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

项目总结/摘要 DNA复制是发生在染色质物理竞技场中的高度调节的过程。的dna 组织这一过程的复制机器，即复制体，面临许多障碍，既有内源性的， 外源的，这威胁到基因组的准确和有效的复制。细胞有多种机制 来应对这些挑战和旁路病变。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方法 应激反应轴，其中蛋白酶体穿梭蛋白DNA损伤诱导1（DDI 1）和DNA 损伤诱导因子2（DDI2），其功能是从停滞的分叉中去除复制终止因子2（RTF 2）， 让他恢复正常相反，DDI 1/2的缺失导致RTF 2保留在复制体中， 导致复制叉重新启动缺陷，导致严重的基因组不稳定和细胞死亡。所述观察细胞 DDI1/2耗尽时的死亡可以通过降低复制叉处的RTF 2水平来挽救。总体而言，我们 因此，实验室已经表明，在复制应激反应期间，复制体被重组。 调节必需复制体组分的去除。 在这项研究中，我们将阐明RTF2从复制叉中移除的机制， 确定RTF2功能在基因组中需要维持基因组稳定性的位置。如果RTF 2是 通过蛋白体穿梭蛋白去除，我们假设RTF2的去除需要泛素 蛋白酶体系统泛素与RTF 2的缀合和泛素标记的RTF 2的降解两者必须是必需的。 监管严格且高效。我们已经表明，DDI 1/2调节实际的去除，但 需要了解DDI 1/2识别RTF 2的机制。我们认为DDI 1/2与一个 在RTF 2上进行泛素缀合以靶向其降解，并旨在鉴定（1）RTF 2上的特异性残基 必须被泛素化才能被DDI 1/2识别;（2）靶向E3连接酶，其泛素化RTF 2。 此外，我们努力了解整个基因组中需要迅速去除的位置。 RTF2和定义染色质生态位，其中RTF2的功能。染色质是一个有序的，但动态的调节 创建由DNA和组蛋白组成的平台和功能不同的染色质环境， 部分通过组蛋白的可逆共价翻译后修饰（PTM）。我们的目标是确定 需要RTF 2功能的基因组位置和染色质环境，并鉴定 需要移除RTF 2。通过这个项目，我们正在研究协调调节的基本事件 泛素蛋白体系统（UPS），染色质和复制体在活跃和停滞复制之间的关系 分叉以促进基因组稳定性。

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.