Abstract
Proteins of the Ard family are specific inhibitors of type I restriction-modification enzymes. The ArdA of R64 is highly homologous to ColIb-P9 ArdA, differing only by four amino acid residues of the overall 166. However, unlike ColIb-P9 ArdA, which inhibits both the endonuclease and the methylase activities of EcoKI, the R64 ArdA protein inhibits only the endonuclease activity of this enzyme. The mutant forms of R64 ArdA—A29T, S43A, and Y75W, capable of partially reversing the protein to ColIb-P9 ArdA form—were produced by directed mutagenesis. It was demonstrated that only Y75W mutation of these three variants essentially influenced the functional activity of ArdA: the antimodification activity was restored to approximately 90%. It is assumed that R64 ArdA inhibits formation of the complex between unmodified DNA and the R subunit of the type I restriction-modification enzyme EcoKI (R2M2S), which translocates and cleaves DNA. ColIb-P9 ArdA protein is capable of forming the DNA complex not only with the R subunit, but also with the S subunit, which contacts sK site (containing modified adenine residues) in DNA. ArdA bound to the specific sK site inhibits concurrently the endonuclease and methylase activities of EcoKI (R2M2S), while ArdA bound to the nonspecific site in the R subunit blocks only its endonuclease activity.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Barcus, V.A and Murray, N.E., Barriers to Recombination: Restriction, Population Genetics of Bacteria (Soc. Gen. Microbiol. Symp. 52), Baumberg, S. et al., Eds., Cambridge: Cambridge Univ. Press, 1995, vol. 52, pp. 31–58.
Loenen, W.A.M., Tracking EcoKI and DNA Fifty Years On: A Golden Story Full of Surprises, Nucleic Acids Res., 2003, vol. 31, pp. 7059–7069.
Murray, N.E., Immigration Control of DNA in Bacteria: Self Versus Non-Self, Microbiology, 2002, vol. 148, pp. 3–20.
Bickle, T.A., DNA Restriction and Modification Systems, Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, Neidhardt, F.C. et al., Eds., Washington, DC: Am. Soc. Microbiol, 1987, pp. 345–358.
Bickle, T.A. and Kruger, D., Biology of DNA Restriction, Microbiol. Rev., 1993, vol. 57, pp. 434–450.
Murray, N.E., Type I Restriction Systems: Sophisticated Molecular Machines, Microbiol. Rev., 2000, vol. 57, pp. 412–434.
Zavilgelsky, G.B., Antirestriktsiya, Mol. Biol. (Moscow), 2000, vol. 34, pp. 854–862.
Zavilgelsky, G.B., Belogurov, A.A., Mershavka, V.Yu., and Yusifov, T.N., Alleviation of Type I Restriction in the Presence of pKM101 (incN), Mol. Biol. (Moscow), 1984, vol. 18, pp. 1590–1596.
Belogurov, A.A., Yussifov, T.N., Kotova, V.Yu., and Zavilgelsky, G.B., The Novel Gene ard of Plasmid pKM101: Alleviation of EcoKI Restriction, Mol. Gen. Genet., 1985, vol. 198, pp. 509–513.
Kotova, V.Yu., Belogurov, A.A., and Zavilgelsky, G.B., Alleviation of Type I Restriction in the Presence of Plasmids of the IncI Group: General Characterization and Molecular Cloning of the ard Gene, Mol. Biol. (Moscow), 1988, vol. 22, pp. 270–276.
Delver, E.P., Kotova, V.Yu., Zavilgelsky, G.B., and Belogurov, A.A., Nucleotide Sequence of the Gene (ard) Encoding the Antirestriction Protein of Plasmid ColIb-P9, J. Bacteriol., 1991, vol. 173, pp. 5887–5892.
Zavilgelsky, G.B., Bakalova, T.L., Duzhii, D.E., and Kotova, V.Yu., The ard Gene Encoding the Type I Restriction Inhibitor Is Present in Conjugative Plasmids of FII, B/O and K Groups of Incompatibility, Rus. J. Genet., 1994, vol. 30, no. 12, pp. 369–372.
Chilley, P.M. and Wilkins, B.M., Distribution of the ardA Family of Antirestriction Genes on Conjugative Plasmids, Microbiology, 1995, vol. 141, pp. 2157–2164.
Dunn, J.J., Elzinga, M., Mark, K.K., and Studier, F.W., Amino Acid Sequence of the Gene 0.3 Protein of Bacteriophage T7 and Nucleotide Sequence of Its Messenger RNA, J. Biol. Chem., 1981, vol. 256, pp. 2579–2585.
Kruger, D.H., Hansen, S., and Reuter, M., The ocr Gene Function of Bacteriophages T3 and T7 Counteracts the Salmonella typhimurium DNA Restriction Systems SA and SB, J. Virol., 1983, vol. 45, pp. 1147–1149.
Iida, S., Hiestand-Nauer, R., Sandmeier, H., et al., Accessory Genes in the darA Operon of Bacteriophage P1 Affect Antirestriction Function Generalized Transduction, Head Morphogenesis, and Host-All Lysis, Virology, 1998, vol. 251, pp. 49–58.
Walkinshaw, M.D., Taylor, P., Sturrock, S.S., et al., Structure of Ocr from Bacteriophage T7, a Protein That Mimics B-Form DNA, Mol. Cell, 2002, vol. 9, pp. 187–194.
Thomas, A.T., Brammar, W.J., and Wilkins, B.M., Plasmid R16 ArdA Protein Preferentially Targets Restriction Activity of the Type I Restriction-Modification System EcoKI, J. Bacteriol., 2003, vol. 185, pp. 2022–2025.
Zavilgelsky, G.B., Letuchaya, E.A., and Rastorguev, S.M., Antirestriction Activity of the ArdA Protein Encoded by incI1 Transmissive Plasmid R64, Mol. Biol. (Moscow), 2004, vol. 38, pp. 904–907.
Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York: Cold Spring Harbor Lab., 1989.
Sanger, F., Nicklen, S., and Coulson, A.R., DNA Sequencing with Chain-Terminating Inhibitors, Proc. Natl. Acad. Sci. USA, 1977, vol. 74, pp. 5463–5467.
Landt, O., Grunert, H.P., and Hahn, U., A General Method for Rapid Site-Directed Mutagenesis Using the Polymerase Chain Reaction, Gene, 1990, vol. 96, pp. 125–128.
Rost, B. and Sanders, C., Prediction of Protein Secondary Structure at Better than 70% Accuracy, J. Mol. Biol., 1993, vol. 232, pp. 584–599.
Belogurov, A.A. and Delver, E.P., A Motif Conserved among the Type I Restriction-Modification Enzymes and Antirestriction Proteins: Basis for Mechanism of Action of Plasmid-Encoded Antirestriction Function, Nucleic Acids Res., 1995, vol. 23, pp. 785–787.
Atanasiu, C., Su, T.-J., Sturrock, S.S., and Dryden, D.T., Interaction of the ocr Gene 0.3 Protein of Bacteriophage T7 with EcoKI Restriction/Modification Enzyme, Nucleic Acids Res., 2002, vol. 30, pp. 3936–3944.
Althorpe, N.J., Chilley, P.M., Thomas, A.T., et al., Transient Transcriptional Activation of the IncI1 Plasmid Antirestriction Gene (ard), and SOS Inhibition Gene (psiB) Early in Conjugating Recipient Bacteria, Mol. Microbiol., 1999, vol. 31, pp. 133–142.
Author information
Authors and Affiliations
Additional information
Original Russian Text © G.B. Zavilgelsky, T.A. Letuchaya, S.M. Rastorguev, 2006, published in Genetika, 2006, Vol. 42, No. 3, pp. 333–338.
Rights and permissions
About this article
Cite this article
Zavilgelsky, G.B., Letuchaya, T.A. & Rastorguev, S.M. Antirestriction and antimodification activities of the ArdA protein encoded by the IncI1 transmissive plasmids R-64 and ColIb-P9. Russ J Genet 42, 252–258 (2006). https://doi.org/10.1134/S1022795406030045
Received:
Issue Date:
DOI: https://doi.org/10.1134/S1022795406030045