Integrases (Int) are enzymes that mediate the integration and excision of viral DNA into or out of their hosts‟ chromosomes and can therefore be exploited to integrate or delete genes in a precise way. In order to establish lysogeny, integrase mediates recombination between the bacterial and phage attachment sites, attB and attP respectively to generate an integrated prophage flanked by attL and attR. This reaction occurs in vitro without any need for accessory proteins prompting the question, how does the prophage excise? Phages use accessory proteins, Recombination Directionality Factors, RDFs to control the directionality of integrase. For the serine integrase family, RDFs have been identified for three phages, TP901-1, φRv1 and Bxb1, and there is no detectable sequence conservation between them. This work has identified the φC31 early protein gp3 as the RDF. Gp3 acts stoichiometrically to activate excision and binds to Int in solution and in complex with DNA. Insight into the mechanism of gp3 action has revealed that it is at the synapse level that gp3 switches the directionality of Int. The properties of the gp3+Int driven reaction was found to be similar to that mediated by a previously characterised mutated Int, IntE449K that triggers gp3 independent excision (Rowley et al., 2008). Despite φC31 and φBT1 Ints being only 21% identical in sequence, the gp3 homologues from these phages cross-react. Both the gp3s bind to the last 200 amino acids of C-terminal domain of φC31 Int to activate excision and inhibit integration. Evidence is presented that gp3, on binding to Int, overcomes an innate mechanism that normally prevents synapsis of the excision substrates. These observations could lead to further exploitation of φC31 system as a tool for genome engineering.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:619161 |
| Date | January 2012 |
| Creators | Khaleel, Thanafez |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=211280 |
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