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A motif in the C-terminal domain of ϕC31 integrase controls the directionality of recombination

By Paul A. Rowley, Matthew C. A. Smith, Ellen Younger and Margaret C. M. Smith

Abstract

Bacteriophage ϕC31 encodes an integrase, which acts on the phage and host attachment sites, attP and attB, to form an integrated prophage flanked by attL and attR. In the absence of accessory factors, ϕC31 integrase cannot catalyse attL x attR recombination to excise the prophage. To understand the mechanism of directionality, mutant integrases were characterized that were active in excision. A hyperactive integrase, Int E449K, gained the ability to catalyse attL x attR, attL x attL and attR x attR recombination whilst retaining the ability to recombine attP x attB. A catalytically defective derivative of this mutant, Int S12A, E449K, could form stable complexes with attP/attB, attL/attR, attL/attL and attR/attR under conditions where Int S12A only complexed with attP/attB. Further analysis of the Int E449K-attL/attR synaptic events revealed a preference for one of the two predicted synapse structures with different orientations of the attL/attR sites. Several amino acid substitutions conferring hyperactivity, including E449K, were localized to one face of a predicted coiled-coil motif in the C-terminal domain. This work shows that a motif in the C-terminal domain of ϕC31 integrase controls the formation of the synaptic interface in both integration and excision, possibly through a direct role in protein–protein interactions

Topics: Nucleic Acid Enzymes
Publisher: Oxford University Press
OAI identifier: oai:pubmedcentral.nih.gov:2475636
Provided by: PubMed Central
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    Citations

    1. (2006). The Bacteriophages. 2nd edn.
    2. (2001). Control of directionality in integrase-mediated recombination: examination of recombination directionality factors (RDFs) including Xis and Cox proteins.

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