Year

2019

Degree Name

Doctor of Philosophy

Department

School of Chemistry and Molecular Bioscience

Abstract

Bacterial resistance to antibiotics has become a major health problem worldwide. The rapid development of drug-resistant strains to clinically used antibiotics and the slow rate of development of new treatment options threaten a healthcare catastrophe. Currently, 700,000 deaths per year are attributable to antimicrobial resistance. By 2050, antibiotic resistance is estimated to contribute to the deaths of 10 million people per year. In recent years, bacteria have emerged resistance to all classes of antibiotics. More than ever, there is a necessity for new antibiotics, exploring new mechanisms of action.

In the search for new drug targets, it is fundamentally important to understand how bacteria battle and develop resistance to antibiotics used in the clinic. It is possible that by understanding the mechanisms of the DNA damage response that promote resistance to antibiotics, new drug targets may be identified. The scope of this thesis is to characterise DNA repair and damage tolerance pathways in Escherichia coli, focusing on the interconnectivity between error-prone DNA polymerases and homologous recombination. Using single-molecule fluorescence live-cell imaging, it has been possible to monitor proteins involved in these pathways directly and in real time.

Monitoring proteins at the single-molecule level in live cells has allowed me to make significant new discoveries. My PhD work challenges long-standing models in the fields of translesion synthesis (TLS) and homologous recombination in Escherichia coli. The error-prone DNA polymerase IV (pol IV) has long been assumed to mainly carry out TLS at stalled replication forks. My live-cell work has revealed that pol IV primarily acts at recombination intermediates and rarely, if ever, binds at replisomes. The resection of DNA double-strand breaks is crucial to pol IV activity in cells, suggesting that pol IV could be a recombination protein. Strikingly, this requirement is shared for cells treated with antibiotics that have different primary targets in cells. The common element appears to be surges in cellular ROS levels, which induce DNA breakage and thus create substrates for pol IV.

My real-time live-cell imaging approach also allowed me to functionally dissect the RecFOR pathway for homologous recombination. Conventionally, the recombination mediators RecF, RecO and RecR have been described to collectively load the RecA recombinase on single-stranded DNA. Contrary to this model, single-molecule imaging revealed that RecF and RecO rarely form a complex in vivo and indicated that RecF and RecO have distinct functions. RecF binds mainly at replisomes while RecO binds to DNA in the region between the nucleoid and membrane, the same region of the cell in which large RecA bundles form in cells carrying DNA damage. Following RecF, I further showed that RecF impacts on pol IV binding at the replisome; a new link connecting the fields of TLS and homologous recombination. This Thesis provides unprecedented single-molecule level insight into the mechanisms of TLS and homologous recombination in Escherichia coli cells suffering DNA damage and, importantly, reveals new and unexpected links between the two processes.

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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.