PROBING THE CONFORMATIONAL STATES OF E-DNA-DNTP COMPLEX USING SAXS

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。DNA复制是细胞繁殖所需的基本生物学过程。DNA复制的中心特征是由DNA聚合酶介导的模板诱导的核苷酸转移反应。近年来，人们对酶促聚合反应的机理进行了结构研究，包括确定几种DNA聚合酶及其与底物和底物类似物的复合物的结构。这些结构表明核苷酸掺入的双金属离子机制，这一特征被认为是所有DNA聚合酶家族所共有的。一种金属与dNTP结合相关，另一种金属参与化学步骤中的金属离子催化。此外，根据Pol β和Klenow片段的晶体结构，提出了在结合dNTP底物时的构象变化步骤。最近有大量的研究致力于研究金属离子间的相互作用，并提出了DNA聚合酶与dNTP结合后的构象变化。这些努力已经利用了无数的技术，包括计算，功能和晶体学研究，即使酶，底物和复合物的构象灵活性使得难以明确预测聚合酶在催化循环期间的动态行为。或者，sAXS已被证明对蛋白质和核酸的构象变化敏感。因此，我们的目标是在这个建议是提供二价金属离子，DNA和dNTP底物之间的相互作用的结构的见解，与三个聚合酶，Pol β，KF，和ASFV Pol X，并进一步将这些研究与我们的生物化学和功能的研究。通过监测DNA和dNTP底物的掺入和二价金属离子的滴定引起的构象变化，我们将实验确定酶-底物相互作用和酶-金属离子相互作用的形式和能量。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. DNA replication is a fundamental biological process required for cellular reproduction. The central feature of DNA replication is the template-induced nucleotidyl transfer reaction mediated by DNA polymerases. Recently, several structural studies have been done in elucidating the mechanism of enzymatic polymerization, including the determination of the structures of several DNA polymerases and their complexes with substrate and substrate analogues. These structures suggest a two-metal ion mechanism of nucleotide incorporation, a feature thought to be shared by all families of DNA polymerases. One metal is associated with dNTP binding and the other involved in metal ion catalysis in the chemical step. In addition, a conformational change step was suggested from crystal structures of Pol beta and Klenow Fragments upon binding of dNTP substrate. There have been major efforts recently to investigate metal ions interactions and proposed conformational change of DNA polymerases upon the binding of the incoming dNTP. These efforts have exploited a myriad of techniques including computational, functional, and crystallographic studies, even though the conformational flexibility of the enzyme, substrates, and complexes makes it difficult to unambiguously predict the dynamic behavior of a polymerase during catalytic cycling. Alternatively, sAXS has been shown to be sensitive to the conformational changes of protein and nucleic acids. Thus, our goal in this proposal is to provide the structural insights into the interactions among divalent metal ions, DNA and dNTP substrates, with three polymerases, Pol beta, KF, and ASFV Pol X, and further correlated these studies to our biochemical and functional studies. By monitoring conformational changes induced by incorporation of DNA and dNTP substrate and by titration of divalent metal ions, we will experimentally determine forms and energetics of enzyme-substrate interactions and enzyme-metal ion interactions.