NMR dynamical characterization of WT and cancer-associated DNA polymerase

描述（由申请人提供）：该项目的长期目标是表征和理解DNA聚合酶中的构象运动？(Pol ?)参与其催化反应。波尔？是DNA修复过程中必不可少的酶，DNA修复过程是维持人类基因组完整性并最终防止癌症的过程。为了推进目前的理解，这种酶，溶液NMR实验结合稳态和预稳态动力学将承担波尔？重点是三个具体目标。 7为了确定WT Pol？APO、二元和三元络合物中的运动与催化反应序列中观察到的速率常数相关，并确定这些运动发生在哪个步骤。 7为了检验肿瘤相关Pol B突变（I260 M、K289 M和E295 K）与WT酶相比具有改变的毫秒运动的假设。 7描述目标2中描述的WT和癌症相关突变体如何与氧化损伤的DNA差异性地相互作用。目标1将满足溶液NMR弛豫分散实验酶复合物，模仿谨慎的步骤上的波尔？催化反应坐标在目标2中，波尔？已知与人类癌症相关的突变将通过酶动力学和NMR动力学测量来研究，以阐明在反应坐标上的哪个阶段发生改变的运动或构象。最后，目标3将比较酶动力学实验，监测掺入dNTPs对氧化损伤的DNA与NMR动力学实验。这些实验将集中在野生型和癌症相关突变之间的差异，在其酶动力学参数和构象变化时，面对受损的DNA。

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.