Role of DNA structural dynamics in mutagenesis and oncogenesis

项目摘要 突变驱动进化，解释了种群中的遗传变异，并且是遗传变异的主要原因。 癌症和其他遗传疾病。然而，我们对生物化学过程的分子理解， 因为变异还很不成熟。对于大多数突变过程，我们不明白为什么 突变概率根据碱基取代的类型而变化很多个数量级， 序列上下文。大多数突变模式不能用1D序列或3D结构来解释。 突变过程中，由于外源基因的突变， 虽然已经广泛地描述了DNA的来源（例如紫外线，吸烟），但越来越多的研究指出DNA复制 错误是致病突变的重要和潜在的主要来源。但 DNA复制错误的分子机制及其对肿瘤发生的作用并不 完全理解此外，在人类癌症中发现的超过一半的突变过程， 未知的生化起源这项提议的主要假设是，改变DNA结构的DNA动力学， 碱基配对模式是突变过程的主要驱动力。该项目将实验性地描述 序列和错配依赖的DNA碱基对动力学具有前所未有的广度和深度， 生成描述形成替代物的序列特异性概率的构象倾向 诱变构象这些知识将被用于发展对复制的预测性理解 由人类聚合酶ε产生的错误，ε是负责真核细胞核DNA的两种聚合酶之一 复制的技术创新是发展的突破口，是关键和必要的 高通量测量DNA结构动力学的技术，使数百项研究成为可能。 在某些情况下有数千个序列变体。目标1将决定各种倾向 错配形成沃森-克里克样构象，测量复制错误的签名， 校正缺陷的人类聚合酶ε，并提出序列和错配的预测模型， 依赖性核苷酸错误掺入。目标2将确定未配对样本的倾向 构象，测量用于校正熟练的人类聚合酶ε的复制错误的签名， 并提出了一个预测模型的序列和错配依赖的复制错误。目标3将 确定形成Hoogsteen碱基对的倾向，并揭示由 Hoogsteen介导的损伤。通过对DNA复制的深入和预测性的理解， 不忠和损害，这项工作将有助于阐明驱动进化的基本过程， 肿瘤发生，同时也提供了一个概念框架和实验工具，可以帮助催化 发现和表征由DNA动力学驱动的其他诱变和生化过程。

Project Summary Mutations drive evolution, account for genetic variants in the population, and are the primary cause of cancer and other genetic disorders. Yet our molecular understanding of the biochemical processes that cause mutations remains rudimentary. For most mutational processes, we do not understand why the mutational probabilities vary by many orders of magnitude depending on the type of base substitution and sequence context. Most mutational patterns cannot be explained by the 1D sequence or 3D structural characteristics of the DNA motif in which they are found. While mutational processes due to exogenous sources (e.g. UV, smoking) have been described extensively, studies increasingly point to DNA replicative errors as an important and potentially dominant source of disease-causing mutations. However, the molecular mechanisms that underlie DNA replicative errors and their contributions to oncogenesis are not fully understood. In addition, over half of the mutational processes identified in human cancers have unknown biochemical origins. The main hypothesis in this proposal is that DNA dynamics that alter the mode of base pairing is a major driver of mutational processes. The project will experimentally characterize sequence and mismatch-dependent DNA base pair dynamics with unprecedented breadth and depth, and generate conformational propensities describing the sequence-specific probabilities of forming alternative mutagenic conformations. This knowledge will be used to develop a predictive understanding of replication errors generated by human polymerase ε, one of two polymerases tasked with eukaryotic nuclear DNA replication. The critical and necessary technological innovation is the development of breakthrough techniques for measuring DNA structural dynamics in high throughput, enabling studies of over hundreds and in some cases thousands of sequence variants. Aim 1 will determine the propensities for various mismatches to form Watson-Crick like conformations, measure the signatures of replicative error for proofreading deficient human polymerase ε, and advance a predictive model for sequence- and mismatch- dependent nucleotide misincorporation. Aim 2 will determine the propensities to sample unpaired conformations, measure the signatures of replicative error for proofreading proficient human polymerase ε, and advance a predictive model for sequence- and mismatch-dependent replicative errors. Aim 3 will determine propensities to form Hoogsteen base pairs, and uncover mutational processes driven by Hoogsteen-mediated damage. By developing a deep and predictive understanding of DNA replication infidelity and damage, this work will help illuminate fundamental processes that drive evolution and oncogenesis while also providing a conceptual framework and experimental tools that can help catalyze the discovery and characterization of other mutagenic and biochemical processes driven by DNA dynamics.