Doctor of Philosophy
School of Chemistry
Hill, Flynn Robert, Macromolecular structure and dynamics in the bacterial replisome, Doctor of Philosophy thesis, School of Chemistry, University of Wollongong, 2013. https://ro.uow.edu.au/theses/4015
The rapid and high-fidelity replication of DNA in bacteria is carried out by a molecular machine called the replisome, which is made up of a dozen different proteins. These include the primosome complex of DnaB6 helicase and DnaG primase, complexed to the Pol III holoenzyme which is made up of two αεθ polymerase-exonuclease cores (one to replicate each parental strand), each in complex with a β2 clamp and held in a pair by the clamp loader complex δγτ2δ’ψχ. Experiments with several of these proteins are reported here. A particular focus of this work is the structure and dynamics of the Bacillus spp. DnaB6 helicase, DnaI helicase loader and their DnaB6-DnaI6 complex.
In this project, I used X-ray and neutron scattering techniques to characterise the structure and dynamics of DNA replication proteins from a variety of species. The recently developed technique of size-exclusion chromatography coupled small-angle Xray scattering (SEC-SAXS) was used extensively for structural measurements. The inline separation of samples into monodisperse species has allowed very high quality SAXS to be measured for samples that would otherwise have given compromised scattering data. The SAXS curves thus obtained have been used for both model building and model validation, as a complement to information from high-resolution structural techniques.