title: Mutations of the domain forming the dimeric interface of the ArdA protein affect dimerization and antimodification activity but not antirestriction activity.
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.
authors.affiliation: EaStCHEM School of Chemistry, University of Edinburgh, UK.
authors.forename: Gareth A
authors.initials: GA
authors.lastname: Roberts
authors.forename: Kai
authors.initials: K
authors.lastname: Chen
authors.forename: Edward K M
authors.initials: EK
authors.lastname: Bower
authors.forename: Julia
authors.initials: J
authors.lastname: Madrzak
authors.forename: Arcadia
authors.initials: A
authors.lastname: Woods
authors.forename: Amy M
authors.initials: AM
authors.lastname: Barker
authors.forename: Laurie P
authors.initials: LP
authors.lastname: Cooper
authors.forename: John H
authors.initials: JH
authors.lastname: White
authors.forename: Garry W
authors.initials: GW
authors.lastname: Blakely
authors.forename: Iain
authors.initials: I
authors.lastname: Manfield
authors.forename: David T F
authors.initials: DT
authors.lastname: Dryden
mesh_terms: D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D022761:Gene Transfer, Horizontal / Q000235:genetics; D009154:Mutation; D055503:Protein Multimerization / Q000235:genetics / Q000502:physiology; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: C096738:ArdA protein, E coli; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D004262:DNA Restriction Enzymes
keywords: ArdA; Tn916; antirestriction; horizontal gene transfer; restriction enzyme
references.citation: EMBO J. 1996 Apr 15;15(8):2003-9
references.pmid: 8617247
references.citation: Nat Rev Microbiol. 2007 Mar;5(3):175-86
references.pmid: 17277795
references.citation: Biophys J. 2000 Mar;78(3):1606-19
references.pmid: 10692345
references.citation: Nucleic Acids Res. 2009 Aug;37(15):4887-97
references.pmid: 19506028
references.citation: Biophys J. 2011 Aug 17;101(4):892-8
references.pmid: 21843480
references.citation: J Bacteriol. 1993 Aug;175(15):4843-50
references.pmid: 8393008
references.citation: Biochemistry. 1993 Apr 27;32(16):4322-9
references.pmid: 8476861
references.citation: Nucleic Acids Res. 2001 Oct 15;29(20):4195-205
references.pmid: 11600708
references.citation: Nucleic Acids Res. 2002 Sep 15;30(18):3936-44
references.pmid: 12235377
references.citation: Mol Cell. 2002 Jan;9(1):187-94
references.pmid: 11804597
references.citation: Lancet Infect Dis. 2006 Oct;6(10):629-40
references.pmid: 17008172
references.citation: Eur Biophys J. 2010 Feb;39(3):423-35
references.pmid: 19234696
references.citation: Curr Opin Microbiol. 2005 Aug;8(4):466-72
references.pmid: 15979932
references.citation: Mol Microbiol. 1995 May;16(4):769-77
references.pmid: 7476171
references.citation: J Bacteriol. 2003 Mar;185(6):2022-5
references.pmid: 12618468
references.citation: Nucleic Acids Res. 2012 Sep;40(16):8129-43
references.pmid: 22684506
references.citation: Anal Biochem. 2003 Sep 1;320(1):104-24
references.pmid: 12895474
references.citation: J Bacteriol. 2006 Aug;188(15):5578-85
references.pmid: 16855248
references.citation: Genetika. 2011 Feb;47(2):159-67
references.pmid: 21516787
references.citation: Mol Gen Genet. 1985;198(3):509-13
references.pmid: 2989658
references.citation: Genetics. 1995 Aug;140(4):1187-97
references.pmid: 7498762
references.citation: Biochemistry (Mosc). 2008 Aug;73(8):906-11
references.pmid: 18774937
references.citation: Nucleic Acids Res. 2004 Oct 08;32(18):e138
references.pmid: 15475385
references.citation: Funct Integr Genomics. 2006 Jul;6(3):186-201
references.pmid: 16453141
references.citation: Environ Microbiol. 2002 Sep;4(9):495-500
references.pmid: 12220405
references.citation: Mol Microbiol. 1999 Jan;31(1):133-42
references.pmid: 9987116
references.citation: J Mol Biol. 2008 Nov 28;383(5):970-81
references.pmid: 18838147
references.citation: Nucleic Acids Res. 2010 Jan;38(Database issue):D234-6
references.pmid: 19846593
references.citation: Nucleic Acids Res. 2003 Apr 1;31(7):1805-12
references.pmid: 12654995
references.citation: Genetika. 2006 Mar;42(3):331-8
references.pmid: 16649659
references.citation: Nucleic Acids Res. 2009 Feb;37(3):762-70
references.pmid: 19074193
references.citation: Microbiol Rev. 1993 Jun;57(2):434-50
references.pmid: 8336674
references.citation: Trends Biotechnol. 2006 Aug;24(8):378-82
references.pmid: 16815576
references.citation: Mol Biol (Mosk). 2009 Mar-Apr;43(2):264-73
references.pmid: 19425495
references.citation: Structure. 2005 May;13(5):723-34
references.pmid: 15893663
references.citation: J Bacteriol. 2004 Nov;186(21):7161-74
references.pmid: 15489427
references.citation: J Mol Biol. 2007 Jan 12;365(2):284-97
references.pmid: 17069852
references.citation: Gene. 1988 Sep 30;69(2):301-15
references.pmid: 3069586
references.citation: Nat Rev Microbiol. 2005 Sep;3(9):711-21
references.pmid: 16138099
references.citation: Anal Biochem. 1989 Nov 1;182(2):319-26
references.pmid: 2610349
references.citation: Science. 2005 Mar 4;307(5714):1463-5
references.pmid: 15746427
references.citation: J Biol Chem. 1993 Jun 25;268(18):13228-36
references.pmid: 8514761