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）：这个项目的长期目标是描述和理解DNA聚合酶的构象运动。（Pol ?）参与其催化反应。波尔吗?是DNA修复过程中必不可少的酶，DNA修复过程维持了人类基因组的完整性，并最终预防癌症。为了进一步加深对这种酶的认识，将结合稳态和预稳态动力学的溶液核磁共振实验应用于Pol ？重点是三个具体目标。7 .确定WT Pol ？载子、二元和三元配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在哪个步骤。为了验证肿瘤相关的Pol b突变（I260M， K289M和E295K）与WT酶相比改变了毫秒运动的假设。描述目的2中描述的WT和癌症相关突变体如何与氧化损伤的DNA发生不同的相互作用。目标1将通过酶配合物的溶液核磁共振弛豫分散实验来满足，该实验模拟了Pol ？催化反应坐标。在第二目标，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来研究，以阐明在反应的哪个阶段坐标改变运动或构象发生。最后，Aim 3将比较酶动力学实验和核磁共振动力学实验，以监测dNTPs对氧化损伤DNA的掺入。这些实验将关注WT和癌症相关突变在面对受损DNA时在酶动力学参数和构象变化方面的差异。