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DNA Polymerase Fidelity Mechanisms: Theory and Experiment

DNA 聚合酶保真机制：理论与实验

基本信息

批准号：
9326179
负责人：
MYRON GOODMAN
金额：
$ 109.35万
依托单位：
UNIVERSITY OF SOUTHERN CALIFORNIA
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-03 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9326179
关键词：
Active Sites Affinity Amino Acids Area Base Excision Repairs Base Pairing Binding Biochemistry Biological Sciences Catalysis Cations Cells Cereals Charge Chemicals Chemistry Chicago Computer Analysis Computer Simulation Coupled Crystallization DNA DNA biosynthesis DNA-Directed DNA Polymerase Data Development Discipline Discrimination Drug Design Electrostatics Enzymes Event Family Free Energy Future Goals Grant Individual Investigation Kinetics Link Malignant Neoplasms Measures Mitochondria Modeling Molecular Molecular Conformation Mutagenesis Myron Nitrogen Nucleotides Oxygen Play Polymerase Principal Investigator Property Proteins Research Personnel Research Project Grants Resolution Roentgen Rays Role Side Specificity Stereoisomer Structure Testing Theoretical model Therapeutic Time United States National Institutes of Health Universities Variant analog atomic interactions base bisphosphonate cancer cell carbene computer studies design divalent metal experimental study human DNA inhibitor/antagonist innovation inorganic phosphate medical schools mutant novel programs scaffold simulation success theories translational approach tumorigenesis

项目摘要

This PPG integrates multiple disciplines to apply X-ray structural studies, presteady state kinetic and theoretical computational analyses and novel chemical probes to elucidate the molecular basis of DNA polymerase catalysis incorporating base-pair discrimination, a fundamental issue in mutagenesis relevant to cancer. The Program Project contains three research projects, structural (Project 1), theoretical computational (Project 2), and kinetics coupled with an approach toward translational paths (Project 3). Our success at synthesizing dNTP substrate analogs, by replacing one or both phosphate bridging oxygen molecules with a large variety of halo-methylene derivatives containing widely differing electrostatic charge and steric properties, allows us to probe fidelity from a transitions state (T) perspective. The use of these substrate analogs is a uniquely powerful aspect of our PPG, and will allow us for the first time to investigate TS effects using stereoisomeric probes, while offering a feasible approach for targeted inhibition of Pol p, on a path toward cancer cell inhibition (Project 3). The objective of Project 1 is to obtain high-resolution structural data for normal and aberrant forms of Pol ¿, using the dNTP analogs designed in Project 3 and synthesized in Core B. The goal of Project 2 is the application of theoretical and computer modeling to perform structure/function analyses of catalytic mechanisms that govern base selection both in the ground-state and TS. The computations are aimed at calculating free energies, which are used to predict individual contributions of amino acid side chains to fidelity, including substrate binding and catalysis in the pol active site. Central to our PPG is that the theory (Project 2) serves as the intellectual framework with which to marry structural analysis (Project 1) with kinetic mechanistic analysis (Project 3). It is atypical for the experimentalist t test a priori computational predictions. A defining aspect of this PPG is its bidirectional interply, where structural data serve as a starting point for computational predictions, which are tested experimentally, and where the experimental data are used to refine the theory.

这PPG集成了多个学科应用X射线结构研究，presteady状态动力学和理论计算分析和新的化学探针，以阐明DNA聚合酶催化的分子基础，结合碱基对歧视，一个基本问题，在诱变相关的癌症。该计划项目包含三个研究项目，结构（项目1），理论计算（项目2）和动力学加上对平移路径的方法（项目3）。我们在合成dNTP底物类似物的成功，通过取代一个或两个磷酸桥氧分子与各种各样的卤代亚甲基衍生物含有广泛不同的静电荷和空间特性，使我们能够探测保真度从过渡态（T）的角度来看。这些底物类似物的使用是我们PPG的一个独特的强大方面，并且将使我们能够首次使用立体异构探针研究TS效应，同时为靶向抑制Pol p提供可行的方法，从而实现癌细胞抑制（项目3）。项目1的目标是获得高分辨率的结构数据，使用项目3中设计并在核心B中合成的dNTP类似物，检测正常和异常形式的Pol？。项目2的目标是应用理论和计算机建模来进行催化机制的结构/功能分析，这些催化机制在基态和TS中都控制着碱基选择。计算的目的是计算自由能，自由能用于预测氨基酸侧链对保真度的个体贡献，包括底物结合和在POL活性位点的催化。我们的PPG的核心是理论（项目2）作为知识框架，将结构分析（项目1）与动力学机理分析（项目3）结合起来。实验主义者检验先验计算预测是不典型的。这个PPG的一个定义方面是它的双向interply，其中结构数据作为计算预测的起点，通过实验进行测试，并且实验数据用于完善理论。

项目成果

期刊论文数量（11）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Uniform Free-Energy Profiles of the P-O Bond Formation and Cleavage Reactions Catalyzed by DNA Polymerases β and λ.

DOI：
10.1021/acs.jpcb.6b08581
发表时间：
2016-12-29
期刊：
JOURNAL OF PHYSICAL CHEMISTRY B
影响因子：
3.3
作者：
Klvana, Martin;Bren, Urban;Florian, Jan
通讯作者：
Florian, Jan

Synthesis of ortho-Formylphenylphosphonic Acids as Covalent Probes of Active Site Lysines.

作为活性位点赖氨酸共价探针的邻甲酰基苯基膦酸的合成。

DOI：
10.1080/10426507.2018.1539996
发表时间：
2019
期刊：
Phosphorus, sulfur, and silicon and the related elements
影响因子：
0
作者：
Duro,MarlonVincentV;Alnajjar,KhadijehS;Sweasy,JoannB;Kashemirov,BorisA;McKenna,CharlesE
通讯作者：
McKenna,CharlesE

Remarkably Stereospecific Utilization of ATP α,β-Halomethylene Analogues by Protein Kinases.

蛋白激酶对 ATP α,β-卤代亚甲基类似物的显着立体特异性利用

DOI：
10.1021/jacs.7b03266
发表时间：
2017-06-14
期刊：
Journal of the American Chemical Society
影响因子：
15
作者：
Ni F;Kung A;Duan Y;Shah V;Amador CD;Guo M;Fan X;Chen L;Chen Y;McKenna CE;Zhang C
通讯作者：
Zhang C

5'-β,γ-CHF-ATP diastereomers: synthesis and fluorine-mediated selective binding by c-Src protein kinase.