Control of serine integrase recombination directionality by fusion with the directionality factor

Olorunniji, F. J., McPherson, A. L., Rosser, S. J., Smith, M. C.M., Colloms, S. D. and Stark, W. M. (2017) Control of serine integrase recombination directionality by fusion with the directionality factor. Nucleic Acids Research, 45(14), pp. 8635-8645. (doi:10.1093/nar/gkx567) (PMID:28666339) (PMCID:PMC5737554)

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Abstract

Bacteriophage serine integrases are extensively used in biotechnology and synthetic biology for assembly and rearrangement of DNA sequences. Serine integrases promote recombination between two different DNA sites, attP and attB, to form recombinant attL and attR sites. The ‘reverse’ reaction requires another phage-encoded protein called the recombination directionality factor (RDF) in addition to integrase; RDF activates attL × attR recombination and inhibits attP × attB recombination. We show here that serine integrases can be fused to their cognate RDFs to create single proteins that catalyse efficient attL × attR recombination in vivo and in vitro, whereas attP × attB recombination efficiency is reduced. We provide evidence that activation of attL × attR recombination involves intra-subunit contacts between the integrase and RDF moieties of the fusion protein. Minor changes in the length and sequence of the integrase–RDF linker peptide did not affect fusion protein recombination activity. The efficiency and single-protein convenience of integrase–RDF fusion proteins make them potentially very advantageous for biotechnology/synthetic biology applications. Here, we demonstrate efficient gene cassette replacement in a synthetic metabolic pathway gene array as a proof of principle.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:McPherson, Miss Arlene and Rosser, Professor Susan and Stark, Professor William and Colloms, Dr Sean and Olorunniji, Dr Femi
Authors: Olorunniji, F. J., McPherson, A. L., Rosser, S. J., Smith, M. C.M., Colloms, S. D., and Stark, W. M.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Nucleic Acids Research
Publisher:Oxford University Press
ISSN:0305-1048
ISSN (Online):1362-4962
Published Online:28 June 2017
Copyright Holders:Copyright © 2017 The Authors
First Published:First published in Nucleic Acids Research 45(14): 86358645
Publisher Policy:Reproduced under a Creative Commons license

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
589141A platform for rapid and precise DNA module rearrangements in Synthetic BiologyWilliam StarkBiotechnology and Biological Sciences Research Council (BBSRC)BB/K003356/1RI MOLECULAR CELL & SYSTEMS BIOLOGY