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首页> 外文期刊>Biochemistry >THE NUCLEOTIDE ANALOG 2-AMINOPURINE AS A SPECTROSCOPIC PROBE OF NUCLEOTIDE INCORPORATION BY THE KLENOW FRAGMENT OF ESCHERICHIA COLI POLYMERASE I AND BACTERIOPHAGE T4 DNA POLYMERASE
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THE NUCLEOTIDE ANALOG 2-AMINOPURINE AS A SPECTROSCOPIC PROBE OF NUCLEOTIDE INCORPORATION BY THE KLENOW FRAGMENT OF ESCHERICHIA COLI POLYMERASE I AND BACTERIOPHAGE T4 DNA POLYMERASE

机译：大肠杆菌类似物聚合酶I和细菌噬菌体T4 DNA聚合酶的Klenow片段将核苷酸类似物2-氨基嘌呤作为核苷酸掺入的光谱探针

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摘要

The fluorescent properties and their sensitivity to the surrounding environment of the nucleotide analog 2-aminopurine (2-AP) have been well documented. In this paper we describe the use of 2-AP as a direct spectroscopic probe of the mechanism of nucleotide incorporation by Escherichia coli Pol I Klenow fragment (KF) and bacteriophage T4 DNA polymerase. The nucleotidyl transfer reaction may be monitored in real time by following the fluorescence of 2-AP, allowing the detection of transient intermediates along the reaction pathway that are inaccessible through traditional radioactive assays. Previous studies with Klenow fragment [Kuchta, R. D., Mizrahi, V., Benkovic, P. A., Johnson, K. A., and Benkovic, S. J. (1987) Biochemistry 26, 8410-8417] have revealed the presence of a nonchemical step prior to chemistry and have identified this conformational change as the rate-limiting step of correct nucleotide incorporation. During correct incorporation, phosphodiester bond formation occurs at a rate greater than the conformational change and has not been measured, However, during misinsertion, the rate of the chemical step becomes partially rate limiting and it becomes possible to detect both steps. We have successfully decoupled the chemical and conformational change steps for nucleotide insertion by KF using the misincorporation reaction, and we present direct spectroscopic evidence for an activated KF'-DNA-dNTP species following the conformational change step which features hydrogen bonding between the incoming and template bases. In addition, we have utilized these same experiments to demonstrate the existence of a similar nonchemical step in the mechanism of dNTP incorporation by bacteriophage T4 DNA polymerase. This study provides the first direct evidence of a conformational change for T4 polymerase and emphasizes the importance of this step in a general polymerase kinetic sequence. [References: 36]
机译：核苷酸类似物2-氨基嘌呤（2-AP）的荧光性质及其对周围环境的敏感性已得到充分证明。在本文中，我们描述了使用2-AP作为直接光谱探针来探究大肠杆菌Pol I Klenow片段（KF）和噬菌体T4 DNA聚合酶掺入核苷酸的机理。可以通过跟踪2-AP的荧光来实时监测核苷酸转移反应，从而可以检测沿反应途径的瞬态中间体，而这些中间体是传统放射性分析无法达到的。以前对Klenow片段的研究[Kuchta，RD，Mizrahi，V.，Benkovic，PA，Johnson，KA，和Benkovic，SJ（1987）Biochemistry 26，8410-8417]揭示了在化学反应之前存在非化学步骤，并且已经将这种构象变化确定为正确核苷酸掺入的限速步骤。在正确掺入期间，磷酸二酯键形成的发生速率大于构象变化，并且尚未被测量。但是，在错误插入期间，化学步骤的速率部分受限于速率，并且可以检测两个步骤。我们已经成功地分离了通过错掺反应通过KF插入核苷酸的化学和构象变化步骤，并且我们提供了在构象变化步骤之后激活的KF'-DNA-dNTP物种的直接光谱证据，其特征在于传入和模板之间存在氢键基地。此外，我们已经利用这些相同的实验来证明在噬菌体T4 DNA聚合酶掺入dNTP的机制中存在类似的非化学步骤。这项研究提供了T4聚合酶构象变化的第一个直接证据，并强调了这一步骤在一般聚合酶动力学序列中的重要性。 [参考：36]

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