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首页> 外文期刊>Biochimica et biophysica acta: BBA: International journal of biochemistry, biophysics and molecular biololgy. Proteins and Proteomics >Spectroscopic analysis of polymerization and exonuclease proofreading by a high-fidelity DNA polymerase during translesion DNA synthesis
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Spectroscopic analysis of polymerization and exonuclease proofreading by a high-fidelity DNA polymerase during translesion DNA synthesis

机译：在转移DNA合成过程中高保真DNA聚合酶对聚合和核酸外切酶校正的光谱分析

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摘要

High fidelity DNA polymerases maintain genomic fidelity through a series of kinetic steps that include nucleotide binding, conformational changes, phosphoryl transfer, polymerase translocation, and nucleotide excision. Developing a comprehensive understanding of how these steps are coordinated during correct and pro-mutagenic DNA synthesis has been hindered due to lack of spectroscopic nucleotides that function as efficient polymerase substrates. This report describes the application of a non-natural nucleotide designated 5-naphthyl-indole-2??-deoxyribose triphosphate which behaves as a fluorogenic substrate to monitor nucleotide incorporation and excision during the replication of normal DNA versus two distinct DNA lesions (cyclobutane thymine dimer and an abasic site). Transient fluorescence and rapid-chemical quench experiments demonstrate that the rate constants for nucleotide incorporation vary as a function of DNA lesion. These differences indicate that the non-natural nucleotide can function as a spectroscopic probe to distinguish between normal versus translesion DNA synthesis. Studies using wild-type DNA polymerase reveal the presence of a fluorescence recovery phase that corresponds to the formation of a pre-excision complex that precedes hydrolytic excision of the non-natural nucleotide. Rate constants for the formation of this pre-excision complex are dependent upon the DNA lesion, and this suggests that the mechanism of exonuclease proofreading is regulated by the nature of the formed mispair. Finally, spectroscopic evidence confirms that exonuclease proofreading competes with polymerase translocation. Collectively, this work provides the first demonstration for a non-natural nucleotide that functions as a spectroscopic probe to study the coordinated efforts of polymerization and exonuclease proofreading during correct and translesion DNA synthesis. ? 2012 Elsevier B.V. All rights reserved.
机译：高保真DNA聚合酶通过一系列动力学步骤（包括核苷酸结合，构象变化，磷酰基转移，聚合酶易位和核苷酸切除）来维持基因组保真度。由于缺乏充当有效聚合酶底物的光谱核苷酸，阻碍了在正确和促突变的DNA合成过程中如何协调这些步骤的全面理解。该报告描述了命名为5-萘基-吲哚-2α-脱氧核糖三磷酸的非天然核苷酸的应用，该荧光粉作为荧光底物，可监测正常DNA与两个不同DNA损伤（环丁烷胸腺嘧啶）的复制过程中核苷酸的掺入和切除二聚体和无碱基位点）。瞬态荧光和快速化学猝灭实验表明，核苷酸掺入的速率常数随DNA损伤而变化。这些差异表明，非天然核苷酸可以用作光谱探针，以区分正常DNA与病变DNA合成。使用野生型DNA聚合酶的研究揭示了荧光恢复阶段的存在，该阶段与非天然核苷酸的水解切除之前的预先切除的复合物的形成相对应。该切除前复合物形成的速率常数取决于DNA损伤，这表明核酸外切酶校对的机制受形成的错配性质的调节。最后，光谱学证据证实核酸外切酶校对与聚合酶易位竞争。总的来说，这项工作为非天然核苷酸提供了第一个证明，该非天然核苷酸可以用作光谱探针，以研究正确的和跨损伤的DNA合成过程中聚合反应和核酸外切酶校对的协调努力。 ？ 2012 Elsevier B.V.保留所有权利。

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