Control of directionality in the site-specific recombination system of the Streptomyces phage phiC31.

Abstract:

:The genome of the Streptomyces temperate phage phiC31 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, phiC31 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.

journal_name

Mol Microbiol

journal_title

Molecular microbiology

authors

Thorpe HM,Wilson SE,Smith MC

doi

10.1046/j.1365-2958.2000.02142.x

subject

Has Abstract

pub_date

2000-10-01 00:00:00

pages

232-41

issue

2

eissn

0950-382X

issn

1365-2958

pii

mmi2142

journal_volume

38

pub_type

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