and galactose-inducible GAL7 and GAL10 mRNAs is also drastically inhibited in the rad3-ts mutant at the restrictive temperature. The RNA polymerase II transcriptional activity in extract from the rad3-ts14 strain is thermolabile, and this in vitro transcriptional defect can be fully corrected by the addition of homogeneous RAD3 protein. These findings indicate that RAD3 protein has a direct and essential role in RNA polymerase II transcription.
酿酒酵母的RAD3基因是紫外线损伤DNA切除修复所必需的，对细胞生存力至关重要。RAD3编码的蛋白质与人类ERCC2（XPD）基因产物具有高度同源性。XPD突变除了会导致易患癌症的着色性干皮病综合征外，还会引发科凯恩综合征和毛发硫营养不良。为了研究RAD3在细胞生存力中的作用，我们在此检测了该基因的隐性温度敏感（ts）条件致死突变对RNA聚合酶II转录的影响。转移到限制温度后，rad3-ts突变体迅速停止生长，多聚腺苷酸（poly(A)+）RNA合成受到极大抑制。在失去RAD3活性后，所有检测基因（HIS3、TRP3、STE2、MET19、RAD23、CDC7、CDC9和ACT1）的信使RNA水平迅速下降。在限制温度下，rad3-ts突变体中热休克诱导的HSP26 mRNA以及半乳糖诱导的GAL7和GAL10 mRNA的合成也受到极大抑制。rad3-ts14菌株提取物中的RNA聚合酶II转录活性具有热不稳定性，添加纯化的RAD3蛋白可完全纠正这种体外转录缺陷。这些发现表明，RAD3蛋白在RNA聚合酶II转录中具有直接且至关重要的作用。
