Mechanisms of DNA Polymerases

描述(由申请人提供)： DNA聚合酶在DNA复制和修复过程中维持基因组秩序，从而对物种的长期生存至关重要。当各种外源性和内源性来源(如环境化学物质和辐射、吸烟、热畸变)引起的DNA损伤得不到准确修复时，它可能会导致人类疾病，如结肠癌、肺癌或皮肤癌和过早衰老。因此，了解DNA合成中聚合酶的保真度机制是一个基本的生物学和生物医学挑战。DNA聚合酶的保真度广泛地指的是它们结合与模板DNA互补的正确的而不是不正确的核苷酸的能力；这种保真度跨越了很大的范围，从1到近10(E6)个误差每掺入一百万个核苷酸。基于大量的结构和动力学数据以及对几种DNA聚合酶的理论研究，我们假设高保真度的酶在核苷酸掺入之前紧密协调活性部位的组装，而低保真度的聚合酶有更灵活的活性部位，因此有不同的组装过程；可能导致静电环境和结合口袋的可塑性的特征差异。由于DNA聚合酶的静态晶体结构和动力学实验研究不能完全描述活性中心的动态和能量效应，动力学模拟是非常合适的，也是迫切需要的，以补充聚合酶的实验结果。在我们的实验团队和理论团队的合作项目中，我们将在原子分辨率下系统地研究高保真(Polbeta)和低保真(Dpo4)聚合酶的构象变化和核苷酸掺入(化学)途径如何决定不同的指导机制，以及模板碱基、传入核苷酸、关键蛋白质残基和病变修饰的DNA如何影响结合口袋静电环境/可塑性，从而影响保真度。这些目标将通过长期分子动力学模拟和新方法(过渡路径抽样、随机路径方法、主成分分析和混合量子经典力学方法)的结合以及理论和实验之间的迭代设计来实现，以测试、验证和扩展这些假设。特别是，通过描绘olβ中正确和不正确碱基对的完整反应轮廓(构象变化和化学)，并将它们与实验确定的催化效率和保真度值相关联，我们将提出限速步骤、活性部位组装的编排和所涉及的保真度机制，并随后通过突变系统的实验对它们进行测试。此外，我们将检验我们的假设，即Dpo4‘S拇指和小指结构域的微小构象变化与Dpo4’S的低保真和病变绕过机制密切相关，这些机制可能与polbeta不同。我们的长期目标是将宏观聚合酶结构与催化效率、保真度和核苷酸结合亲和力的动力学测量联系起来，以更好地了解DNA聚合酶的保真机制，包括对氧化损伤和其他损伤的反应。这类研究立即应用于诊断，并最终通过聚合酶抑制剂治疗由DNA修复缺陷引起的人类疾病，如各种癌症和过早衰老。

DESCRIPTION (provided by applicant): DNA polymerases are essential for maintaining genomic order during DNA replication and repair and thus for the long-term survival of a species. When DNA damage arising from a variety of exogenous and endogenous sources (e.g. environmental chemicals and radiation, smoking, thermal aberrations) is not accurately repaired, it can lead to human diseases like colon, lung, or skin cancer and premature aging. Thus, understanding polymerase fidelity mechanisms in DNA synthesis represents a fundamental biological and biomedical challenge. The fidelity of DNA polymerases broadly refers to their ability to incorporate correct rather than incorrect nucleotides complementary to the template DNA; such fidelities span a wide range, from 1 to nearly 10(E6) errors per one million nucleotides incorporated. Based on extensive structural and kinetic data as well as theoretical studies for several DNA polymerases, we hypothesize that high fidelity enzymes tightly orchestrate the assembly of the active site prior to nucleotide incorporation, while lower fidelity polymerases have a more flexible active site and thus a distinct assembly process; characteristic differences in the electrostatic environment and plasticity of the binding pocket likely result. Since static crystallographic structures and kinetic experimental studies of DNA polymerases cannot describe complete dynamic and energetic effects of the active site, dynamics simulations are well poised, and critically needed, to complement polymerase experimental results. In our collaborative project between an experimental and theoretical team, we will investigate systematically at atomic resolution how the conformational changes and nucleotide incorporation (chemical) pathways for higher-fidelity (pol beta) and low-fidelity (Dpo4) polymerases dictate different steering mechanisms, and how the template base, incoming nucleotide, key protein residues, and lesion-modified DNA affect the binding pocket electrostatic environment/plasticity and thus fidelity. These aims will be achieved by a combination of long-time molecular dynamics simulations and novel methodologies (transition path sampling, stochastic path approach, principal component analysis, and mixed quantum-classical mechanics methods) and an iterative design between theory and experimentation for testing, validating, and expanding these hypotheses. In particular, by delineating complete reaction profiles (conformational change and chemistry) for correct and incorrect basepairs in pol beta and relating them to experimentally-determined catalytic efficiencies and fidelity values, we will propose the rate-limiting step, orchestration of the active site assembly, and fidelity mechanisms involved and subsequently test them by experiments on mutant systems. Moreover, we will test our hypothesis that subtle conformational changes in Dpo4's thumb and little finger domains are closely associated with Dpo4's low-fidelity and lesion bypassing mechanisms, which are likely distinct than pol beta's. Our long term goals are to bridge macroscopic polymerase structures and kinetic measurements regarding catalytic efficiency, fidelity, and nucleotide binding affinity to better understand fidelity mechanisms of DNA polymerases, including response to oxidative damage and other lesions. Such studies have immediate applications to the diagnostics, and eventually treatment via polymerase inhibitors, of human diseases caused by defective repair of DNA, like various cancers and premature aging.