PROBING THE CONFORMATIONAL STATES OF E?DNA COMPLEX UPON THE INCORPORATION OF DNT

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 DNA复制是细胞繁殖所必需的基本生物学过程。DNA复制的中心特征是DNA聚合酶介导的模板诱导的核苷酸转移反应。近年来，人们在阐明酶促聚合机理方面进行了一些结构研究，包括几种DNA聚合酶及其与底物和底物类似物的络合物的结构测定。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聚合酶家族的共同特征。一种金属与dNTP结合，另一种金属参与化学步骤中的金属离子催化。此外，Polβ和Pol I的晶体结构在与dNTP底物结合时发生了构象变化。最近，人们在研究金属离子相互作用和DNA聚合酶与dNTP结合时的构象变化方面做了大量的工作。这些努力利用了无数的技术，包括计算、功能和结晶学研究，尽管酶、底物和络合物的构象灵活性使得很难明确预测聚合酶在催化循环中的动态行为。另外，小角X射线散射(SAXS)被证明对蛋白质和核酸的构象变化很敏感。因此，我们在这个建议中的目标是提供关于二价金属离子、DNA和dNTP底物与三种聚合酶Polβ、Pol I和ASFV Pol X之间相互作用的结构见解，并进一步将这些研究与我们的生化和功能研究联系起来。通过监测DNA和dNTP底物的掺入以及二价金属离子的滴定引起的构象变化，我们将从实验上确定酶-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 Pol I 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, small-angle x-ray scattering (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, Pol I 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.