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对2-氨基嘌呤与胞嘧啶及2-氨基嘌呤与胸腺嘧啶碱基对进行动力学测量，以此作为DNA聚合酶保真机制的一项测试。

Kinetic measurement of 2-aminopurine X cytosine and 2-aminopurine X thymine base pairs as a test of DNA polymerase fidelity mechanisms.

作者信息

Watanabe S M, Goodman M F

出版信息

Proc Natl Acad Sci U S A. 1982 Nov;79(21):6429-33. doi: 10.1073/pnas.79.21.6429.

DOI:10.1073/pnas.79.21.6429
PMID:6959128
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC347139/
Abstract

Enzyme kinetic measurements are presented showing that Km rather than maximum velocity (Vmax) discrimination governs the frequency of forming 2-aminopurine X cytosine base mispairs by DNA polymerase alpha. An in vitro system is used in which incorporation of dTMP or dCMP occurs opposite a template 2-aminopurine, and values for Km and Vmax are obtained. Results from a previous study in which dTTP and dCTP were competing simultaneously for insertion opposite 2-aminopurine indicated that dTMP is inserted 22 times more frequently than dCMP. We now report that the ratio of Km values KCm/KTm = 25 +/- 6, which agrees quantitatively with the dTMP/dCMP incorporation ratio obtained previously. We also report that VCmax is indistinguishable from VTmax. These Km and Vmax data are consistent with predictions from a model, the Km discrimination model, in which replication fidelity is determined by free energy differences between matched and mismatched base pairs. Central to this model is the prediction that the ratio of Km values for insertion of correct and incorrect nucleotides specifies the insertion fidelity, and the maximum velocities of insertion are the same for both nucleotides.

摘要

酶动力学测量结果表明，决定DNA聚合酶α形成2-氨基嘌呤与胞嘧啶碱基错配频率的是米氏常数（Km）而非最大反应速度（Vmax）。研究采用了一个体外系统，其中dTMP或dCMP在模板2-氨基嘌呤的对应位置掺入，并获得了Km和Vmax的值。之前一项研究的结果表明，dTTP和dCTP同时竞争在2-氨基嘌呤对应位置插入时，dTMP的插入频率比dCMP高22倍。我们现在报告，Km值的比率KCm/KTm = 25 ± 6，这与之前获得的dTMP/dCMP掺入比率在数量上一致。我们还报告，VCmax与VTmax没有差异。这些Km和Vmax数据与一个模型——Km辨别模型的预测一致，在该模型中，复制保真度由匹配和错配碱基对之间的自由能差异决定。该模型的核心预测是，正确和错误核苷酸插入的Km值比率决定了插入保真度，并且两种核苷酸的最大插入速度相同。

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9
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Proc Natl Acad Sci U S A. 1980 Sep;77(9):5248-52. doi: 10.1073/pnas.77.9.5248.
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