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

TitleMutations of the domain forming the dimeric interface of the ArdA protein affect dimerization and antimodification activity but not antirestriction activity.
Publication TypeJournal Article
Year of Publication2013
AuthorsRoberts GA, Chen K, Bower EKM, Madrzak J, Woods A, Barker AM, Cooper LP, White JH, Blakely GW, Manfield I, Dryden DTF
JournalFEBS J
Volume280
Issue19
Pagination4903-14
Date Published2013 Oct
ISSN1742-4658
KeywordsDNA Restriction Enzymes, Escherichia coli Proteins, Gene Transfer, Horizontal, Mutation, Protein Multimerization, Protein Structure, Secondary, Repressor Proteins
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. As 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 SA 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 investigated the role of the domain forming the dimer interface for ArdA activity via site-directed mutagenesis. The antirestriction activity of ArdA was maintained when up to seven mutations per monomer were made or the interface was disrupted such that the protein could only exist as a monomer. The antimodification activity of ArdA was lost upon mutation of this domain. The ability of the monomeric form of ArdA to function in antirestriction suggests, first, that it can bind independently to the restriction subunit or the modification subunits of the RM enzyme, and second, that the many ArdA homologues with long amino acid extensions, present in sequence databases, may be active in antirestriction.

DOI10.1111/febs.12467
Alternate JournalFEBS J.
PubMed ID23910724
PubMed Central IDPMC3906837
Grant List090288/Z/09/ZA / / Wellcome Trust / United Kingdom
BB/C511599/1 / / Biotechnology and Biological Sciences Research Council / United Kingdom
GR080463MA / / Wellcome Trust / United Kingdom
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