Understanding the regulatory mechanisms for Replication Termination Factor 2 (RTF2) removal and function during DNA replication

项目摘要/摘要 DNA复制是一个高度受调控的过程，发生在染色质的物理领域。DNA 组织这一过程的复制机制，即复制体，面临着许多障碍，既有内源性的，也有 外源，这威胁到基因组的准确和有效复制。细胞有多种机制 以应对这些挑战并绕过病变。尽管如此，DNA复制压力已经成为一个强有力的来源 导致基因组不稳定和癌症的内源性损伤。我们的实验室发现了一种新的复制 蛋白酶体穿梭蛋白DNA损伤诱导1(DDI1)和DNA的应激反应轴 损伤诱导2(DDI2)，功能是将复制终止因子2(RTF2)从停滞的叉子移至 调解一个适当的恢复。相反，DDI1/2的耗尽导致RTF2保留在复制体中，这 导致复制分叉重新启动缺陷，从而导致严重的基因组不稳定和细胞死亡。观察到的细胞 DDI1/2耗尽时的死亡可以通过降低复制分叉处的RTF2水平来挽救。总的来说，我们的 因此，实验室已经证明，在复制应激反应过程中，复制体通过 一个重要的复制体成分的受调控的移除。 在这项研究中，我们将阐明RTF2从复制分叉中移除的机制和 确定维持基因组稳定性所需的RTF2功能。假设RTF2是 被蛋白体穿梭蛋白移除，我们假设RTF2的移除需要泛素 蛋白酶体系统。泛素与RTF2的结合和泛素标记的RTF2的降解必须 监管严密，效率高。我们已经表明，DDI1/2规范了实际的删除，但 需要了解DDI1/2识别RTF2的机制。我们建议DDI1/2与一个 RtF2上泛素偶联靶向降解及鉴定(1)RtF2上的特异性残基(S) 它必须被泛素化才能被DDI1/2识别，以及(2)泛素化RTF2的靶向E3连接酶。 此外，我们努力了解基因组中需要迅速移除的位置 RTF2，并定义RTF2起作用的染色质生态位。染色质是一种有序的、动态的调节 由DNA和组蛋白组成的平台和功能不同的染色质环境被创建， 部分通过可逆组蛋白的共价翻译后修饰(PTM)。我们的目标是确定 需要RTF2功能的基因组位置和染色质环境，并确定 需要删除RTF2。通过这个项目，我们正在研究协调监管的基本事件 泛素蛋白小体系统(UPS)、染色质和复制体之间活跃和停滞的复制 分叉以促进基因组的稳定。

Project Summary/ Abstract DNA replication is a highly regulated process that occurs in the physical arena of chromatin. The DNA replication machinery organizing this process, the replisome, confronts many obstacles, both endogenous and exogenous, that threaten the accurate and efficient duplication of the genome. Cells have multiple mechanisms to respond to these challenges and bypass lesions. Still, DNA replication stress has emerged as a potent source of endogenous damage driving genome instability and cancer. Our laboratory has identified a novel replication stress response axis in which the proteasome shuttle proteins DNA Damage Inducible 1 (DDI1) and DNA Damage Inducible 2 (DDI2), function to remove Replication Termination Factor 2 (RTF2) from stalled forks to mediate a proper recovery. Conversely, depletion of DDI1/2 results in retention of RTF2 in the replisome which causes a replication fork restart defect that leads to severe genomic instability and cell death. The observed cell death upon DDI1/2 depletion can be rescued by reducing levels of RTF2 at the replication fork. Overall, our laboratory has therefore shown that the replisome is reorganized during the replication stress response by the regulated removal of an essential replisome component. In this study, we will elucidate the mechanism by which RTF2 is removed from the replication fork and determine where RTF2 functions are required in the genome to maintain genome stability. Given that RTF2 is removed by a proteosome shuttle protein, we hypothesize that the removal of RTF2 requires the ubiquitin proteasome system. Both ubiquitin conjugation to RTF2 and degradation of the ubiquitin tagged RTF2 must be tightly regulated and efficient. We have already shown that DDI1/2 regulates the actual removal, but the mechanism of recognition of RTF2 by DDI1/2 needs to be understood. We propose that DDI1/2 interacts with a ubiquitin-conjugation on RTF2 to target its degradation and aim to identify (1) the specific residue(s) on RTF2 that must be ubiquitinated for recognition by DDI1/2 and (2) the targeting E3 ligase that ubiquitinates RTF2. Furthermore, we strive to understand the locations throughout the genome that require the prompt removal of RTF2 and define the chromatin niche wherein RTF2 functions. Chromatin is an ordered, yet dynamic regulatory platform composed of DNA and histone proteins and functionally distinct chromatin environments are created, in part, through reversible covalent post-translational modifications (PTMs) of histones. Our goal is to determine the genomic locations and chromatin environment that require RTF2 function and identify genomic loci that require RTF2 removal. With this project, we are studying the fundamental events that coordinate regulation between the ubiquitin proteosome system (UPS), chromatin, and the replisome at active and stalled replication forks to promote genome stability.