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Control of directionality in the site‐specific recombination system of the Streptomyces phage φC31
Author(s) -
Thorpe Helena M.,
Wilson Stuart E.,
Smith Margaret C. M.
Publication year - 2000
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.2000.02142.x
Subject(s) - integrase , integrases , biology , recombinase , site specific recombination , recombination , holliday junction , temperateness , tn3 transposon , dna , genetics , microbiology and biotechnology , streptomyces , bacteriophage , gene , transposable element , mutant , bacteria , escherichia coli
The genome of the Streptomyces temperate phage φC31 integrates into the host chromosome via a recombinase belonging to a novel group of phage integrases related to the resolvase/invertase enzymes. Previously, it was demonstrated that, in an in vitro recombination assay, φC31 integrase catalyses integration ( attP/attB recombination) but not excision ( attL/attR ). The mechanism responsible for this recombination site selectivity was therefore investigated. Purified integrase was shown to bind with similar apparent binding affinities to between 46 bp and 54 bp of DNA at each of the attachment sites, attP , attB , attL and attR . Assays using recombination sites of 50 bp and 51 bp for attP and attB , respectively, showed that these fragments were functional in attP/attB recombination and maintained strict site selectivity, i.e. no recombination between non‐permissive sites, such as attP/attP , attB/attL , etc., was observed. Using bandshifts and supershift assays in which permissive and non‐permissive combinations of att sites were used in the presence of integrase, only the attP/attB combination could generate supershifts. Recombination products were isolated from the supershifted complexes. It was concluded that these supershifted complexes contained the recombination synapse and that site specificity, and therefore directionality, is determined at the level of stable synapse formation.