NMR dynamical characterization of WT and cancer-associated DNA polymerase

WT 和癌症相关 DNA 聚合酶的 NMR 动态表征

• 批准号: 8221655
• 负责人: JOSEPH P LORIA
• 金额: $ 27.64万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-01-15 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8221655
• 关键词: Base Excision Repairs; Biochemical; Biochemical Reaction; Biological Assay; Cancer Etiology; Cells; Chemistry; Complex; DNA; DNA Damage; DNA lesion; DNA-Directed DNA Polymerase; Data; Enzyme Kinetics; Enzymes; Event; Genome; Genomic Instability; Genomics; Goals; Guanine; Human; Human Genome; Kinetics; Lead; Maintenance; Malignant Neoplasms; Measurement; Molecular Conformation; Monitor; Motion; Multienzyme Complexes; Mutation; Nucleotides; Pharmaceutical Preparations; Polymerase; Positioning Attribute; Process; Reaction; Relaxation; Research; Site; Solutions; Staging; Substrate Specificity; Tertiary Protein Structure; Testing; Time; Ursidae Family; Variant; Work; base; enzyme activity; human DNA; insight; meetings; millisecond; mutant; prevent; repaired; research study; tumor

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.

描述（由申请人提供）：该项目的长期目标是表征和了解DNA聚合酶中的构象运动如何？ （POL？）参与其催化反应。 pol？是DNA修复过程中必不可少的酶，该过程维持人类基因组的完整性并最终预防癌症。为了促进当前对这种酶的理解，将使溶液NMR实验与稳态和稳态的动力学结合使用？专注于三个特定目标。 7确定WT POL是否？ Apo，二元和三元复合物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的步骤。 7为了测试与WT酶相比，与肿瘤相关的POL B突变（I260M，K289M和E295K）的假设改变了毫秒。 7为了表征AIM 2中描述的WT和与癌症相关的突变体如何与氧化损坏的DNA相互作用。 AIM 1将通过在模仿POL上逐步的酶复合物上的溶液NMR松弛色散实验来满足目标1？催化反应坐标。在AIM 2中，POL？已知与人类癌症相关的突变将通过酶动力学研究，NMR动力学测量值以阐明反应坐标阶段的变化运动或构象发生的阶段。最后，AIM 3将比较酶动力学实验，该酶实验通过NMR动力学实验来监测与氧化型DNA相反的DNTP的掺入。这些实验将集中在其酶动力学参数中WT和癌症相关突变之间的差异以及面临损坏的DNA时的构象变化。 公共卫生相关性：必须对人DNA不断损害这种损害，以防止导致癌症的突变。 DNA聚合酶？是固定受损DNA的酶。该提案旨在了解聚合酶B的工作原理，以提供将带来更好药物的见解。