NMR dynamical characterization of WT and cancer-associated DNA polymerase

描述(由申请人提供)：这个项目的长期目标是描述和理解DNA聚合酶中的构象运动？(波尔？)参与其催化反应。波尔？是DNA修复过程中的一种必不可少的酶，DNA修复过程是维持人类基因组完整性并最终预防癌症的过程。为了加深对该酶的认识，将结合稳态和稳态前动力学的溶液核磁共振实验对POL？重点关注三个具体目标。7以确定WT Pol是否？脱脂蛋白、二元和三元络合物中的运动与观察到的催化反应序列的速率常数相关，并确定这些运动发生在什么步骤。7检验与肿瘤相关的Pol b突变(I260M、K289M和E295K)与WT酶相比改变了毫秒运动的假设。7表征目标2中描述的WT和癌症相关突变体如何与氧化损伤的DNA发生差异相互作用。目标1将通过模拟POL？上谨慎步骤的酶复合体的溶液核磁共振弛豫分散实验来实现。催化反应坐标。在目标2里，波尔？已知与人类癌症相关的突变将通过酶动力学和核磁共振动力学测量进行研究，以阐明在反应坐标上的哪个阶段发生运动或构象变化。最后，Aim 3将比较监控dNTPs掺入相对氧化损伤DNA的酶动力学实验和核磁共振动力学实验。这些实验将集中于WT和癌症相关突变在酶动力学参数和面对DNA损伤时的构象变化方面的差异。 与公共卫生相关：人类DNA不断受到破坏，必须修复这种损害，以防止可能导致癌症的突变。DNA聚合酶？是一种修复受损DNA的酶。这项提议旨在了解聚合酶b是如何工作的，以提供将导致更好药物的洞察力。

DESCRIPTION (provided by applicant): The long-term goals of this project are to characterize and understand how conformational motions in DNA polymerase ? (Pol ?) participate in its catalytic reaction. Pol ? is an essential enzyme in the repair process of DNA, a process that maintains the integrity of the human genome and ultimately protects against cancer. To advance the current understanding of this enzyme, solution NMR experiments in combination with steady- state and pre-steady-state kinetics will be brought to bear on Pol ? with focus on three specific aims. 7 To determine if WT Pol ? motions in the apo, binary, and ternary complexes correlate with rate constants observed for the catalytic reaction sequence and to determine at what step these motions occur. 7 To test the hypothesis that tumor-associated Pol b mutations (I260M, K289M & E295K) have altered millisecond motions compared to the WT enzyme. 7 To characterize how WT and cancer-associated mutants, described in Aim 2, differentially interact with oxidatively damaged DNA. Aim 1 will be met by solution NMR relaxation dispersion experiments on enzyme complexes that mimic discreet steps on the Pol ? catalytic reaction coordinate. In Aim 2, Pol ? mutations that are known to be associated with human cancer will be investigated by enzyme kinetics, and NMR dynamics measurements to elucidate at what stage on the reaction coordinate altered motions or conformations occur. Lastly, Aim 3 will compare enzyme kinetics experiments that monitor the incorporation of dNTPs opposite oxidatively damaged DNA with NMR dynamics experiments. These experiments will focus on differences between WT and cancer- related mutations in their enzyme kinetics parameters and conformational changes when faced with damaged DNA. PUBLIC HEALTH RELEVANCE: Human DNA is constantly being damaged this damage must be repaired to prevent mutations that would lead to cancer. DNA polymerase ? is an enzyme that fixes damaged DNA. This proposal aims to understand how polymerase b works, to provide insight that will lead to better drugs.