Mutation of cysteine-88 in the Saccharomyces cerevisiae RAD6 protein abolishes its ubiquitin-conjugating activity and its various biological functions.
Mutation of cysteine-88 in the Saccharomyces cerevisiae RAD6 protein abolishes its ubiquitin-conjugating activity and its various biological functions.
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DOI：
10.1073/pnas.87.7.2695
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发表时间：
1990-04
影响因子：
11.1
通讯作者：
P. Sung;S. Prakash;L. Prakash
中科院分区：
文献类型：
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作者：
P. Sung;S. Prakash;L. Prakash
酿酒酵母的 RAD6 基因是 DNA 修复、DNA 损伤诱导的诱变和孢子形成所必需的。 RAD6 蛋白是一种泛素结合酶 (E2)，已被证明可将多个泛素分子附着到组蛋白 H2A 和 H2B 上。我们现在已经检查了 RAD6 的 E2 活性是否与其各种生物学功能有关。由于 E2 和泛素之间形成硫酯加合物对于 E2 活性是必要的，因此 RAD6 中存在的单个半胱氨酸残基 (Cys-88) 被更改为丙氨酸或缬氨酸。突变蛋白在酵母细胞中过量产生并纯化至接近均质。我们发现 rad6 Ala-88 和 rad6 Val-88 突变蛋白缺乏与泛素形成硫酯的能力，因此完全没有任何 E2 活性。 rad6 Ala-88 和 rad6 Val-88 突变导致 DNA 修复、诱变和孢子形成缺陷，与 rad6 无效等位基因中的缺陷相同。我们认为RAD6的生物学功能需要其E2活性。
The RAD6 gene of Saccharomyces cerevisiae is required for DNA repair, DNA damage-induced mutagenesis, and sporulation. RAD6 protein is a ubiquitin-conjugating enzyme (E2) that has been shown to attach multiple molecules of ubiquitin to histones H2A and H2B. We have now examined whether the E2 activity of RAD6 is involved in its various biological functions. Since the formation of a thioester adduct between E2 and ubiquitin is necessary for E2 activity, the single cysteine residue (Cys-88) present in RAD6 was changed to alanine or valine. The mutant proteins were overproduced in yeast cells and purified to near homogeneity. We show that the rad6 Ala-88 and rad6 Val-88 mutant proteins lack the capacity for thioester formation with ubiquitin and, as a consequence, are totally devoid of any E2 activity. The rad6 Ala-88 and rad6 Val-88 mutations confer a defect in DNA repair, mutagenesis, and sporulation equivalent to that in the rad6 null allele. We suggest that the biological functions of RAD6 require its E2 activity.