Simulating Large-Scale Conformational Rearrangements and Reaction Kinetics Profiles in DNA Polymerase Beta to Interpret DNA Synthesis Fidelity Mechanisms

模拟 DNA 聚合酶 Beta 中的大规模构象重排和反应动力学曲线，以解释 DNA 合成保真度机制

• 批准号: 0316771
• 负责人: Tamar Schlick
• 金额: $ 83.88万
• 依托单位: New York University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0316771&HistoricalAwards=false
• 关键词: Simulating; Large; Scale; Conformational; Rearrangements

Studying large-scale, long-time biological processes such as enzyme catalysis, protein folding, and macromolecular assembly is a challenging task in computational biophysics. Since these processes occur over microseconds to seconds, much beyond the scope of traditional dynamics simulations, new techniques are needed to provide insights into detailed, local motions to supplement experiments. In this project, funded jointly by the Molecular Biophysics Program in the Division of Molecular and Cellular Biosciences and the Computational Math Program in the Division of Mathematical Sciences, the PI will develop, compare, and apply two rigorous and complementary path-generation methods, Elber's stochastic path approach (SPA) and Chandler's transition path sampling (TPS), to study the conformational transitions between closed and open states for human DNA polymerase beta (pol beta) complexed with DNA template/primer. This millisecond process is thought to be key in maintaining DNA synthesis fidelity. With these new tools, the PI will pursue several fundamental biological questions related to DNA synthesis fidelity, including the identification of slow conformational steps that steer the enzyme toward the chemistry-competent state and determination of rate-limiting steps in the enzyme's pathway. Atomic-level mechanistic insights, as well as associated free-energy barriers, will be delineated and related to enzyme function. The methodology developed is widely applicable to many other fundamental processes in molecular biophysics, and the biological findings will provide atomic-level interpretations to puzzling experimental variations in catalytic rates and error frequencies. Thus, the biological findings will help interpret fundamental fidelity mechanisms employed by DNA polymerases to replicate and repair DNA faithfully from one generation to the next.DNA polymerases maintain genomic integrity in the cell by replicating DNA and repairing damages in the genome from generation to generation. The goal of this project is to dissect the conformational aspects of the selectivity and fidelity of DNA repair process. Fidelity refers to the ability of DNA polymerases to discriminate among the various nucleotide building blocks as each base unit is synthesized and choose the correct base (parent strand's partner) for insertion and extension. The PI will employ modeling and simulation by novel path-generation schemes that can capture large-scale long-time processes to study these polymerase mechanisms to explain fidelity. Such information has important ramifications to our understanding the fundamental DNA synthesis and repair fidelity processes. This project represents a collaboration with theoreticians and experimentalists with expertise in nucleic-acid structure, polymerase mechanisms, and simulation methodology, and relies on solid groundwork in both methodology for biomolecular modeling. The tools developed are also widely applicable to many other important problems in biology. Involving undergraduate and graduate students and postdoctoral fellows, the project offers important multidisciplinary educational and training opportunities to young scientists, including women and minorities, in molecular modeling and computational biology, fields of growing importance to science and society.

研究酶催化、蛋白质折叠和大分子组装等大规模、长时间的生物过程是计算生物物理学中的一项具有挑战性的任务。 由于这些过程发生在微秒到秒的时间内，远远超出了传统动力学模拟的范围，因此需要新技术来提供对详细的局部运动的洞察，以补充实验。 在该项目中，由分子和细胞生物科学部的分子生物物理学计划和数学科学部的计算数学计划联合资助，PI 将开发、比较和应用两种严格且互补的路径生成方法，Elber 的随机路径方法 (SPA) 和钱德勒转变路径采样 (TPS)，研究人类 DNA 聚合酶 β (pol beta) 复合物的闭合状态和开放状态之间的构象转变与 DNA 模板/引物。 这一毫秒过程被认为是维持 DNA 合成保真度的关键。 借助这些新工具，PI 将解决与 DNA 合成保真度相关的几个基本生物学问题，包括识别引导酶进入化学活性状态的缓慢构象步骤，以及确定酶途径中的限速步骤。原子水平的机制见解以及相关的自由能障碍将被描述并与酶功能相关。所开发的方法广泛适用于分子生物物理学中的许多其他基本过程，并且生物学发现将为催化速率和误差频率中令人费解的实验变化提供原子水平的解释。因此，生物学发现将有助于解释 DNA 聚合酶用于忠实地复制和修复 DNA 的基本保真机制。DNA 聚合酶通过一代又一代地复制 DNA 和修复基因组中的损伤来维持细胞中的基因组完整性。 该项目的目标是剖析 DNA 修复过程的选择性和保真度的构象方面。 保真度是指 DNA 聚合酶在合成每个碱基单元时区分各种核苷酸构件并选择正确碱基（母链的伴侣）进行插入和延伸的能力。 PI 将通过新颖的路径生成方案进行建模和模拟，这些方案可以捕获大规模的长时间过程，以研究这些聚合酶机制以解释保真度。这些信息对于我们理解基本的 DNA 合成和修复保真过程具有重要影响。该项目代表了与在核酸结构、聚合酶机制和模拟方法方面具有专业知识的理论家和实验家的合作，并依赖于生物分子建模这两种方法的坚实基础。开发的工具也广泛适用于生物学中的许多其他重要问题。该项目涉及本科生、研究生和博士后研究员，为包括女性和少数族裔在内的年轻科学家提供重要的多学科教育和培训机会，涉及分子建模和计算生物学这些对科学和社会日益重要的领域。