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
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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DOI：
10.1021/bi00028a031
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发表时间：
1995-07-18
期刊：
影响因子：
2.9
通讯作者：
BENKOVIC, SJ
中科院分区：
文献类型：
--
作者：
FREY, MW;SOWERS, LC;BENKOVIC, SJ
核苷酸类似物2-氨基嘌呤（2-AP）的荧光特性及其对周围环境的敏感性已经得到了很好的记录。在本文中，我们描述了使用2-AP作为大肠杆菌Pol I know片段（KF）和噬菌体T4 DNA聚合酶的核苷酸结合机制的直接光谱探针。通过跟踪2-AP的荧光，可以实时监测核苷酸转移反应，从而可以检测到传统放射性检测方法无法检测到的反应途径中的瞬态中间体。先前对Klenow片段的研究[Kuchta， R. D, Mizrahi， V., Benkovic， P. A., Johnson， K. A., and Benkovic， S. J. (1987) Biochemistry 26,8410 -8417]揭示了在化学之前存在一个非化学步骤，并确定了这种构象变化是正确核苷酸结合的限速步骤。在正确插入过程中，磷酸二酯键形成的速率大于构象变化的速率，并且没有被测量到。然而，在错误插入过程中，化学步骤的速率变得部分限速，并且可以检测到两个步骤。我们利用错配反应成功地解耦了KF插入核苷酸的化学和构象变化步骤，并且我们提供了直接的光谱证据，证明在构象变化步骤之后激活了KF'-DNA-dNTP物种，其特征是进入的碱基和模板碱基之间的氢键。此外，我们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，并强调了这一步在一般聚合酶动力学序列中的重要性。
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.