The homodimeric Ocr protein of bacteriophage T7 is a molecular mimic of a bent double-stranded DNA molecule ~24 bp in length. As such, Ocr is a highly effective competitive inhibitor of the bacterial Type I restriction modification (R/M) system. Thus, Ocr facilitates phage infection of the bacterial cell to proceed unhindered by the action of the R/M defense system. The main aim of this work was to understand the basis of the DNA mimicry displayed by Ocr. The surface of the protein is replete with acidic residues, most or all of which mimic the phosphate backbone of DNA. Aspartate and glutamate residues on the surface of Ocr were either mutated or chemically modified in order to investigate their contribution to the tight binding between Ocr and the EcoKI Type I R/M enzyme. Single or double mutations of Ocr had no discernable effect on binding to EcoKI or its methyltransferase component (M.EcoKI). Chemical modification was then used to specifically modify the carboxyl moieties of Ocr, thereby neutralizing the negative charges on the protein surface. Ocr samples modified to varying degrees were analysed to establish the extent of derivatisation prior to extensive biophysical characterisation to assess the impact of these changes in terms of binding to the EcoKI R/M system. The results of this analysis revealed that the electrostatic mimicry of Ocr increases the binding affinity for its target enzyme by at least ~800-fold. In addition, based on the known 3-D structure of the protein, a set of multiple mutations were introduced into Ocr aimed at eliminating patches of negative charge from the protein surface. Specifically, between 5 and 17 acidic residues were targeted for mutation (Asp and Glu to Asn and Gln, respectively). Analysis of the in vivo activity of the mutant Ocr along with biophysical characterisation of the purified proteins was then performed. Results from these studies identified regions of the Ocr protein that were critical in forming a tight association with the EcoKI R/M system. Furthermore by comparing the relative contribution of different groups of acidic residues to the free energy of binding, the actual mechanism by which Ocr mimics the charge distribution of DNA has been delineated.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:562870 |
| Date | January 2010 |
| Creators | Stephanou, Augoustinos S. |
| Contributors | Dryden, David |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/4348 |
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