Zinc-finger recombinase activities in vitro

Prorocic, M.M., Wenlong, D., Olorunniji, F.J., Akopian, A., Schloetel, J.-G., Hannigan, A., McPherson, A.L. and Stark, W.M. (2012) Zinc-finger recombinase activities in vitro. Nucleic Acids Research, 39(21), pp. 9316-9328. (doi: 10.1093/nar/gkr652) (PMID:21849325) (PMCID:PMC3241657)

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Abstract

Zinc-finger recombinases (ZFRs) are chimaeric proteins comprising a serine recombinase catalytic domain linked to a zinc-finger DNA binding domain. ZFRs can be tailored to promote site-specific recombination at diverse -sites, which each comprise a central core sequence flanked by zinc-finger domain-binding motifs. Here, we show that purified ZFRs catalyse efficient high-specificity reciprocal recombination between pairs of Z-sites in vitro. No off-site activity was detected. Under different reaction conditions, ZFRs can catalyse Z-site-specific double-strand DNA cleavage. ZFR recombination activity in Escherichia coli and in vitro is highly dependent on the length of the Z-site core sequence. We show that this length effect is manifested at reaction steps prior to formation of recombinants (binding, synapsis and DNA cleavage). The design of the ZFR protein itself is also a crucial variable affecting activity. A ZFR with a very short (2 amino acids) peptide linkage between the catalytic and zinc-finger domains has high activity in vitro, whereas a ZFR with a very long linker was less recombination-proficient and less sensitive to variations in Z-site length. We discuss the causes of these phenomena, and their implications for practical applications of ZFRs

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:McQue, Mrs Arlene and Stark, Professor Marshall and Olorunniji, Dr Femi and Prorocic, Dr Marko
Authors: Prorocic, M.M., Wenlong, D., Olorunniji, F.J., Akopian, A., Schloetel, J.-G., Hannigan, A., McPherson, A.L., and Stark, W.M.
College/School:College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
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:17 August 2011
Copyright Holders:Copyright © 2011 The Authors
First Published:First published in Nucleic Acids Research 39(21): 9316-9328
Publisher Policy:Reproduced under a Creative Commons License

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