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.