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Title: PcrA helicase and RepD initiator protein in rolling circle replication
Author: Gray, L. F.
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2016
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This thesis investigates rolling circle replication. In particular it investigates the activity of the initiation complex with proteins all from the same species and the biochemical steps in the termination. In rolling circle replication, the two strands are asynchronously copied, one simultaneously with unwinding of the parent plasmid. An initiator, RepD, binds specifically to the double-stranded origin, nicks one strand, forming a covalent bond with the 5'-end. This nicking opens up a short singlestranded region, allowing PcrA helicase and a polymerase to bind and begin unwinding and replication. After travelling all around the plasmid, probably as a single protein complex, RepD then does a series of strand exchanges to close the two plasmid circles. In previous work, PcrA was from B. stearothermophilus, while the polymerase and RepD were S. aureus. Successful preparation of S. aureus PcrA and a B. stearothermophilus-Staph PcrA chimera enabled characterisation of activity when all proteins in the initiation complex are from the same species. This is achieved using fluorescent biosensors in both steady-state and real time assays, along with agarose gel assays. Initiation of rolling circle replication and the movement of the complex along DNA are well understood, however little has been known about the termination. At termination RepD plays a vital role. This thesis proposes and tests a mechanism for termination, involving a series of nicking and ligation reactions, mediated by RepD. A combination of measurements with whole plasmids and oligonucleotide models were used to elucidate the series of events at each stage. In particular, by following individual processes in real time, the sequence and identity of biochemical steps could be defined. Quench-flow rapid mixing enabled real-time measurements. The identity of species was determined by combinations of gel assays, Mass Spectrometry and CD spectrometry, in particular.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available