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Nucleic Acids Res. 2016 Sep 6;44(15):7360-72. doi: 10.1093/nar/gkw616. Epub 2016 Jul 7.

The mechanism of ϕC31 integrase directionality: experimental analysis and computational modelling.

Author information

  • 1Institute of Molecular, Cell and Systems Biology, Bower Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.
  • 2Cluster of Excellence on Plant Sciences (CEPLAS), Heinrich-Heine-University, Universitätsstraße 1, D-40225 Düsseldorf, Germany Institute for Complex Systems and Mathematical Biology, University of Aberdeen, AB24 3UE, UK.
  • 3Department of Biology, University of York, Wentworth Way, York YO10 5DD, UK.
  • 4Institute of Molecular, Cell and Systems Biology, Bower Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK Marshall.Stark@glasgow.ac.uk.
  • 5Institute of Molecular, Cell and Systems Biology, Bower Building, University of Glasgow, Glasgow G12 8QQ, Scotland, UK Sean.Colloms@glasgow.ac.uk.

Abstract

Serine integrases, DNA site-specific recombinases used by bacteriophages for integration and excision of their DNA to and from their host genomes, are increasingly being used as tools for programmed rearrangements of DNA molecules for biotechnology and synthetic biology. A useful feature of serine integrases is the simple regulation and unidirectionality of their reactions. Recombination between the phage attP and host attB sites is promoted by the serine integrase alone, giving recombinant attL and attR sites, whereas the 'reverse' reaction (between attL and attR) requires an additional protein, the recombination directionality factor (RDF). Here, we present new experimental data on the kinetics and regulation of recombination reactions mediated by ϕC31 integrase and its RDF, and use these data as the basis for a mathematical model of the reactions. The model accounts for the unidirectionality of the attP × attB and attL × attR reactions by hypothesizing the formation of structurally distinct, kinetically stable integrase-DNA product complexes, dependent on the presence or absence of RDF. The model accounts for all the available experimental data, and predicts how mutations of the proteins or alterations of reaction conditions might increase the conversion efficiency of recombination.

PMID:
27387286
PMCID:
PMC5009753
DOI:
10.1093/nar/gkw616
[PubMed - in process]
Free PMC Article
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