Published online 9 December 2009 Nucleic Acids Research, 2010, Vol. 38, No. 5 1723–1737 doi:10.1093/nar/gkp1144 The structure of the KlcA and ArdB proteins reveals a novel fold and antirestriction activity against Type I DNA restriction systems in vivo but not in vitro Dimitra Serfiotis-Mitsa 1 , Andrew P. Herbert1, Gareth A. Roberts1, Dinesh C. Soares1,2, John H. White1, Garry W. Blakely3, Dusˇ an Uhr´ın1,* and David T. F. Dryden1,* 1EaStChem School of Chemistry, University of Edinburgh, The King’s Buildings, Edinburgh, EH9 3JJ, 2Medical Genetics Section, Molecular Medicine Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU and 3School of Biological Sciences, The University of Edinburgh, The King’s Buildings, Edinburgh EH9 3JR, UK Received August 25, 2009; Revised November 17, 2009; Accepted November 18, 2009 ABSTRACT Plasmids, conjugative transposons and phage frequently encode anti-restriction proteins to enhance their chances of entering a new bacterial host that is highly likely to contain a Type I DNA restriction and modification (RM) system. The RM system usually destroys the invading DNA. Some of the anti-restriction proteins are DNA mimics and bind to the RM enzyme to prevent it binding to DNA. In this article, we characterize ArdB anti-restriction proteins and their close homologues, the KlcA proteins from a range of mobile genetic elements; including an ArdB encoded on a pathogenicity island from uropathogenic Escherichia coli and a KlcA from an IncP-1b plasmid, pBP136 isolated from Bordetella pertussis. We show that all the ArdB and KlcA act as anti-restriction proteins and inhibit the four main families of Type I RM systems in vivo, but fail to block the restriction endonuclease activity of the archetypal Type I RM enzyme, EcoKI, in vitro indicating that the action of ArdB is indirect and very different from that of the DNA mimics. We also present the structure determined by NMR spectroscopy of the pBP136 KlcA protein. The structure shows a novel protein fold and it is clearly not a DNA structural mimic. INTRODUCTION DNA restriction and modiﬁcation (RM) systems are wide- spread in bacteria and archaea, and function as defence systems to reduce the inﬂux of foreign DNA on mobile genetic elements via transduction, transformation and conjugation (1–3). The RM systems can be classiﬁed into four types, I–IV, depending upon their complexity of structure and function (2) with the Type I RM enzymes being the most complex exhibiting both restric- tion endonuclease and modiﬁcation methyltransferase activities in one large complex although a sub- assembly can act as a monofunctional modiﬁcation methyltransferase (4). These enzymes are composed of three subunits encoded by the genes hsdR, hsdM and hsdS (hsd denotes ‘host speciﬁcity of DNA’). HsdM ( 50–60 kDa) and HsdS ( 50 kDa) subunits comprise a methyltransferase (Mtase, M2S1), which in association with HsdR forms an endonuclease (R2M2S1). The HsdR ( 140 kDa) subunits are required for restriction, the HsdS subunit speciﬁes the DNA target sequence and the HsdM subunits catalyses the methylation reaction. Depending upon the methylation state of the DNA, the RM complex can function as either an endonuclease (REase) or an Mtase. The REase will cleave incoming DNA that has not been appropriately modiﬁed by the Mtase. If the target sequence is unmodiﬁed (unmethylated) then it is targeted for restriction. If the target sequence is hemi-methylated, the unmethylated target strand also becomes methylated as well. Fully methylated DNA is immune to restriction. The Type I RM systems are further subdivided into families, ﬁve of which are deﬁned at present by DNA hybridization, subunit complementation, antibody cross-reactivity and sequence conservation. These complex Type I systems are found in over 50% of bacterial species (5) and this defence can be extremely strong (6). They therefore place a considerable *To whom correspondence should be addressed. Tel: 0131 650 7061; Fax: 0131 650 7155; Email: dusan.uhrin@ed.ac.uk Correspondence may also be addressed to David Dryden. Tel: 0131 650 4735; Fax: 0131 650 6453; Email: david.dryden@ed.ac.uk The Author(s) 2009. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.