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首页> 外文期刊>Journal of Molecular Biology >Reversal of a mutator activity by a nearby fidelity-neutral substitution in the RB69 DNA polymerase binding pocket.
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Reversal of a mutator activity by a nearby fidelity-neutral substitution in the RB69 DNA polymerase binding pocket.

机译：RB69 DNA聚合酶结合口袋中附近保真-中性取代引起的突变体活性逆转。

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摘要

Phage RB69 B-family DNA polymerase is responsible for the overall high fidelity of RB69 DNA synthesis. Fidelity is compromised when conserved Tyr567, one of the residues that form the nascent polymerase base-pair binding pocket, is replaced by alanine. The Y567A mutator mutant has an enlarged binding pocket and can incorporate and extend mispairs efficiently. Ser565 is a nearby conserved residue that also contributes to the binding pocket, but a S565G replacement has only a small impact on DNA replication fidelity. When Y567A and S565G replacements were combined, mutator activity was strongly decreased compared to that with Y567A replacement alone. Analyses conducted both in vivo and in vitro revealed that, compared to Y567A replacement alone, the double mutant mainly reduced base substitution mutations and, to a lesser extent, frameshift mutations. The decrease in mutation rates was not due to increased exonuclease activity. Based on measurements of DNA binding affinity, mismatch insertion, and mismatch extension, we propose that the recovered fidelity of the double mutant may result, in part, from an increased dissociation of the enzyme from DNA, followed by the binding of the same or another polymerase molecule in either exonuclease mode or polymerase mode. An additional antimutagenic factor may be a structural alteration in the polymerase binding pocket described in this article.
机译：噬菌体RB69 B族DNA聚合酶负责RB69 DNA合成的总体高保真度。当保守的Tyr567（形成新生聚合酶碱基对结合口袋的残基之一）被丙氨酸替代时，保真度受到损害。 Y567A突变体突变体具有扩大的结合口袋，可以有效地整合和延伸错配。 Ser565是附近的保守残基，也有助于结合口袋，但S565G替代品对DNA复制保真度影响很小。当Y567A和S565G替代品组合使用时，突变体活性与单独的Y567A替代品相比大大降低。在体内和体外进行的分析表明，与单独的Y567A替代相比，双突变体主要减少碱基取代突变，并在较小程度上减少移码突变。突变率的下降并不是由于核酸外切酶活性的增加。基于对DNA结合亲和力，错配插入和错配延伸的测量，我们建议恢复双突变体的保真度可能部分是由于酶与DNA的解离增加，然后是相同或另一种酶的结合核酸外切酶模式或聚合酶模式的聚合酶分子。另外的抗诱变因子可能是本文所述的聚合酶结合口袋中的结构改变。

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