Gated rotation mechanism of site-specific recombination by ϕC31 integrase

Olorunniji, F. J., Buck, D. E., Colloms, S. D. , McEwan, A. R., Smith, M. C. M., Stark, W. M. and Rosser, S. J. (2012) Gated rotation mechanism of site-specific recombination by ϕC31 integrase. Proceedings of the National Academy of Sciences of the United States of America, 109(48), pp. 19661-19666. (doi:10.1073/pnas.1210964109)

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Publisher's URL: http://dx.doi.org/10.1073/pnas.1210964109

Abstract

Integrases, such as that of the Streptomyces temperate bacteriophage ϕC31, promote site-specific recombination between DNA sequences in the bacteriophage and bacterial genomes to integrate or excise the phage DNA. ϕC31 integrase belongs to the serine recombinase family, a large group of structurally related enzymes with diverse biological functions. It has been proposed that serine integrases use a “subunit rotation” mechanism to exchange DNA strands after double-strand DNA cleavage at the two recombining att sites, and that many rounds of subunit rotation can occur before the strands are religated. We have analyzed the mechanism of ϕC31 integrase-mediated recombination in a topologically constrained experimental system using hybrid “phes” recombination sites, each of which comprises a ϕC31 att site positioned adjacent to a regulatory sequence recognized by Tn3 resolvase. The topologies of reaction products from circular substrates containing two phes sites support a right-handed subunit rotation mechanism for catalysis of both integrative and excisive recombination. Strand exchange usually terminates after a single round of 180° rotation. However, multiple processive “360° rotation” rounds of strand exchange can be observed, if the recombining sites have nonidentical base pairs at their centers. We propose that a regulatory “gating” mechanism normally blocks multiple rounds of strand exchange and triggers product release after a single round.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Rosser, Professor Susan and Stark, Professor William and Colloms, Dr Sean and Olorunniji, Dr Femi
Authors: Olorunniji, F. J., Buck, D. E., Colloms, S. D., McEwan, A. R., Smith, M. C. M., Stark, W. M., and Rosser, S. J.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Proceedings of the National Academy of Sciences of the United States of America
Journal Abbr.:Proc. Natl. Acad. Sci.
Publisher:National Academy of Sciences
ISSN:0027-8424
ISSN (Online):1091-6490
Published Online:12 November 2012
Copyright Holders:Copyright © 2012 National Academy of Sciences
First Published:First published in Proceedings of the National Academy of Sciences of the United States of America 109(48):19661-19666
Publisher Policy:Reproduced in accordance with the copyright policy of the publisher

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
519111Sandpit: Synthetic integrons for continuous directed evolution of complex genetic ensemblesSusan RosserEngineering & Physical Sciences Research Council (EPSRC)EP/H019154/1RI MOLECULAR CELL & SYSTEMS BIOLOGY