The proofreading exonuclease activity of replicative DNA polymerase excises misincorporated nucleotides during DNA synthesis, but these events are rare. Therefore, we were surprised to find that T7 replisome excised nearly 7% of correctly incorporated nucleotides during leading and lagging strand syntheses. Similar observations with two other DNA polymerases establish its generality. We show that excessive excision of correctly incorporated nucleotides is not due to events such as processive degradation of nascent DNA or spontaneous partitioning of primer-end to the exonuclease site as a "cost of proofreading". Instead, we show that replication hurdles, including secondary structures in template, slowed helicase, or uncoupled helicase-polymerase, increase DNA reannealing and polymerase backtracking, and generate frayed primer-ends that are shuttled to the exonuclease site and excised efficiently. Our studies indicate that active-site shuttling occurs at a high frequency, and we propose that it serves as a proofreading mechanism to protect primer-ends from mutagenic extensions.

中文翻译：

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复制障碍解释了 DNA 合成过程中正确核苷酸的过度切除。

复制性 DNA 聚合酶的校对核酸外切酶活性可在 DNA 合成过程中切除错误掺入的核苷酸，但这些事件很少见。因此，我们惊讶地发现 T7 复制体在前导和滞后链合成过程中切除了近 7% 的正确掺入的核苷酸。与其他两种 DNA 聚合酶的类似观察确立了其普遍性。我们表明，正确掺入的核苷酸的过度切除不是由于诸如新生 DNA 的持续降解或引物末端自发分配到核酸外切酶位点等事件，作为“校对成本”。相反，我们表明复制障碍，包括模板中的二级结构、解旋酶减慢或解旋酶-聚合酶解偶联，增加了 DNA 重退火和聚合酶回溯，并产生磨损的引物末端，这些引物末端穿梭到核酸外切酶位点并有效切除。我们的研究表明，活性位点穿梭发生的频率很高，我们建议将其作为一种校对机制，以保护引物末端免受诱变延伸。