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

研究大规模、长时间的生物过程，如酶催化、蛋白质折叠和大分子组装，是计算生物物理学中一项具有挑战性的任务。由于这些过程发生在微秒到几秒之间，远远超出了传统动力学模拟的范围，因此需要新的技术来提供对详细的局部运动的洞察，以补充实验。在分子与细胞生物科学系分子生物物理计划和数学科学部计算数学计划的共同资助下，PI将发展、比较和应用两种严格和互补的路径生成方法：Elber的随机路径方法(SPA)和Chandler的转移路径抽样(TPS)，以研究与DNA模板/引物复合的人DNA聚合酶β(Pol Beta)的闭合和开放状态之间的构象转变。这一毫秒级的过程被认为是维持DNA合成保真度的关键。利用这些新工具，PI将解决几个与DNA合成保真度相关的基本生物学问题，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。原子级的机械论见解，以及相关的自由能障碍，将被描绘出来并与酶的功能相关。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，生物学发现将为令人困惑的催化速率和误差频率的实验变化提供原子水平的解释。因此，这些生物学发现将有助于解释DNA聚合酶用来从一代到下一代忠实地复制和修复DNA的基本保真机制。DNA聚合酶通过复制DNA和修复基因组中的损伤来维持细胞中基因组的完整性。这个项目的目标是剖析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位时区分不同的核苷酸组成单元，并选择正确的碱基(亲链的伴侣)进行插入和延伸的能力。PI将通过新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来研究这些聚合酶机制，以解释保真度。这些信息对我们理解基本的DNA合成和修复保真度过程有重要的影响。这个项目代表了与具有核酸结构、聚合酶机制和模拟方法学专业知识的理论家和实验学家的合作，并依赖于生物分子建模的两种方法学的坚实基础。所开发的工具也广泛适用于生物学中的许多其他重要问题。该项目涉及本科生和研究生以及博士后研究员，为包括女性和少数民族在内的年轻科学家提供了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，这两个领域对科学和社会的重要性越来越大。

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.