Arora Karunesh, Schlick Tamar
Department of Chemistry and Courant Institute of Mathematical Sciences, New York University, New York, New York 10012, USA.
Biophys J. 2004 Nov;87(5):3088-99. doi: 10.1529/biophysj.104.040915.
Structural information for mammalian DNA pol-beta combined with molecular and essential dynamics studies have provided atomistically detailed views of functionally important conformational rearrangements that occur during DNA repair and replication. This conformational closing before the chemical reaction is explored in this work as a function of the bound substrate. Anchors for our study are available in crystallographic structures of the DNA pol-beta in "open" (polymerase bound to gapped DNA) and "closed" (polymerase bound to gapped DNA and substrate, dCTP) forms; these different states have long been used to deduce that a large-scale conformational change may help the polymerase choose the correct nucleotide, and hence monitor DNA synthesis fidelity, through an "induced-fit" mechanism. However, the existence of open states with bound substrate and closed states without substrates suggest that substrate-induced conformational closing may be more subtle. Our dynamics simulations of two pol-beta/DNA systems (with/without substrates at the active site) reveal the large-scale closing motions of the thumb and 8-kDa subdomains in the presence of the correct substrate--leading to nearly perfect rearrangement of residues in the active site for the subsequent chemical step of nucleotidyl transfer--in contrast to an opening trend when the substrate is absent, leading to complete disassembly of the active site residues. These studies thus provide in silico evidence for the substrate-induced conformational rearrangements, as widely assumed based on a variety of crystallographic open and closed complexes. Further details gleaned from essential dynamics analyses clarify functionally relevant global motions of the polymerase-beta/DNA complex as required to prepare the system for the chemical reaction of nucleotide extension.
哺乳动物DNA聚合酶β的结构信息,结合分子动力学和主成分动力学研究,提供了在DNA修复和复制过程中发生的功能重要的构象重排的原子细节视图。在这项工作中,我们研究了化学反应前这种构象闭合与结合底物的关系。我们研究的依据来自DNA聚合酶β处于“开放”(聚合酶与缺口DNA结合)和“闭合”(聚合酶与缺口DNA和底物dCTP结合)形式的晶体结构;长期以来,这些不同状态被用于推断大规模构象变化可能通过“诱导契合”机制帮助聚合酶选择正确的核苷酸,从而监控DNA合成保真度。然而,存在结合底物的开放状态和没有底物的闭合状态表明,底物诱导的构象闭合可能更为微妙。我们对两个聚合酶β/DNA系统(活性位点有无底物)的动力学模拟表明,在存在正确底物的情况下,拇指和8 kDa亚结构域会发生大规模闭合运动,导致活性位点的残基几乎完美重排,以进行随后的核苷酸转移化学反应步骤;而当没有底物时则呈现开放趋势,导致活性位点残基完全解离。因此,这些研究提供了计算机模拟证据,证明了底物诱导的构象重排,这与基于各种晶体学开放和闭合复合物广泛假设的情况一致。从主成分动力学分析中获得的进一步细节阐明了聚合酶β/DNA复合物功能相关的整体运动,这是为核苷酸延伸化学反应准备系统所必需的。