Mechanisms of DNA Polymerases

描述（由申请人提供）： DNA聚合酶在DNA复制和修复过程中对维持基因组秩序至关重要，因此对于物种的长期生存至关重要。当由各种外源性和内源性来源（例如环境化学品和辐射，吸烟，热畸变）引起的DNA损伤不能准确修复时，它可能导致人类疾病，如结肠癌，肺癌或皮肤癌和过早衰老。因此，理解DNA合成中的聚合酶保真度机制代表了基本的生物学和生物医学挑战。DNA聚合酶的保真度广义上是指它们掺入与模板DNA互补的正确而不是不正确的核苷酸的能力;这样的保真度跨越宽范围，从每一百万个掺入的核苷酸1个到接近10个（E6）错误。基于广泛的结构和动力学数据，以及理论研究的几种DNA聚合酶，我们假设高保真酶紧密编排的组装前的活性位点的核苷酸掺入，而较低的保真度聚合酶有一个更灵活的活性位点，因此一个独特的组装过程，在静电环境和可塑性的结合口袋可能导致的特征差异。由于DNA聚合酶的静态晶体结构和动力学实验研究不能描述完整的动态和充满活力的活性位点的影响，动力学模拟是很好的准备，迫切需要，以补充聚合酶的实验结果。在我们的实验和理论团队之间的合作项目中，我们将系统地研究原子分辨率下的构象变化和核苷酸掺入高保真度的（化学）途径（pol β）和低保真（Dpo 4）聚合酶决定不同的操纵机制，以及模板碱基、进入的核苷酸、关键蛋白残基，和损伤修饰的DNA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（过渡路径采样，随机路径方法，主成分分析和混合量子经典力学方法）的组合以及理论和实验之间的迭代设计来实现，用于测试，验证和扩展这些假设。特别是，通过描绘完整的反应配置文件（构象变化和化学）正确和不正确的碱基对在聚合酶β和相关的实验确定的催化效率和保真度值，我们将提出的限速步骤，编排的活性位点组装，和保真度机制，并随后测试它们的突变体系统的实验。此外，我们将测试我们的假设，即Dpo 4的拇指和小指域的细微构象变化与Dpo 4的低保真度和病变绕过机制密切相关，这可能与pol β不同。我们的长期目标是弥合宏观聚合酶结构和动力学测量有关的催化效率，保真度，和核苷酸结合亲和力，以更好地了解DNA聚合酶的保真度机制，包括对氧化损伤和其他病变的反应。这些研究可立即应用于诊断，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.