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

描述(由申请人提供)： 该领域的长期目标集中在DNA损伤检查点和DNA复制的分子机制上，这对癌症、衰老、免疫缺陷和神经退行性疾病等生物和病理过程的研究具有重要意义。所有活着的生物体的基因组都面临着各种威胁，但基因组的DNA损伤检查点作为一种监测机制，监测基因组中的损伤，然后通过传感器、介体和效应器蛋白直接做出细胞反应来解决问题。如果检查点在必要时未能激活，结果是无法修复的损伤，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。具体地说，目前尚不清楚激活的传感器激酶ATR如何磷酸化其下游效应蛋白Chk1。TopBP1除了在DNA复制起始和DNA复制应激反应中发挥重要作用外，还通过其C末端区域在DNA损伤检查点信号中发挥重要作用。然而，其潜在的机制尚不清楚。在非洲爪蛙卵提取液中的初步数据表明，WDR18是一种与TopBP1相互作用的蛋白，它与TopBP1的C末端相互作用，并且它们的相互作用是Chk1在响应双链断裂时ATR激活所必需的。这个R15项目的目标是测试TopBP1合作伙伴WDR18在DNA损伤检查点和DNA复制中发挥关键作用的假设。这个R15项目将在非洲爪哇卵子提取物中进行，这是一个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。其具体目的是：(1)通过各种生化和分子生物学方法，阐明TopBP1的合作伙伴WDR18在非洲爪哇卵提取液中DNA损伤检查点信号中的作用；以及(2)通过监测当内源性WDR18被免疫耗竭去除时，大量DNA合成产物的存在或不存在，来确定在正常或应激条件下，DNA复制是否需要WDR18。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损伤检查点如何工作以保护基因组的完整性，以及当DNA损伤得不到修复或未引发适当反应时癌症是如何发展的。 公共卫生相关性： 项目叙述准确复制遗传信息并通过监测机制感知任何可能的DNA损伤DNA损伤检查点对于维持基因组完整性和抑制肿瘤至关重要。我们将使用一个简单的模型系统，从青蛙卵中提取非洲爪哇卵提取物，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提供更好的了解癌症是如何发展的，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.