Role of DNA structural dynamics in mutagenesis and oncogenesis

项目摘要 突变推动了进化，解释了种群中的遗传变异，是 癌症和其他遗传性疾病。然而，我们对生化过程的分子理解 导致突变的原因仍然是初步的。对于大多数突变过程，我们不理解为什么 突变概率根据碱基替换的类型和 序列上下文。大多数突变模式不能用一维序列或三维结构解释 它们所在的DNA基序的特征。而外源基因引起的突变过程 来源(如紫外线、吸烟)已经被广泛描述，研究越来越多地指向DNA复制 错误是致病突变的一个重要和潜在的主导来源。然而， DNA复制错误背后的分子机制及其对肿瘤发生的贡献 完全理解。此外，在人类癌症中发现的突变过程中，超过一半的人 未知的生化来源。这一提议中的主要假设是，改变DNA动力学的 碱基配对模式是突变过程的主要驱动因素。该项目将试验性地描述 序列和错配相关的DNA碱基对动力学具有前所未有的广度和深度，以及 生成描述序列特定的形成替代的概率的构象倾向 诱变构象。这些知识将用于发展对复制的预测性理解 人类聚合酶ε产生的错误，这是负责真核核DNA的两种聚合酶之一 复制。关键和必要的技术创新是突破性的发展 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，还有数千种序列变体。目标1将确定各种不同的倾向 失配以形成类似Watson-Crick的构象，测量复制误差的特征 校对缺失的人类聚合酶ε，并提出序列和错配的预测模型 依赖的核苷酸错误结合。目标2将确定未配对样本的倾向 构象，测量复制错误的特征以校对熟练的人聚合酶ε， 并提出了序列和失配相关复制错误的预测模型。目标3将 确定形成Hoogsteen碱基对的倾向，并发现由 胡格斯汀介导的损害。通过发展对DNA复制的深入和预测性理解 不忠和破坏，这项工作将有助于阐明推动进化和 肿瘤发生，也提供了一个概念框架和实验工具，可以帮助催化 由DNA动力学驱动的其他诱变和生化过程的发现和表征。

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.