Structural basis of replisome mediated DNA replication and repair

DNA是生命的蓝图，DNA的复制和维护对人类健康至关重要。 内源性或环境攻击造成的DNA损伤会阻碍DNA复制和 诱发基因组异常和人类疾病。复制体是一个多蛋白质复合体，负责 用于DNA复制。此外，作为第一个遇到任何复制障碍的人，复制体发挥着作用 在信号传递复制压力和促进DNA修复方面的重要作用。然而，结构性基础 复制体操作和复制体介导的复制、应激反应和修复的关系尚不清楚。 我们建议使用生化重组和低温EM结构测定来研究如何 复制体在正常和应激条件下的作用以及复制体的功能障碍如何导致 基因变异和人类疾病。首先，我们将利用T7噬菌体的复制体作为 研究应激条件下复制体操作的结构基础的模型系统。我 最近获得了在其DNA底物上工作的T7复制体的第一个结构，这 说明了复制体的组织和工作机制。我们将进一步调查如何 T7复制体对各种复制障碍作出反应以触发适当的复制偶联 修理。其次，我们将研究线粒体DNA复制的结构基础。线粒体 是细胞的动力所在，线粒体DNA因错误的DNA复制而产生的错误 与许多人类遗传病有关。我们将重建线粒体复制体和 研究线粒体DNA是如何复制的，以及线粒体复制体上的突变是如何导致 人类疾病。我们的调查将提供第一个结构框架 复制体介导DNA复制、应激反应和修复。结构信息将 有助于开发新的治疗方法，以靶向或避免靶向DNA复制。

DNA is the blueprint of life and DNA replication and maintenance are essential for human health. Damages on DNA due to endogenous or environmental assaults can hamper DNA replication and induce genome abnormities and human diseases. A replisome is a multi-protein complex responsible for DNA replication. Moreover, as the first one to meet any replication barrier, the replisome plays essential roles in signaling replication stress and facilitating DNA repair. However, the structural basis of replisome operation and replisome mediated replication stress response and repair remains unclear. We propose to use biochemical reconstitution and cryo-EM structural determination to investigate how the replisome acts under normal and stressed conditions and how disfunction of the replisome leads to genetic variations and human diseases. First, we will employ replisome from bacteriophage T7 as a model system to investigate the structural basis of replisome operation under stressed conditions. I have recently obtained the first structure of the T7 replisome operating on its DNA substrate, which illustrated the organization and the working mechanism of a replisome. We will further investigate how the T7 replisome responses to various replication barriers to trigger appropriate replication-coupled repair. Second, we will investigate the structural basis of mitochondria DNA replication. Mitochondria is the power house of a cell and errors on mitochondria DNA due to faulty DNA replication are connected to many human genetic diseases. We will reconstitute the mitochondria replisome and investigate how mitochondria DNA is replicated and how mutations on mitochondria replisome lead to human diseases. Our investigations will provide the first structural framework for understanding replisome mediated DNA replication, stress response and repair. The structural information will contribute to the development of novel therapies to target or avoid targeting DNA replication.