Switching the polarity of a bacteriophage integration system.
During lysogenic growth many temperate bacteriophage genomes are integrated into the host's chromosome and efficient integration and excision are therefore an essential part of the phage life cycle. The Streptomyces phage phiC31 encodes an integrase related to the resolvase/invertases and is evolutionarily and mechanistically distinct from the integrase of phage lambda. We show that during phiC31 integration the polarity of the recombination sites, attB and attP, is dependent on the sequences of the two base pairs (bp) where crossover occurs. A loss or switch in polarity of the recombination sites can occur by mutation of this dinucleotide, leading to incorrectly joined products. The properties of the mutant sites implies that phiC31 integrase interacts symmetrically with the substrates, which during synapsis can align apparently freely in either of two alternative forms that lead to correct or incorrect joining of products. Analysis of the topologies of the reaction products provided evidence that integrase can synapse and activate strand exchange even when recombinant products cannot form due to mismatches at the crossover site. The topologies of the recombination products are complex and indicative of multiple pathways to product formation. The efficiency of integration of a phiC31 derivative, KC859, into an attB site with switched polarity was assayed in vivo and shown to be no different from integration into a wild-type attB. Thus neither the host nor KC859 express a factor that influences the alignment of the recombination sites at synapsis.
Institute of Genetics, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK.
Matthew C A
MC
Smith
Rob
R
Till
Margaret C M
MC
Smith
D001435:Bacteriophages / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D001483:Base Sequence; D017931:DNA Primers; D016679:Genome, Viral; D019426:Integrases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011995:Recombination, Genetic; D013302:Streptomyces / Q000821:virology; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016662:Virus Integration
D017931:DNA Primers; D014764:Viral Proteins; D019426:Integrases