- Title
- The rescue of replication forks stalled by nucleoproteins in E. coli
- Creator
- Weaver, Georgia M.
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2019
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- DNA replication is a vital process for all forms of life and is required for the accurate transfer of the chromosome to progeny cells. The replication machinery, the replisome, is a dynamic multi-protein complex whose subunits are frequently exchanged to maintain progressive DNA synthesis. Despite being a highly accurate mechanism for cell duplication, stalls in replication at the replication fork are extremely common. Proteins tightly bound to DNA, such as RNA polymerase, arethe majority of impediments to replication fork progression, inhibiting replisome movement and causing the replication forks to stall. If cells are to maintain viability, it is crucial that replication stalls are overcome so replication can reinitiate. The stalling of a replisome can lead to its dissociation from the chromosome, either in part or in its entirety, causing the demise of the replication fork structure. However, replisome disengagement also enables the access of repair protein to the stalled fork to restore it to a functional structure before the replisome can reassemble and restart. Homologous recombination pathways have evolved that remodel and repair the DNA to ensure its integrity is maintained and to enable replication progression. Thus, replication and recombination are closely linked with a myriad of proteins implicated in DNA repair pathways. Population-based investigations into cellular repair processes are fundamental to the understanding of general cellular responses to DNA replication impediments. This study focused on the repair mechanisms employed at persistent nucleoprotein blocks with an aim to elucidate the contributions of proteins previously implicated in fork processing including RecG, RuvABC, RecA and the RecFOR pathway proteins. Accordingly, this study utilised a Fluorescent Reporter Operator System (FROS) technique for the controlled induction of replication fork stalling and restart at a specific location on the E. coli chromosome. The status of replication could be visualised in single living cells using fluorescence microscopy and DNA replication intermediates analysed by 2-dimensional agarose gel electrophoresis. The system was used extensively to evaluate the effect of replisome components and repair proteins on replication fork stability, processing and recovery at nucleoprotein blocks. Temperature sensitive mutants of replisome components were incorporated into the system to determine the stability of a replisome at a blocked fork. At the non-permissive temperature, dnaBts and dnaCts alleles led to the collapse of replication forks within the time scale examined (15 minutes) at which the replisome could not be reloaded, leading to extensive DNA processing of the DNA at the fork. In contrast, inactivation of dnaNts, the β-clamp, led to an initial processing event moving the fork away, but then forked DNA recovered at the blockage which was unexpected. The study determined that replication fork regression, the combination of replication fork reversal leading to formation of a Holliday Junction along with exonuclease digestion, is the preferred pathway for dealing with a collapsed fork in Escherichia coli. Direct endonuclease activity at the replication fork by proteins with this function was not observed. Indeed, a mixture of forked DNA and HJ structures were present over the course of an hour and showed no sign of endonuclease digestion. The roles of the recombinational repair proteins that are involved in fork rescue were investigated using genetic knockouts within FROS, both individually and in mutant combination. The proteins that were seen to have the greatest impact on fork processing events were the RecQ helicase and the RecJ exonuclease, evidenced by incomplete replication fork processing in each mutant background. A model for this principal repair pathway at blocked forks is proposed where RecQ and RecJ work in synergy to enlarge the ssDNA region of a lagging nascent strand. Their actions facilitate RecFOR-mediated RecA filament formation and subsequent fork reversal. Relatively minor roles of RecG and RuvABC in initial fork regression were revealed while stronger evidence was provided for the key role of RecG in the processing of Holliday junctions back into replication forked structures. Stalled forks were subjected to exonuclease driven DNA degradation when RecA was not present. Lastly, several methods were employed to determine the influence of the SOS response when forks are stalled by nucleoproteins. The SOS response was found not to be induced by the protein-DNA roadblock during the period of analysis, and, therefore, did not affect fork processing. The fork remodelling pathways used to rescue forks stalled by persistent nucleoproteins have been elucidated. This study has contributed further understanding to the functions of recombinational repair proteins at stalled forks. Overall, a broad picture of the multiple pathways that fork rescue can be shunted down in response to a nucleoprotein blockage has been realised. The ability for a cell to substitute one repair process with another may allow fork reactivation to avoid mutagenesis or activation of SOS. These multiple repair options may also reflect either the stochastic nature of choice of repair pathway, or that subtle underlying differences in the exact DNA structure at the fork may lead to preferences for processing by different pathways.
- Subject
- microbiology; E. coli; DNA replication; replication forks; nucleoproteins
- Identifier
- http://hdl.handle.net/${Handle}
- Identifier
- uon:35350
- Rights
- Copyright 2019 Georgia M. Weaver
- Language
- eng
- Full Text
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