Arbore, C; (2013) Mechanisms and Functions of Molecular Interactions during Plasmid Rolling Circle Replication. Doctoral thesis , UCL (University College London).

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Abstract

The system under investigation in this project is the replication of plasmid DNA belonging to the pT181 family from the Gram-positive Staphylococcus aureus. This plasmid replicates through an asymmetric rolling circle mechanism, initiated by a plasmid-encoded protein that nicks the supercoiled plasmid allowing unidirectional unwinding by the helicase and elongation by a polymerase. The proteins involved in this process are the replication initiator protein, Staphylococcus aureus RepD, the ATP-driven 3’ to 5’ helicase, Bacillus Stearothermophilus PcrA, and the S. aureus DNA polymerase III. The project is mainly focused on three different aspects of plasmid replication including the formation of the initiation complex, the involvement of DNA polymerase III during plasmid elongation and the analysis of plasmid replication dynamics using AFM imaging. The kinetic mechanism of RepD initiation is examined here. Plasmid nicking occurs at a rate > 25 s-1 (30 °C). Without RepD, PcrA is a poor helicase as it is unable to unwind as short as 20 bp DNA junctions. The function of nicking is also investigated as requirement of PcrA processivity and Rep-PcrA translocation complex. The inclusion of DNA polymerase in these in vitro experiments generates a full in vitro plasmid replication system. The kinetics of PcrA-mediated unwinding has been studied previously, but the involvement of polymerase is little understood. PcrA is able to unwind plasmid DNA at a rate of ~30 bp s-1 (30 °C), however the inclusion of polymerase increased the unwinding rate to ~71 bp s-1 (30 °C). Using a fluorescence-based kinetic approach combined with rapid-mix techniques and AFM imaging, a variety of processes are investigated during RepD, PcrA and PolC mediated DNA replication. These in vitro data would provide an understanding of kinetics and dynamics of several complex processes during plasmid replication.

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