- Title
- Processing of DNA replication forks after encountering a protein roadblock
- Creator
- Graham, Adam
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2018
- Description
- Masters Research - Master of Philosophy (MPhil)
- Description
- It is imperative that DNA is replicated without error. Organisms face multiple challenges that can compromise the integrity of their DNA. To counter these threats, organisms have pathways capable of removing obstacles and maintaining efficient and stable DNA replication. Despite significant effort, these pathways remain poorly characterised. The first aim of this thesis was to clarify what pathways are utilised in E. coli upon encountering an inducible artificial protein roadblock in E. coli. We used chromosome dimers as a proxy for crossover frequency at Holliday junctions before the roadblock was induced and following its induction and removal. Chromosome dimers were measured by viability loss associated with a dif knockout strain, which renders the strain incapable of resolving chromosome dimers. It was found that chromosome dimer formation does not increase when the roadblock is present and then removed. This result is despite 2D gels that show Holliday junctions were formed by DNA replication fork collisions with the roadblock. No relationship was found between chromosome dimer formation and the presence of the roadblock in several recombination mutants. However, we identified gene knockouts that influence the frequency of chromosome dimer formation. These measurements of chromosome dimer formation before and after induction of a protein roadblock demonstrate that DNA replication forks that encounter a protein roadblock will form a Holliday junction, but will resolve that Holliday junction without exchange of DNA. Additionally, measurements in single gene knockouts show that crossover free resolution of Holliday junctions occurs either independently of several genes suspected to be involved in crossover events. Or that E. coli are able to utilise secondary pathways which also favour crossover-free Holliday junction resolution in the absence of the primary pathway or protein. The second aim of this thesis was to study the behaviour of the E. coli replisome via visualisation with fluorescently tagged replisome components, both before and after encountering the artificial roadblock. It was observed that E. coli replisomes will persist in cells if the artificial roadblock is present. Replisomes will proceed to copy the rest of the chromosome immediately after the roadblock is removed. After removal of the roadblock, replisomes from the other replichore will remain at Tus-ter complexes and replication will be completed when the replisome previously trapped at the artificial roadblock reaches the ter region. These results demonstrate that the E. coli replisome can tolerate a persistent artificial roadblock and complete DNA replication after the removal of the roadblock. The rapid restart of the DNA replication, with other evidence, suggests rapid processing of the DNA to form a functional replisome which likely occurs through a continuous cycle of disassembly and reformation.
- Subject
- DNA replication; genomic integrity; replisome; genetic recombination; XerCD
- Identifier
- http://hdl.handle.net/1959.13/1395228
- Identifier
- uon:33836
- Rights
- Copyright 2018 Adam Graham
- Language
- eng
- Full Text
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| Thumbnail | File | Description | Size | Format | |||
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| View Details Download | ATTACHMENT01 | Thesis | 4 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 856 KB | Adobe Acrobat PDF | View Details Download |