Mutations of the domain forming the dimeric interface of the ArdA protein affect dimerisation and antimodification activity but not antirestriction activity

Gareth A Roberts, Kai Chen, Edward K M Bower, Julia Madrzak, Arcadia Woods, Amy M Barker, Laurie P Cooper, John H White, Garry W Blakely, Iain Manfield, David T F Dryden

Research output: Contribution to journalArticlepeer-review

Abstract

ArdA antirestriction proteins are encoded by genes present in many conjugative plasmids and transposons within bacterial genomes. Antirestriction is the ability to prevent cleavage of foreign incoming DNA by restriction-modification (RM) systems. Antimodification, the ability to inhibit modification by the RM system, can also be observed with some antirestriction proteins. Since these mobile genetic elements can transfer antibiotic resistance genes, the ArdA proteins assist their spread. The consequence of antirestriction is therefore the enhanced dissemination of mobile genetic elements. ArdA proteins cause antirestriction by mimicking the DNA structure bound by Type I RM enzymes. The crystal structure of ArdA showed it to be a dimeric protein with a highly elongated curved cylindrical shape [McMahon et al., 2009. Nucleic Acids Res. 37, 4887-4897]. Each monomer has three domains covered with negatively charged side chains and a very small interface with the other monomer. We have investigated the role of the domain forming the dimer interface for ArdA activity via site-directed mutagenesis. The antirestriction activity of ArdA is maintained when up to seven mutations per monomer are made or the interface is disrupted such that the protein can only exist as a monomer. The antimodification activity of ArdA is lost upon mutation of this domain. The ability of the monomeric form of ArdA to function in antirestriction suggests firstly that it can bind independently to the restriction subunit or the modification subunits of the RM enzyme and secondly that the many ArdA homologues with long amino acid extensions, present in sequence databases, may be active in antirestriction. This article is protected by copyright. All rights reserved.
Original languageEnglish
Pages (from-to)4903–4914
JournalFebs Journal
Volume280
Issue number19
DOIs
Publication statusPublished - 2 Aug 2013

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