The Structure and Dynamics of the Bacterial Replisome

Thesis (Ph.D.)--University of Washington, 2017-08Cellular reproduction relies on DNA replication. Many bacteria, including Bacillus subtilis and Escherichia coli, possess only a single circular chromosome which is replicated bi-directionally from a single origin. It has remained controversial whether replication is achieved by mobile protein complexes that translocate along the DNA like a track, or by a stationary “replication factory” through which the DNA is pulled. In the first part of this thesis, we used time-lapse imaging to observe and quantify the position of fluorescently-labeled processivity-clamp (DnaN) complexes throughout the cell cycle. We find that the two replication forks maintain diffraction limited separation during the majority of the cell cycle, suggesting a factory-like model for replication. These data unify the models for replication across several domains of life. The canonical model of DNA replication describes a highly-processive and largely contin- uous process by which the genome is duplicated. However, many simultaneously-occurring processes compete with replication for the DNA substrate, potentially causing disruptions that could decrease the timeliness of the replication process and leave DNA vulnerable to mutations. In the second part of this thesis, we characterize the dynamics of the replisome in individual cells using fluorescence microscopy with single-molecule sensitivity. We find that the replisome is disassembled multiple times per cell cycle. Furthermore, this instability is caused by transcription