Resolving stalled replication forks in Escherichia coli

DNA replication is essential to successful cell proliferation. Inheritance of traits during cell propagation relies on the accurate duplication of the parental double-stranded DNA (dsDNA) to form two identical daughter copies. This process is carried out by a multi-protein complex referred to as the replisome. Decades of investigations using the model Escherichia coli (E. coli) replisome have provided an overall picture of the process of DNA replication initiation, elongation and termination. However, DNA replication in cells occurs on template DNA coated in DNA-binding proteins that can act as roadblocks and stall the replisome, often resulting in drastic effects on the chromosome. However, the fate of the replisome at these sites remains poorly understood. Stalled DNA replication has been linked to the emergence of antimicrobial resistance in prokaryotes, and the development of severe physical disorders and diseases in eukaryotes. Therefore, understanding the underlying mechanisms of stalled DNA replication can inform future investigations into the maintenance of genome integrity. This thesis focuses on the development and use of single-molecule tools to investigate stalled replication and the resolution of protein roadblocks. Single-molecule tools provide the ability to watch one molecule at a time. Extensive use of these techniques has revealed the heterogeneity that exists within complex biological pathways. Specifically, this thesis highlights the myriad of previously unknown behaviors of proteins on DNA as revealed by single-molecule tools.