- Title
- When forks collide: analysis of recombination-dependent stable DNA replication (RSDR)
- Creator
- Corocher, Taylor
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2020
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- It is well established that bacteria rely on the accurate and complete replication and segregation of chromosomal DNA to create viable progeny cells. In most bacteria, chromosome replication proceeds bidirectionally from the single origin towards the terminus. In E. coli, the terminus region contains specific DNA sequences, known as ter sites, which when bound by the Tus protein create polarized nucleoprotein barriers to replication. The purpose of these traps is to contain the replication forks within the terminus, ensuring termination occurs within this region. If one replication fork is delayed, the other fork could potentially progress through the terminus and directly conflict the replication fork moving in the opposite direction. As most genes are oriented so that transcription is co-directional with replication, forks that escape the terminus and progress into the opposite arm of the chromosome will frequently suffer head-to-head conflicts with transcription complexes. Conflicts between two replication forks, as well as head-to-head conflicts between replication forks and transcription complexes can have severe consequences and can reduce the viability of bacterial cells. When two replication forks do converge, the helicase of one fork can displace the leading strand polymerase of the oncoming fork. This convergence can also displace the 3′ end of the nascent leading strand creating a 3′ flap of single-stranded DNA (ssDNA). It is thought that this flap is usually processed by the RecG protein. In the absence of RecG, the presence of the unannealed 3′ ssDNA flap can lead to the formation of a D-loop structure in a RecA-dependent reaction. This D-loop DNA structure is acted upon by the PriA replication restart pathway to reload the replication machinery, thus allowing reinitiation of replication in the terminus region, which travels bidirectionally back towards the origin. This process is referred to as recombination-dependent stable DNA replication (RSDR) throughout this study. Replication initiated via RSDR is usually contained in the terminus by Tus-ter traps. Removal of the Tus/ter traps allows replication forks that initiate within the terminus to proceed back towards the origin. This RSDR allows cells to survive even when the normal replication origin, oriC, is inactivated. The overall viability of these cells, as a population, is greatly increased with a mutation in RNA Polymerase that reduces the stability of transcription complexes. Cells lacking recG and tus, and carrying mutant rpoB and dnaA alleles are capable of robust RSDR-dependent growth. The first aim of this research was to investigate how replication solely from the terminus affected other processes in the cell. This was accomplished through the analysis of growth curves, cell viability and fluorescent microscopy. A variety of defects were evident in cells undertaking RSDR-dependent growth. Chromosome segregation and cell division defects were particularly evident, with DNA tending to accumulate at mid-cell. Viability also showed a dependence on chromosome dimer resolution, but the introduction of a mutation that decreased overall transcription caused the viability defect to recovery almost entirely. This recovery of cell viability implies that the majority of recombination events causing chromosome dimer formation stemmed from transcription-replication machinery collisions. Marker Frequency Analysis (MFA) has shown that when RSDR is initiated, there is a distinct increase in the copy number of the terminus between terA and terB/C, with the peak situated nearby the dif site. This study aimed to analyse the specific DNA replication intermediates that are visible when RSDR occurred (in the absence of recG) through the use of 2-Dimensional Gel Electrophoresis at terC and the dif site. The effect of a number of mutations (recQ, ruvABC, priA300) that have been indicated to function in RSDR was also investigated. A variety of novel DNA structures were observed at the dif and terC loci in a ΔrecG strain. There was an accumulation of replication forks at terC, as expected when RSDR caused replication to reinitiate nearby dif. The presence of Holliday Junctions (HJs) at terC suggested a reduction in the ability to process replication forks from Tus/ter in the absence of RecG. Further, there was an accumulation of replication forks, HJs and fork convergences at the dif site, supporting the model that in the absence of RecG, fork convergences lead to the formation of HJs and ultimately the reloading of replication forks at that point. In the absence of the RecQ helicase, there was an accumulation of replication forks at terC, but no other discernable DNA intermediates could be visualised at terC or the dif site. Interestingly, there were no visible DNA intermediates at the dif site within the ΔrecQ ΔrecG mutant, and this indicated that even in the absence of RecG, RSDR did not occur if RecQ was also absent. As RSDR does not occur in the absence of recQ alone, the accumulation of replication forks at terC was specifically indicative of the inability to process these forks. Visualisation of DNA replication intermediates in the absence of the RuvABC resolvase/translocase highlighted the need for HJ cleavage by RuvC when replication forks were reversed from nucleoprotein blockages, as demonstrated by the accumulation of HJs at terC. Furthermore, a novel structure was visualised in the absence of the ruvABC gene. This structure was hypothesised to result from the encounter of a replication fork with an unprocessed HJ in the terminus. The appearance of this signal at terC and the dif site indicates that this type of collision can occur anywhere within the region. Lastly, the involvement of the priA helicase activity on the DNA structures in the terminus was investigated. While there was only a small increase of replication forks at terC in the priA300 mutant background, there was the appearance of HJs at the dif site, indicating that without the helicase activity of PriA, the replication forks are unable to be loaded here. The final aim of this project was to produce lists of genes that are essential in cells undertaking RSDR-dependent growth to elucidate what other proteins could be involved in containing/processing these structures or maintaining cell viability due to an increase in DNA copy number at the terminus region. This involved the use of a novel ISY100 transposon delivered via conjugation, that is both economical, affordable and applicable to a variety of strains where traditional electroporation has low efficiency. This research demonstrated that the ISY100 conjugative TraDIS system holds great potential for future use to create TraDIS libraries, as the insertions were relatively uniformly distributed, with a high percentage of reads mapping to the reference chromosome. This project has highlighted the complexity of the process of RSDR and demonstrated the complications that arise in the cell cycle as a result of such an increase in DNA copy number, related to chromosome organisation, segregation and ultimately cell division. Further, this research investigated and identified the specific replication/recombination DNA structures in the terminus and noted how a variety of proteins influence the structures that are seen. The conclusions made were supportive of the current model of RSDR in the literature, including the visualisation of a novel 2-D gel electrophoresis structure predicted by previously published work.
- Subject
- recG recombination; DNA replication; E. coli; Tus Ter; RTP; over-replication
- Identifier
- http://hdl.handle.net/1959.13/1419596
- Identifier
- uon:37473
- Rights
- Copyright 2020 Taylor Corocher
- Language
- eng
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