Investigations into the Architecture and Function of the Bacterial Replisome

The accurate and efficient replication of Escherichia coli DNA is catalysed by a 17‐subunit assembly of proteins termed the DNA polymerase III holoenzyme (Pol III HE). The Pol III HE is further organised into three sub‐assemblies: the clamp‐loader complex, the β2 sliding clamp and the Pol III core complex (Pol III core). The Pol III core is comprised of the α polymerase, the ε exonuclease and the small θ subunit of an as yet undefined function. The β2 subunit encircles dsDNA and locks the Pol III core onto a template DNA by interaction with clamp‐binding motifs (CBMs) located in the α and ε subunits. A detailed understanding of how the β2αεθ complex interacts with DNA and transforms to accommodate both DNA synthesis and proofreading functions is of great interest to researchers in the field. In this project, I used a combination of experimental techniques including protein crystallography, small‐angle X‐ray scattering (SAXS), nuclear magnetic resonance (NMR) and a variety of additional biochemical methods to build and validate an atomic resolution model of the structure of the β2αεθ complex. I further investigated a strategy for the fluorescent labelling of Pol III core proteins for use in future single‐molecule experiments.