Mechanisms of replication fork degradation in vertebrates

项目总结 DNA复制不断受到来自环境的各种遗传毒素的挑战。这些 遗传毒素可由环境(如紫外线和电离辐射)直接产生或间接产生 对环境因素(如多环芳烃、活性氧类)的反应。 新生链降解(NSD)和叉状反转通过以下方式促进基因组对遗传毒素的稳定 促进复制分叉重启。尽管新生的链降解和叉子颠倒很重要， 关于这条途径，还有许多悬而未决的问题。例如，降级太多或太少都会导致 基因组的稳定性。因此，重要的是要了解如何有效地触发新生的链降解 当需要的时候，但要有足够的专一性，以避免虚假的退化。然而，我们目前不支持 理解新生的链降解是如何被触发的。此外，目前的新生钢丝绳模型 降解的程度太有限，无法解释目前涉及的数十种蛋白质。几个人的遗传缺陷 这些蛋白质直接与人类疾病有关(例如SMARCAL1、BRCA1、BRCA2)，这表明 这一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 为新生的链降解和叉子反转开发一个健壮的范例，以准确地确定这一点 途径导致复制重新开始和基因组的稳定。当前研究新生链条的方法 退化和叉形颠倒缺乏解决这些问题的特异性和敏感性。要克服 针对这些局限性，我们开发了一种新的针对特定地点、高度敏感和同步的方法来研究 利用非洲爪哇卵提取液在体外进行初生链降解和叉子反转。该系统包含完整的 一组参与DNA复制和DNA修复的细胞蛋白质，为 观察和操纵这些过程。我们的新方法已经揭示了对 对新生链降解的要求及其发生的机制。拟议中的工作 将结合生化和单分子方法，在非洲爪哇卵子提取液和人类细胞中都是如此。我们 将利用我们现有的洞察力并利用我们新系统的力量来确定新股如何 降解和分叉反转被触发，潜在的分子机制涉及到这些 流程。这项工作将加强我们对应答的主要细胞通路之一的理解 环境来源的基因毒素，使我们能够更好地了解这一途径中的缺陷可能会如何改变 个体对环境基因毒素的易感性。

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.