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 Res37, 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.Structured digital abstractArdA and ArdA bind by molecular sieving (1, 2)ArdA and ArdA bind by cosedimentation in solution (1, 2)
Highlights
The majority of eubacteria contain the genes for active or putative DNA restriction-modification (RM) systems [1,2,3]
The model of ArdA bound to the EcoKI MTase suggests that these amino acid substitutions occur at positions equivalent to the region of DNA recognised by the S subunit of the RM enzyme (Fig. 1)
Our results show that mutations in domain 3 of ORF18 ArdA affect the ability of the protein to form the dimer observed in the crystal structure
Summary
The majority of eubacteria contain the genes for active or putative DNA restriction-modification (RM) systems [1,2,3]. The identification of potential antirestriction and antimodification (anti-RM) genes within the mobile elements [9,10] suggests a mechanism by which the mobile elements can overcome the RM systems These anti-RM systems have occasionally been acquired and maintained by the host organism, and the occasional activation of such genes weakens or negates the RM defence system, allowing further HGT [11,12]. Depending on the methylation status of the DNA substrate, this complex functions as either a restriction endonuclease or an MTase These enzymes recognise an asymmetric, bipartite sequence (for example, EcoKI recognises AACNNNNNNGTGC), and require ATP to affect cleavage at a distant site reached via extensive DNA translocation. We investigated the effect of mutagenesis in domain 3 of ORF18 ArdA, which forms the dimer interface and is predicted to interact with the MTase core of a Type I RM enzyme [18]. These data indicate that antirestriction activity resides in domains 1 and 2 of ArdA, and that antimodification activity resides in domain 3
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