The role of the checkpoint clamp in DNA repair

暴露于活性氧（ROS）和辐射导致DNA损伤，损害基因组 完整8-氧代鸟嘌呤是最常见的和高度致突变的氧化损伤之一，因为它错配 与腺嘌呤结合氧化基底病变主要通过基底切除修复消除 (BER)通路BER与DNA损伤反应（DDR）紧密协调，以维持基因组的稳定。 稳定性和细胞存活。虽然BER和DDR已经分别得到了很好的研究， 过程不被理解。异源三聚体9-1-1（Rad 9-Rad 1-Hus 1）检查点钳在细胞凋亡中起双重作用。 激活DDR和DNA修复过程。我们已经确定了BER和DDR之间独特的相互作用 蛋白质，证明了9-1-1对BER的新的和关键的贡献。我们假设9-1-1提供了一个 平台协调BER过程，以避免有毒中间体的积累。这个项目的目标 是为了确定9-1-1和两种酶之间的生物化学和功能关系，介导的初始 BER的步骤MYH/MUTYH DNA糖基化酶切除与8-氧代- 鸟嘌呤，以防止基因突变的第一步BER。APE 1核酸内切酶随后在 在BER的第二步中的无碱基位点。这种协调的研究是重要的，因为这些蛋白质是 端粒维持和人类疾病预防等过程中的关键参与者。已经显示 人类MYH基因突变与结直肠癌相关，而APE 1和9-1-1是结直肠癌的关键基因。 细胞活力和发育。提出了以下三个具体目标。(1)我们将定义一个函数 DNA修复复合物由MYH、APE 1和9-1-1组成。我们将研究这种修复的形成是否 复合物对于它们的生物学功能是至关重要的，它们通过中断蛋白质来维持基因组稳定性， 蛋白质相互作用(2)我们将测试一个模型，9-1-1的每个子单元在BER和DDR中扮演不同的角色。 我们认为Rad 9稳定了DNA上的BER机制，而Hus 1促进了DNA的平稳转移。 从MYH到APE 1的毒性中间体。(3)我们将采用新的诱导活性氧系统， 对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BER率， 在两个位置的BER因子关联的顺序。我们将检查BER因子的相互依赖性 使用敲低和敲除方法与DNA损伤位点相关。这些研究将揭示 确切地说，BER复合物是如何在病变部位以精细的尺度组装的，以及BER对 维持端粒的完整性。从这些研究中获得的见解将大大促进我们的 了解BER和DDR在致癌作用、癌症治疗和衰老中的作用。因为DNA 修复和9-1-1介导的信号传导与癌症的发展和治疗有关， 协调一致的进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.