|Institution:||University of York|
|Full text PDF:||http://etheses.whiterose.ac.uk/13168/|
DNA replication initiation is strictly controlled to maintain chromosome copy number. Over- or under-replication of an organism's genome is detrimental to survival, thus initiation events are tightly regulated to ensure only one round of DNA replication occurs per cell cycle. In prokaryotes, DNA replication commences when a protein initiator, DnaA, forms a helical filament at the origin of replication, oriC, inducing localised DNA unwinding and the recruitment of the replication machinery. The work described here sought to elucidate how DNA replication initiation is regulated in the Gram-positive model organism Bacillus subtilis. In particular, this work aimed to offer insight into the mechanisms of two negative regulators of DNA replication initiation, SirA and YabA; proteins that play significant roles in regulating replication initiation in sporulating and vegetatively growing cells, respectively. Both SirA and YabA interact directly with the initiator DnaA, and YabA has additionally been shown to interact with the DNA polymerase β-clamp, DnaN (an essential component of the replication machinery). Detailed here are structural and biophysical studies of SirA and YabA. This includes the X-ray crystal structure of SirA bound to the N-terminal domain of DnaA in an inhibitory complex, and characterisation of the SirA-DnaADI interface using an in vitro assay. When coupled with in vivo localisation studies carried out by our collaborators, the work on SirA suggests a mechanism for its inhibition of DNA replication. Also detailed are biophysical studies used to characterise the architecture of YabA, guided by the use of in silico models, and the X-ray crystal structure of YabA's N-terminal domain. These results are discussed alongside structural and mutational studies of YabA carried out by our collaborators; collectively the results delineate the full-length structure of YabA and offer insight into its interactions with DnaA and DnaN.