Fingers-closing and other rapid conformational changes in DNA polymerase I (Klenow fragment) and their role in nucleotide selectivity
Fingers-closing and other rapid conformational changes in DNA polymerase I (Klenow fragment) and their role in nucleotide selectivity
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DOI：
10.1021/bi7021848
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发表时间：
2008-06-10
期刊：
影响因子：
2.9
通讯作者：
Grindley, Nigel D. F.
中科院分区：
文献类型：
--
作者：
Joyce, Catherine M.;Potapova, Olga;Grindley, Nigel D. F.
我们开发了一种基于fret的分析方法，用于当DNA聚合酶I （KIenow片段）的二元复合物与引物模板结合时发生的指状闭合构象转变。一种互补的dNTP，并使用这种和其他荧光分析来确定反应途径中的指合步骤。由于指状闭合的速率远远快于化学猝灭实验中测量到的核苷酸掺入速率，因此指状闭合不可能是早期动力学研究定义的限速化学前步骤。用Ca2+代替Mg2+作为金属辅因子的实验表明，化学预处理步骤可能涉及到聚合酶活性位点金属离子占用的变化。核糖核苷酸底物的使用表明在指合之前有一个碱基鉴别步骤。2-AP荧光检测到的这一早期步骤由互补核苷酸（核糖-和脱氧核糖-）促进，但被错配阻断。互补的rNTP阻断了随后的手指闭合步骤。因此，对rNTPs的歧视发生在从开放构象到封闭构象的转变过程中，而对不匹配碱基的选择则在通路的早期，在开放复合体中开始。错配的dNTPs加速了DNA从聚合酶的释放，这表明存在一个早期的中间体，在这个中间体中DNA的结合相对于二元复合物是不稳定的；这可能对应于允许传入dNTP预览模板基的构造。本研究确定的早期动力学检查点为不匹配的碱基和核糖糖的排斥提供了有效的机制，从而提高了聚合酶的吞吐量。
We have developed a FRET-based assay for the fingers-closing conformational transition that occurs when a binary complex of DNA polymerase I (KIenow fragment) with a primer-template binds. a complementary dNTP and have used this and other fluorescence assays to place the fingers-closing step within the reaction pathway. Because the rate of fingers-closing was substantially faster than the rate of nucleotide incorporation measured in chemical quench experiments, fingers-closing cannot be the rate-limiting prechemistry step defined by earlier kinetic studies. Experiments using Ca2+ instead of Mg2+ as the metal cofactor suggest instead that the prechemistry step may involve a change in metal ion occupancy at the polymerase active site. The use of ribonucleotide substrates shows there is a base discriminating step that precedes fingers-closing. This earlier step, detected by 2-AP fluorescence, is promoted by complementary nucleotides (ribo- as well as deoxyribo-) but is blocked by mismatches. The complementary rNTP blocks the subsequent fingers-closing step. Thus, discrimination against rNTPs occurs during the transition from open to closed conformations, whereas selection against mismatched bases is initiated earlier in the pathway, in the open complex. Mismatched dNTPs accelerate DNA release from the polymerase, suggesting the existence of an early intermediate in which DNA binding is destabilized relative to the binary complex; this could correspond to a conformation that allows an incoming dNTP to preview the template base. The early kinetic checkpoints identified by this study provide an efficient mechanism for the rejection of mismatched bases and ribose sugars and thus enhance polymerase throughput.