DNA Replication Checkpoint in Fission Yeast

项目摘要/摘要。 DNA复制检查点是在所有真核生物中运行的一条细胞信号通路，它监控正常的S期 进展和对干扰的DNA复制的反应，激活细胞反应以防止不可逆转 复制分叉停滞、基因组不稳定和细胞死亡。检查点感测到受干扰的复制， 维持基因组在应激状态下的稳定性，从而起到重要的抗癌屏障作用。许多 抗癌药物通过干扰DNA复制发挥作用，因此其疗效受到 癌症的检查站状态。尽管它在疾病预防和癌症化疗中很重要，但我们仍然 不完全了解复制分叉的检查点启动过程，也不确切知道 检查点在压力下保护叉子的功能。作为研究细胞的既定模型 在人类中保守的机制，分裂酵母为这项研究提供了几个好处。的目标是 本课题旨在研究新筛选出的裂殖酵母检查点突变体。 两个目标：(1)了解在受扰的岔路口启动检查站的机制基础，以及 (2)揭示检查点调控的叉子保护的基本分子细节。作为起点，我们 已经开发了一种正向遗传学和生化分析的组合方法，并确定了几个 在复制胁迫下具有各种检查点启动缺陷的新突变体。我们还放映了一部大型 叉子保护有缺陷的突变体的集合。在我们强大的初步和公布的数据的指导下， 我们将在第一个目标下进行体内和体外研究，以研究检查点传感器如何 RAD3(ATR)信号受RAD3-RAD26复合体、RECQ解旋酶Rqh1、RQH1、RQH3、RQH1、RQH1、RQH3、RQH1、 SMC5/6复合体和RPA复合体。在第二个目标下，我们将调查如何激活 Checkpoint调节前导链上的DNA聚合酶e和其他尚未识别的叉子目标 保护。这项研究计划的长期目标是提供对 复制检查点，涉及三个主要调查领域：第一，通过使用我们新改进的基因 方法，鉴定具有保守检查点功能的复制蛋白。第二，重组 使用能够正确概括WE和其他人体内数据的纯化蛋白的体外检查点途径 已经获得了。第三，正如我们在Rqh1的研究中所表明的，人类检查点机制的保守性 将对单元格进行评估。总体而言，这项研究计划将大大提高分子的清晰度 在分裂酵母和哺乳动物细胞中复制检查点的机制。拟议的研究 因为它与基因组不稳定、肿瘤发生和癌症化疗有关，所以意义重大。

PROJECT SUMMARY/ABSTRACT . DNA replication checkpoint is a cell signaling pathway operating in all eukaryotes that monitors normal S phase progression and in response to perturbed DNA replication, activates cellular responses to prevent irreversible replication fork arrest, genomic instability, and cell death. The checkpoint senses the perturbed replication, maintains the genomic stability under stress and thus functions as an important anticancer barrier. Many anticancer drugs work by interfering with DNA replication and their efficacy is therefore influenced by the checkpoint status of cancer. Despite its importance in disease prevention and cancer chemotherapies, we still do not fully understand the checkpoint initiation process at the replication forks, nor do we know exactly how the checkpoint protects the fork functions under stress. As an established model for studying the cellular mechanisms that are conserved in humans, fission yeast offers several benefits for this research. The goal of this project is to investigate the newly screened checkpoint mutants in fission yeast with particular emphasis on two objectives: (1) understand the mechanistic underpinnings of checkpoint initiation at the perturbed forks, and (2) uncover the essential molecular details of the checkpoint-regulated fork protection. As a starting point, we have developed a combined approach of forward genetics and biochemical analysis and have identified several new mutants with various checkpoint initiation defects under replication stress. We have also screened a large collection of mutants that are defective in fork protection. Guided by our strong preliminary and published data, we will conduct in vivo and in vitro studies under the first objective to investigate how the checkpoint sensor kinase Rad3(ATR) signaling is affected by mutations in the Rad3-Rad26 complex, the RecQ helicase Rqh1, the Smc5/6 complex, and the RPA complex. Under the second objective, we will investigate how the activated checkpoint regulates DNA polymerase e on the leading strand and other yet-to-be identified targets for fork protection. The long-term goal of this research program is to provide a comprehensive understanding of the replication checkpoint that involves three primary areas of inquiry: First, by using our newly improved genetic method, replication proteins with conserved checkpoint functions will be identified. Second, reconstitution of the checkpoint pathway in vitro using purified proteins that can properly recapitulate the in vivo data we and others have obtained. Third, as we show in the studies on Rqh1, conservation of the checkpoint mechanisms in human cells will be evaluated. Overall, this research program will bring much improved clarity to the molecular mechanisms of the replication checkpoint in fission yeast as well as in mammalian cells. The proposed research is significant because of its relevance to genome instability, oncogenesis, and cancer chemotherapies.