All living organisms need to accurately replicate their genome to survive. Genomic replication occurs in three phases; initiation, elongation, and completion. While initiation and elongation have been extensively characterized, less is known about how replication completes. In Escherichia coli completion occurs at sites where two replication forks converge and is proposed to involve the transiently bypass of the forks, before the overlapping sequences are resected and joined. The reaction requires RecBCD, and involves several other gene products including RecG, ExoI, and SbcDC but can occur independent of recombination or RecA. While several proteins are known to be involved, how they promote this reaction and the intermediates that arise remain uncharacterized.
In the first part of this work, I describe the construction of plasmid "mini-chromosomes" containing a bidirectional origin of replication that can be used to examine the intermediates and factors required for the completion reaction. I verify that these substrates can be used to study the completion reaction by demonstrating that these plasmids require completion enzymes to propagate in cells. The completion enzymes are required for plasmids containing two-replisomes, but not one replisome, indicating that the substrate these enzymes act upon in vivo is specifically created when two replication forks converge.
Completion events in E. coli are localized to one of the six termination (ter) sequences within the 400-kb terminus region due to the autoregulated action of Tus, which binds to ter and inhibits replication fork progression in an orientation-dependent manner. In the second part of this work, I examine how the presence of ter sequences affect completion on the 2-replisome plasmid. I show that addition of ter sequences modestly decreases the stability of the two-replisome plasmid and that this correlates with higher levels of abnormal, amplified molecules. The results support the idea that ter sites are not essential to completion of DNA replication; similar to what is seen on the chromosome.
Rec-B-C-D forms a helicase-nuclease complex that, in addition to completion, is also required for double-strand break repair in E. coli. RecBCD activity is altered upon encountering specific DNA sequences, termed chi, in a manner that promotes crossovers during recombinational processes. In the third part of this work, I demonstrate that the presence of chi in a bidirectional plasmid model promotes the appearance of over-replicated linear molecules and that these products correlate with a reduced stability of the plasmid. The effect appears specific to plasmids containing two replisomes, as chi on the leading or lagging strand of plasmids containing one replisome had no-effect. The observation implies chi promotes a reaction that may encourage further synthesis during the completion reaction, and that at least on the mini-chromosomes substrates, this appears to be a destabilizing force.
| Identifer | oai:union.ndltd.org:pdx.edu/oai:pdxscholar.library.pdx.edu:open_access_etds-6136 |
| Date | 19 July 2019 |
| Creators | Hamilton, Nicklas Alexander |
| Publisher | PDXScholar |
| Source Sets | Portland State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Dissertations and Theses |
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