MECHANISMS OF MUTAGENESIS WITH YEAST REPLICATION AND REPAIR PROTEINS

简单的真核生物酿酒酵母提供了很好的 遗传系统和高度纯化的DNA复制和来源 修复蛋白在分子机制研究中的应用 诱变。过去两年的主要努力是 用主要复制体确定DNA合成的保真度 酵母菌中的聚合酶，DNA聚合酶I，以检测这种可能性 聚合酶催化亚基以外的亚基可以 影响保真度，我们测量了yPolI纯化的准确性 传统的方法，产生140kD的聚合酶 催化亚基和NO相关的底物酶活性，以及 用免疫亲和层析法纯化yPolI，得到 催化亚基约为180kd的聚合酶 以及三个额外的多肽和底物酶活性。这个 两种聚合酶的平均保真度相似 证明，就像高等真核生物中的等价物一样 (聚合酶α)，既有聚合酶I，也有聚合酶I-Primase复合体 产生三种主要的错误类别，单一基数 替换、单碱基移码和更大的删除。为 聚合酶的两种形式--特定错误和模板位置 确实表现出了有趣的保真度差异。尽管如此 差异，当考虑总体错误频率和 光谱的DNA合成错误，结果表明 聚合酶I-Primase复合体不是很准确，就像 仅对聚合酶来说，它的保真度不足以解释 体内的低自发突变率。突变体的专一性 数据还建议用模型来解释两类错误。第一, 我们认为，在适当的序列上下文中，某些碱基 错误插入事件可以重新排列以形成稳定的未对齐 导致-1移码，即使在非重复序列中也是如此。结果 特定设计不匹配的DNA聚合酶反应 模板-引子支持这一模式。第二，在删除的内容中， 其中许多是在直接重复之间，是包含 不寻常的交汇点。这些可以用异常的“环回”来解释。 用yPoli合成通过直接重复产生复杂缺失 机制。

The simple eukaryote Saccharomyces cerevisiae provides a good genetic system and a source of highly purified DNA replication and repair proteins for the study of molecular mechanisms of mutagenesis. A major effort of the past two years has been to determine the fidelity of DNA synthesis by the major replicative polymerase in yeast, DNA polymerase I. To examine the possibility that subunits other than the polymerase catalytic subunit may influence fidelity, we measured the accuracy of yPolI purified by conventional procedures, which yields polymerase with a 140 Kd catalytic subunit and no associated primase activity, and that of yPolI purified by immunoaffinity chormatography, which yields polymerase having a catalytic subunit of approximately 180 kd as well as three additional polypeptides and primase activity. The average fidelity was similar for both forms of polymerase and demonstrate that, like its equivalent in higher eukaryotes (polymerase alpha), both yPolI and the polymerase I-primase complex produce three predominant classes of errors, single-base substitutions, single-base frameshifts and larger deletions. For specific errors and template positions, the two forms of polymerase do exhibit interesting fidelity differences. Despite these differences, when considering the overall error frequency and the spectrum of DNA synthesis errors, the results suggest that the polymerase I-primase complex is not highly accurate, and, just as for the polymerase alone, its fidelity is not sufficient to account for low spontaneous mutation rates in vivo. The mutant specificity data also suggest models to explain two subsets of errors. First, we propose that in the appropriate sequence context certain base misinsertion events can rearrange to form stable misalignments that lead to -1 frameshifts even in nonreiterated sequences. Results of DNA polymerase reactions with specifically designed mismatched template-primers support this model. Second, among the deletions, many of which are between direct repeats, is a subset that contain unusual junctions. These can be explained by aberrant "loop-back" synthesis by yPolI to generate complex deletions by a direct repeat mechanism.