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首页> 外文期刊>Molecular and Cellular Biology >Identification of rad27 Mutations That Confer Differential Defects in Mutation Avoidance, Repeat Tract Instability, and Flap Cleavage
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Identification of rad27 Mutations That Confer Differential Defects in Mutation Avoidance, Repeat Tract Instability, and Flap Cleavage

机译：rad27突变的鉴定，该突变可在避免突变，重复道不稳定性和瓣裂中提供差异性缺陷

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In eukaryotes, the nuclease activity of Rad27p (Fen1p) is thought to play a critical role in lagging-strand DNA replication by removing ribonucleotides present at the 5′ ends of Okazaki fragments. Genetic analysis of Saccharomyces cerevisiae also has identified a role for Rad27p in mutation avoidance. rad27Δ mutants display both a repeat tract instability phenotype and a high rate of forward mutations to canavanine resistance that result primarily from duplications of DNA sequences that are flanked by direct repeats. These observations suggested that Rad27p activities in DNA replication and repair could be altered by mutagenesis and specifically assayed. To test this idea, we analyzed two rad27alleles, rad27-G67S and rad27-G240D, that were identified in a screen for mutants that displayed repeat tract instability and mutator phenotypes. In chromosome stability assays,rad27-G67S strains displayed a higher frequency of repeat tract instabilities relative to CAN1 duplication events; in contrast, the rad27-G240D strains displayed the opposite phenotype. In biochemical assays, rad27-G67Sp displayed a weak exonuclease activity but significant single- and double-flap endonuclease activities. In contrast, rad27-G240Dp displayed a significant double-flap endonuclease activity but was devoid of exonuclease activity and showed only a weak single-flap endonuclease activity. Based on these observations, we hypothesize that the rad27-G67S mutant phenotypes resulted largely from specific defects in nuclease function that are important for degrading bubble intermediates, which can lead to DNA slippage events. Therad27-G240D mutant phenotypes were more difficult to reconcile to a specific biochemical defect, suggesting a structural role for Rad27p in DNA replication and repair. Since the mutants provide the means to relate nuclease functions in vitro to genetic characteristics in vivo, they are valuable tools for further analyses of the diverse biological roles of Rad27p.
机译：在真核生物中，Rad27p（Fen1p）的核酸酶活性被认为通过消除冈崎片段5'端存在的核糖核苷酸在滞后链DNA复制中起关键作用。酿酒酵母的遗传分析也确定了Rad27p在避免突变中的作用。 rad27 Δ突变体既表现出重复道不稳定性表型，又显示出高的对犬小鸟碱抗性的正向突变，这主要是由直接重复序列两侧的DNA序列重复引起的。这些观察结果表明，可以通过诱变和特异性测定来改变Rad27p在DNA复制和修复中的活性。为了验证这一想法，我们分析了两个 rad27 等位基因， rad27-G67S 和 rad27-G240D ，这些突变体在显示突变体的屏幕中被识别出重复道不稳定和突变表型。在染色体稳定性测定中，相对于 CAN1 重复事件， rad27-G67S 菌株表现出更高的重复道不稳定性频率。相反， rad27-G240D 菌株表现出相反的表型。在生化分析中，rad27-G67Sp显示出较弱的核酸外切酶活性，但具有显着的单瓣和双瓣核酸内切酶活性。相反，rad27-G240Dp显示出显着的双瓣核酸内切酶活性，但没有核酸外切酶活性，而仅显示出弱的单瓣核酸内切酶活性。基于这些观察结果，我们假设 rad27-G67S 突变表型主要是由核酸酶功能的特定缺陷引起的，这些缺陷对于降解泡沫中间体非常重要，这可能导致DNA滑移事件。 rad27-G240D 突变表型更难与特定的生化缺陷协调一致，表明Rad27p在DNA复制和修复中的结构作用。由于突变体提供了将核酸酶功能与体外遗传特性相关联的手段，因此它们是进一步分析Rad27p的多种生物学作用的有价值的工具。

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