Role of TopBP1 partner WDR18 in DNA damage checkpoint and DNA replication

描述（由申请人提供）： 项目概要/摘要本AREA项目的长期目标是研究DNA损伤检查点和DNA复制的分子机制，这对癌症、衰老、免疫缺陷和神经退行性疾病等生物学和病理学过程的研究具有重要意义。所有生物体的基因组都暴露于各种威胁，但基因组的DNA损伤检查点作为一种监视机制，监测基因组中的损伤，然后通过传感器，介体和效应蛋白直接细胞反应来解决问题。如果检查点在必要时未能激活，结果是未修复的损伤，导致基因组不稳定和肿瘤形成。理解这一过程的主要障碍是确定检查点如何感知DNA损伤以及蛋白质如何传递损伤信号。具体而言，目前尚不清楚激活的传感激酶ATR如何磷酸化其下游效应蛋白Chk 1。除了在DNA复制起始和DNA复制应激反应中的关键作用外，TopBP 1通过其C-末端区域在DNA损伤检查点信号传导中起重要作用。然而，其潜在机制尚不清楚。在爪蟾卵提取物中的初步数据显示，WDR 18，TopBP 1相互作用蛋白，与TopBP 1的C-末端相互作用，并且它们的相互作用是ATR激活Chk 1在响应双链断裂所必需的。这个R15项目的目标是测试TopBP 1伴侣WDR 18在DNA损伤检查点和DNA复制中起重要作用的假设。这个R15项目将在非洲爪蟾卵提取物中进行，这是一种可靠的无细胞生化系统，已用于细胞周期，DNA复制和检查点激活的各种研究。具体目标是：（1）通过各种生物化学和分子生物学方法阐明TopBP 1配偶体WDR 18在非洲爪蟾卵提取物中的DNA损伤检查点信号传导中的作用;（2）通过监测当通过免疫耗竭去除内源性WDR 18时存在或不存在损伤剂的大量DNA合成产物，确定在正常或应激条件下DNA复制是否需要WDR 18。这些研究将确定一种新的检查点和复制蛋白，并将使人们能够更好地了解DNA损伤检查点如何保护基因组完整性，以及当DNA损伤未修复或未触发适当反应时癌症如何发展。 公共卫生相关性： 通过监测机制准确复制遗传信息并检测任何可能的DNA损伤DNA损伤检查点对于维持基因组完整性和肿瘤抑制至关重要。我们将使用一个简单的模型系统，从青蛙卵中提取的非洲爪蟾卵提取物，来研究DNA损伤检查点信号是如何启动和传递的，以及DNA是如何高保真复制的。揭示这些基本过程的细节将更好地了解癌症如何发展，并最终通过操纵新的检查点和复制蛋白来设计抗癌药物的新途径。

DESCRIPTION (provided by applicant): Project Summary/Abstract The long-term goal of this AREA project focuses on the molecular mechanisms of DNA damage checkpoint and DNA replication, which has significant implications for the study of biological and pathological processes such as cancer, aging, immune deficiency and neurodegenerative disorders. Genomes of all living organisms are exposed to a variety of threats, but the genome's DNA damage checkpoint functions as a surveillance mechanism, monitoring damage in a genome and then directly cellular responses through sensor, mediator, and effector proteins to resolve the problem. If a checkpoint fails to activate when necessary, the result is unrepaired damage that leads to genomic instability and tumor formation. The main barriers to understanding this process are in identifying how checkpoints sense DNA damage and how the proteins relay the damage signal. Specifically, it is not known how the activated sensor kinase ATR phosphorylates its downstream effector protein Chk1. In addition to the critical roles in DNA replication initiation and DNA replication stress response, TopBP1 plays an important role in DNA damage checkpoint signaling through its C-terminus region. However, the underlying mechanism is not known. Preliminary data in Xenopus egg extracts show that WDR18, a TopBP1-interacting protein, interacts with TopBP1 C-terminus, and their interaction is required for ATR activation of Chk1 in the response to double-stranded breaks. The goal of this R15 project is to test the hypothesis that TopBP1 partner WDR18 plays essential roles in DNA damage checkpoint and DNA replication. This R15 project will be carried out in Xenopus egg extracts, a reliable cell-free biochemical system, that has been used for a wide variety of studies in cell cycle, DNA replication, and checkpoint activation. The specific aims are: (1) To elucidate the role of WDR18, a TopBP1 partner, in DNA damage checkpoint signaling in Xenopus egg extract through various biochemical and molecular biology approaches; and (2) To determine if WDR18 is required for DNA replication under normal or stressful conditions, by monitoring bulk DNA synthesis product with the presence or absence of damaging agents when endogenous WDR18 is removed by immunodepletion. These studies will identify a novel checkpoint and replication protein, and will enable a better understanding of how a DNA damage checkpoint works to protect genomic integrity and how cancer develops when DNA damage is not repaired or not triggering appropriate responses. PUBLIC HEALTH RELEVANCE: Project Narrative Accurate duplication of genetic information and sensing any possible DNA damage by a surveillance mechanism DNA damage checkpoint are vital for the maintenance of genomic integrity and tumor suppression. We will use a simple model system, Xenopus egg extracts derived from frog eggs, to examine how DNA damage checkpoint signaling is initiated and relayed, and how DNA is copied with high fidelity. Revealing the details of these essential processes will provide a better understanding of how cancer develops and ultimately novel avenues to design anti-cancer drugs by manipulating novel checkpoint and replication proteins.