Mechanisms of replication fork degradation in vertebrates

脊椎动物复制叉降解机制

• 批准号: 10564777
• 负责人: James M Dewar
• 金额: $ 48.87万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2027-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10564777
• 关键词: Address; Aromatic Polycyclic Hydrocarbons; BRCA1 gene; BRCA2 gene; Back; Behavior; Biochemical; Cancer Etiology; Cell Death; Cells; Chemicals; Complex; Cytoprotection; DNA; DNA Damage; DNA Repair; DNA Repair Enzymes; DNA biosynthesis; DNA lesion; DNA replication fork; Data; Defect; Disease; Ensure; Environment; Event; Exposure to; Genome Stability; Genomic Instability; Human; In Vitro; Individual; Inherited; Ionizing radiation; Learning; Lesion; Malignant Neoplasms; Modeling; Molecular; Mutagens; Mutation; Pathway interactions; Predisposition; Process; Proteins; Radiation; Reactive Oxygen Species; Sensitivity and Specificity; Site; Source; Specificity; System; Time; Ultraviolet Rays; Vertebrates; Work; Xenopus; disease-causing mutation; egg; environmental agent; environmental chemical; helicase; human disease; improved; insight; novel strategies; nuclease; protein function; response; single molecule

PROJECT SUMMARY DNA replication is constantly challenged by a variety of genotoxins that arise from the environment. These genotoxins can be directly produced by the environment (e.g. UV and ionizing radiation) or can arise indirectly in response to environmental agents (e.g. polycyclic aromatic hydrocarbons, reactive oxygen species). Nascent strand degradation (NSD) and fork reversal promote genome stability in response to genotoxins by facilitating replication fork restart. Despite the importance of nascent strand degradation and fork reversal, there are many open questions about this pathway. For example, too much or too little degradation results in genome stability. It is therefore important to understand how nascent strand degradation is efficiently triggered when needed but with enough specificity that spurious degradation is avoided. However, we do not currently understand how nascent strand degradation is triggered. Additionally, current models for nascent strand degradation are too limited to explain the dozens of proteins currently implicated. Inherited defects in several of these proteins are directly implicated in human diseases (e.g. SMARCAL1, BRCA1, BRCA2) suggesting that defects in this pathway may alter individuals’ susceptibility to environmental genotoxins. Thus, it is crucial to develop a robust paradigm for nascent strand degradation and fork reversal to establish exactly how this pathway leads to replication restart and genome stability. Current approaches to study nascent strand degradation and fork reversal lack the specificity and sensitivity to address these questions. To overcome these limitations, we have developed a new site-specific, highly sensitive, and synchronous approach to study nascent strand degradation and fork reversal in vitro using Xenopus egg extracts. This system contains the full set of cellular proteins involved in DNA replication and DNA repair and provides unparalleled opportunities to observe and manipulate these processes. Our new approach has already revealed key insights into the requirements for nascent strand degradation and the mechanism by which it takes place. The proposed work will combine biochemical and single molecule approaches, both in Xenopus egg extracts and human cells. We will leverage our existing insights and exploit the power of our new system to determine how nascent strand degradation and fork reversal are triggered and the underlying molecular mechanisms involved in these processes. This work will enhance our understanding of one of the major cellular pathways that responds to environmentally sourced genotoxins and allow us to better understand how defects in this pathway may alter individuals’ susceptibility to environmental genotoxins.

项目概要 DNA 复制不断受到环境中产生的各种基因毒素的挑战。这些 基因毒素可以由环境直接产生（例如紫外线和电离辐射），也可以间接产生 响应环境因素（例如多环芳烃、活性氧）。 新生链降解（NSD）和叉逆转通过以下方式促进基因组稳定性以应对基因毒素： 促进复制叉重新启动。尽管新生链降解和分叉逆转很重要， 关于这条途径还有许多悬而未决的问题。例如，降解过多或过少都会导致 基因组稳定性。因此，了解如何有效触发新生链降解非常重要 在需要时但具有足够的特异性以避免虚假降解。然而，我们目前不 了解新生链降解是如何触发的。此外，当前新生链模型 降解太有限，无法解释目前涉及的数十种蛋白质。一些遗传缺陷 这些蛋白质与人类疾病直接相关（例如 SMARCAL1、BRCA1、BRCA2），表明 该途径的缺陷可能会改变个体对环境基因毒素的敏感性。因此，至关重要的是 开发一个强大的新生链降解和分叉逆转范例，以确定这是如何发生的 途径导致复制重启和基因组稳定性。目前研究新生链的方法 降解和分叉反转缺乏解决这些问题的特异性和敏感性。克服 针对这些限制，我们开发了一种新的针对特定地点、高度敏感和同步的研究方法 使用非洲爪蟾卵提取物进行体外新生链降解和叉逆转。该系统包含完整的 一组参与 DNA 复制和 DNA 修复的细胞蛋白，并提供了无与伦比的机会 观察并操纵这些过程。我们的新方法已经揭示了对以下问题的关键见解： 新生链降解的要求及其发生的机制。拟议的工作 将在非洲爪蟾卵提取物和人类细胞中结合生物化学和单分子方法。我们 将利用我们现有的见解并利用我们新系统的力量来确定新生链如何 降解和分叉反转被触发，并且涉及这些的潜在分子机制 流程。这项工作将增强我们对响应的主要细胞途径之一的理解 环境来源的基因毒素，使我们能够更好地了解该途径中的缺陷如何可能改变 个体对环境基因毒素的易感性。