φC31 integrase (Int) and other site-specific recombinases enable controlled and precise genetic manipulations of complex genomes. Int mediates integration of the φC31 genome into the genome of its Streptomyces host. Recombination occurs between specific attachment sites; attB and attP. Int binds attP and attB with similar affinities, despite significant sequence differences. The mechanism through which Int recognises its substrates is not fully understood. To study DNA binding in vivo in the absence of recombination, we employed the challenge phage assay. In this assay, binding by Int to attP or attB results in a high frequency of P22-1000 lysogen formation in Salmonella. When Int has lost binding activity, fewer lysogens are generated. A randomly mutated integrase library has been screened using this assay. A number of the mutants showed a reduction in binding to both attB and attP or just to attB. Point mutations in these integrases largely clustered either a putative zinc finger or to the pfam07508 ‘recombinase' domain. To validate the phage challenge assay data, the binding defective Int mutants were purified and tested in in vitro DNA binding experiments. Int mutants displayed reduced binding to attB and/or attP compared to attL or attR. The purified proteins were used in in vitro recombination assays. Mutants in the recombinase domain generally showed reduced integration whilst demonstrating almost wild type gp3 dependant excision. These data combined with data from others suggested two DNA binding domains in Int; the recombinases domain and the zinc finger. A truncated mutant Int, IntV371SUGA had previously been shown to bind DNA with low affinity. The mutations in the recombinase domain were transferred to IntV371SUGA to test their effect on DNA binding. I suggest that the recombinase motif is intimately involved in DNA recognition and discrimination between the att sites required for phage integratation and excision.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:606460 |
| Date | January 2014 |
| Creators | Paget, Jane Elizabeth |
| 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=210800 |
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