The role of the checkpoint clamp in DNA repair

暴露在活性氧簇(ROS)和辐射下会导致DNA损伤，危及基因组 正直。8-氧鸟嘌呤是最常见和高度诱变的氧化损伤之一，因为它错配 在DNA复制过程中与腺嘌呤结合。氧化的碱基损伤主要通过碱基切除修复来消除 (BER)途径。BER与DNA损伤反应(DDR)紧密协调，以维持基因组 稳定性和细胞存活率。虽然BER和DDR已经分别进行了很好的研究，但两者的协调 流程不被理解。异三聚体9-1-1(Rad9-Rad1-Hus1)检查点钳在 启动DDR和DNA修复过程。我们已经确定了BER和DDR之间的独特交互作用 蛋白质，展示了9-1-1对误码率的新的和关键的贡献。我们假设9-1-1提供了 协调误码率流程的平台，以避免有毒中间体的积累。这个项目的目标是 是确定9-1-1和两种酶之间的生化和功能关系，这些酶介导最初的 误码率的阶梯。MYH/MUTYH DNA糖基化酶将错配的腺嘌呤与8-氧代-8-氧代-2 鸟嘌呤防止基因突变的第一步是误码率。APE1核酸内切酶随后在DNA上缺口 BER第二步中的基本站点。对这种协调作用的研究很重要，因为这些蛋白质 在端粒维持和人类疾病预防等过程中发挥关键作用。事实证明， 人类MYH基因突变与结直肠癌相关，而APE1和9-1-1是结直肠癌的关键因素 细胞的存活和发育。提出了以下三个具体目标。(1)我们将定义一个功能 由MYH、APE1和9-1-1组成的DNA修复复合体。我们将检查是否形成了这种修复 复合体对它们的生物学功能至关重要，通过干扰蛋白质来维持基因组的稳定- 蛋白质的相互作用。(2)我们将测试9-1-1的每个亚基在误码率和DDR中扮演不同角色的模型。 我们认为Rad9稳定了DNA上的误码率机制，Hus1促进了 从MYH到APE1的有毒中间体。(3)我们将使用新的可诱导的ROS系统来限制DNA 单个基因组位置和端粒的损伤。我们将使用这些系统来比较误码率和 两个位置的误码率因素关联顺序。我们将检验误码率因素的相互依赖关系 使用基因敲除和基因敲除方法与DNA损伤部位相关联。这些研究将揭示 BER复合体是如何在病变部位精细组装的，以及为什么BER对 保持端粒的完整性。从这些研究中获得的见解将极大地推动我们的 了解BER和DDR在癌症发生、癌症治疗和衰老中的作用。因为DNA 修复和9-1-1介导的信号与癌症的发展和治疗有关，从而阻断这些 协调的过程可以为抗癌药物的开发提供一种创新战略。

Exposure to reactive oxygen species (ROS) and radiation leads to DNA damage that compromise genomic integrity. 8-oxo-guanine is one of the most frequent and highly mutagenic oxidative lesions because it mispairs with adenine during DNA replication. Oxidized base lesions are primarily eliminated by the base excision repair (BER) pathway. BER is tightly coordinated with DNA damage response (DDR) in order to maintain genomic stability and cell survival. Although BER and DDR have been well studied separately, the coordination of both processes is not understood. The heterotrimeric 9-1-1 (Rad9-Rad1-Hus1) checkpoint clamp plays dual roles in activation of DDR and DNA repair processes. We have identified unique interactions among BER and DDR proteins, demonstrating a novel and critical contribution of 9-1-1 to BER. We hypothesize that 9-1-1 provides a platform to coordinate BER processes to avoid the accumulation of toxic intermediates. The goal of this project is to define the biochemical and functional relationships between 9-1-1 and two enzymes that mediate initial steps of BER. The MYH/MUTYH DNA glycosylase excises misincorporated adenines paired with 8-oxo- guanine to prevent gene mutation in the first step of BER. APE1 endonuclease subsequently nicks DNA at abasic sites in the second step of BER. Studies of this coordination are important because these proteins are key players in processes as telomere maintenance and human disease prevention. It has been shown that mutations in human MYH gene are associated with colorectal cancer while APE1 and 9-1-1 are essential for cell viability and development. The following three specific aims are proposed. (1) We will define a functional DNA repair complex consisting of MYH, APE1, and 9-1-1. We will examine whether the formation of this repair complex is critical for their biological functions in maintaining genomic stability by interrupting the protein- protein interactions. (2) We will test a model that each subunit of 9-1-1 plays a distinct role in BER and DDR. We propose that Rad9 stabilizes BER machinery on DNA and that Hus1 promotes the smooth transfer of the toxic intermediate from MYH to APE1. (3) We will employ novel inducible ROS systems to confine DNA damage to a single genomic location and telomeres. We will use these systems to compare the BER rates and the order of BER factor association at both locations. We will examine the mutual dependence of BER factor association with sites of DNA damage using knockdown and knockout approaches. These studies will reveal exactly how the BER complex is assembled at lesion sites on fine scale and why BER is important for maintaining telomere integrity. The insights gained from these studies will significantly advance our understanding of the roles of BER and DDR in carcinogenesis, cancer treatment, and aging. Because DNA repair and 9-1-1 mediated signaling are associated with cancer development and treatment, interrupting these coordinated processes could provide an innovative strategy for the development of anticancer drugs.