Year

2021

Degree Name

Doctor of Philosophy

Department

School of Chemistry and Molecular Bioscience

Abstract

Bacterial DNA replication is a potential novel antibiotic target in the fight against increasing anti-microbial resistance. To fully explore those opportunities, a full understanding of the molecular mechanisms underlying DNA replication is required. The components of the bacterial replisome, the multi-protein machinery responsible for replication, have been characterised extensively over the past 60 years, largely relying on the E. coli model system. However, the intricacies of the nucleo-protein network of the replisome and the complexity of its molecular steps have impeded our ability to produce a complete picture of the replication process. One E. coli replisomal component, the DnaB replicative helicase, has as a clear function to catalyse the unwinding of duplex DNA at the front of the replication fork. This component is also suspected to play other regulatory roles, given its extensive contacts with the primase and polymerase enzymes within the replisome. This thesis focusses on the role of the DnaB helicase in DNA replication with new insights provided by single molecule techniques.

As part of this thesis, novel single-molecule fluorescence methods were developed to visualise previously inaccessible molecular details of DnaB helicase activity. These methods revealed new properties of the DnaB helicase that change our picture of how the replisome works. Specifically, the work detailed in this thesis demonstrates that in contrast to other replisomal factors, the DnaB helicase is stably associated with active replisomes. These results suggest a key role for the DnaB stability in providing the replisome with high processivity in replication. Furthermore, evidence is presented in this thesis that unwinding during replication does not require nucleotide hydrolysis by the DnaB helicase. Nucleotide incorporation by the DNA polymerase as part of the DNA-synthesis process is proposed to provide the energy for DNA unwinding at the replication fork. The presence of DnaB is still required, but in the absence of ATP, the helicase likely acts as a passive wedge.

This thesis sheds new light on the roles of the DnaB helicase in E. coli DNA replication. The introduction of single-molecule approaches to study the replisome is rapidly changing our view of the dynamic molecular transactions that underpin DNA replication. Future work will need to focus on understanding the role of the many dynamic interactions within the replisome and assessing which of these molecular ”pressure points” could be targets for antibiotic development.

FoR codes (2008)

030406 Proteins and Peptides, 060107 Enzymes, 060199 Biochemistry and Cell Biology not elsewhere classified, 060408 Genomics

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Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.