MECHANISMS OF MUTAGENESIS WITH YEAST REPLICATION AND REPAIR PROTEINS

简单的真核生物酿酒酵母提供了一个很好的 遗传系统和高度纯化的DNA复制的来源， 修复蛋白的分子机制的研究 诱变 过去两年的一项重大努力是 确定DNA合成的保真度的主要复制 酵母中的聚合酶，DNA聚合酶I。 审查是否可能 除了聚合酶催化亚基以外的亚基可以 为了影响保真度，我们测量了通过以下方法纯化的yPolI的准确性： 常规程序，其产生具有140 Kd的聚合酶， 催化亚基和无相关引发酶活性，以及 通过免疫亲和层析纯化yPolI，得到 具有约180 kD的催化亚基的聚合酶， 以及三种另外的多肽和引发酶活性。 的 两种形式的聚合酶的平均保真度相似， 证明了，就像高等真核生物中的等价物一样， （聚合酶α），yPolI和聚合酶I-引发酶复合物 产生三类主要的错误，单碱基 取代、单碱基移码和较大的缺失。 为 特异性错误和模板位置，聚合酶的两种形式 确实表现出有趣的保真度差异。 尽管有这些 差异，当考虑到整体错误频率和 DNA合成错误的光谱，结果表明， 聚合酶I-引物酶复合物的准确性不高， 对于单独的聚合酶，其保真度不足以解释 体内自发突变率低。 突变体特异性 数据还提出了解释两类错误的模型。 第一、 我们提出，在适当的序列背景下，某些碱基 错误插入事件可以重新排列以形成稳定的未对准， 甚至在非重复序列中也导致-1移码。 结果 DNA聚合酶反应， 模板引物支持该模型。 第二，在删除的内容中， 其中许多是在直接重复之间，是包含 不寻常的连接 这些可以用异常的“环回”来解释 通过yPolI合成以通过同向重复序列产生复杂缺失 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.