ArdB, ArdA, and Ocr proteins inhibit the endonuclease activity of the type I restriction-modification enzymes (RMI). In this study, we evaluated the ability of ArdB, ArdA, and Ocr to inhibit different subtypes of Escherichia coli RMI systems (IA, IB, and IC) as well as two Bacillus licheniformis RMI systems. Furthermore we explored, the antirestriction activity of ArdA, ArdB, and Ocr against a type III restriction-modification system (RMIII) EcoPI and BREX. We found that DNA-mimic proteins, ArdA and Ocr exhibit different inhibition activity, depending on which RM system tested. This effect might be linked to the DNA mimicry nature of these proteins. In theory, DNA-mimic might competitively inhibit any DNA-binding proteins; however, the efficiency of inhibition depend on the ability to imitate the recognition site in DNA or its preferred conformation. In contrast, ArdB protein with an undescribed mechanism of action, demonstrated greater versatility against various RMI systems and provided similar antirestriction efficiency regardless of the recognition site. However, ArdB protein could not affect restriction systems that are radically different from the RMI such as BREX or RMIII. Thus, we assume that the structure of DNA-mimic proteins allows for selective inhibition of any DNA-binding proteins depending on the recognition site. In contrast, ArdB-like proteins inhibit RMI systems independently of the DNA recognition site.

Center of Cellular and Molecular Biology Skolkovo Institute of Science and Technology Moscow Russia

Faculty of Physics HSE University Moscow Russia

Kurchatov Genomic Center National Research Center Kurchatov Institute Moscow Russia

Laboratory for Microbiology BIOTECH University Moscow Russia

Laboratory for Molecular Genetics Moscow Institute of Physics and Technology Dolgoprudny Russia

Laboratory of Structural Biology and Bioinformatics Institute of Microbiology Academy of Sciences of the Czech Republic Nové Hrady Czechia

Zobrazit více v PubMed

Andriianov A., Triguis S., Drobiazko A., Sierro N., Ivanov N. V., Selmer M., et al. . (2023). Phage T3 overcomes the BREX defence through SAM cleavage and inhibition of SAM synthesis. bioRxiv. 10.1101/2023.02.27.530186 PubMed DOI

Balabanov V. P., Kudryavtseva A. A., Melkina O. E., Pustovoit K. S., Khrulnova S. A., Zavilgelsky G. B. (2019). ArdB protective activity for unmodified λ phage against EcoKI restriction decreases in UV-treated Escherichia coli. Curr. Microbiol. 76, 1374–1378. 10.1007/s00284-019-01755-z PubMed DOI

Bazhenov S. V., Scheglova E. S., Utkina A. A., Kudryavtseva A. A., Al Ebrahim R., Manukhov I. V. (2023). New temperature-switchable acyl homoserine lactone-regulated expression vector. Appl. Microbiol. Biotechnol. 107, 807–818. 10.1007/s00253-022-12341-y PubMed DOI

Belogurov A. A., Delver E. P., Rodzevich O. V. (1993). Plasmid pKM101 encodes two nonhomologous antirestriction proteins (ArdA and ArdB) whose expression is controlled by homologous regulatory sequences. J. Bacteriol. 175, 4843–4850. 10.1128/jb.175.15.4843-4850.1993 PubMed DOI PMC

Belogurov A. A., Yussifov T. N., Kotova V. U., Zavilgelsky G. B. (1985). The novel gene(s) ARD of plasmid pKM101: alleviation of EcoK restriction. MGG Mol. Gen. Genet. 198, 509–513. 10.1007/BF00332948 PubMed DOI

Csefalvay E., Lapkouski M., Guzanova A., Csefalvay L., Baikova T., Shevelev I., et al. . (2015). Functional coupling of duplex translocation to DNA cleavage in a Type I restriction enzyme. PLoS ONE. 10. 10.1371/journal.pone.0128700 PubMed DOI PMC

Delver E. P., Kotova V. U., Zavilgelsky G. B., Belogurov A. A. (1991). Nucleotide sequence of the gene (ard) encoding the antirestriction protein of plasmid ColIb-P9. J. Bacteriol. 173, 5887–5892. 10.1128/jb.173.18.5887-5892.1991 PubMed DOI PMC

Droettboom M., Caswell T. A., Hunter J., Firing E., Nielsen J. H., Root B., et al. . (2017). Matplotlib/Matplotlib: V2.0.0. Genève. Switzerland: Zenodo.

Goldfarb T., Sberro H., Weinstock E., Cohen O., Doron S., Charpak-Amikam Y., et al. . (2015). BREX is a novel phage resistance system widespread in microbial genomes. EMBO J. 34, 169–183. 10.15252/embj.201489455 PubMed DOI PMC

Gordeeva J., Morozova N., Sierro N., Isaev A., Sinkunas T., Tsvetkova K., et al. . (2019). BREX system of Escherichia coli distinguishes self from non-self by methylation of a specific DNA site. Nucleic Acids Res. 47, 253–265. 10.1093/nar/gky1125 PubMed DOI PMC

Gubler M., Bickle T. A. (1991). Increased protein flexibility leads to promiscuous protein - DNA interactions in type IC restriction - modification systems. EMBO J. 10, 951–957. 10.1002/j.1460-2075.1991.tb08029.x PubMed DOI PMC

Isaev A., Drobiazko A., Sierro N., Gordeeva J., Yosef I., Qimron U., et al. . (2020). Erratum: Phage T7 DNA mimic protein Ocr is a potent inhibitor of BREX defence. Nucleic Acids Res. 48, 5297–5406. 10.1093/nar/gkaa290 PubMed DOI PMC

Janscak P., Abadjieva A., Firman K. (1996). The type I restriction endonuclease R.EcoR124I: over-production and biochemical properties. J. Mol. Biol. 257, 977–991. 10.1006/jmbi.1996.0217 PubMed DOI

Janscak P., Bickle T. A. (1998). The DNA recognition subunit of the type IB restriction-modification enzyme EcoAI tolerates circular permutions of its polypeptide chain. J. Mol. Biol. 284, 937–948. 10.1006/jmbi.1998.2250 PubMed DOI

Jindrova E., Schmid-Nuoffer S., Hamburger F., Janscak P., Bickle T. A. (2005). On the DNA cleavage mechanism of Type I restriction enzymes. Nucleic Acids Res. 33, 1760–1766. 10.1093/nar/gki322 PubMed DOI PMC

Krüger D. H., Reuter M., Hansen S., Schroeder C. (1982). Influence of phage T3 and T7 gene functions on a type III (EcoP1) DNA restriction-modification system in vivo. MGG Mol. Gen. Genet. 185, 457–461. 10.1007/BF00334140 PubMed DOI

Kudryavtseva A. A., Alekhin V. A., Lebedeva M. D., Csefalvay E., Weiserova M., Manukhov I. V. (2023). Anti-restriction activity of ArdB protein against EcoAI endonuclease. Mol. Biol. 57, 101–104. 10.1134/S0026893323010053 PubMed DOI

Kudryavtseva A. A., Okhrimenko I. S., Didina V. S., Zavilgelsky G. B., Manukhov I. V. (2020). Antirestriction protein ArdB (R64) interacts with DNA. Biochem. 85, 318–325. 10.1134/S0006297920030074 PubMed DOI

León L. M., Park A. E., Borges A. L., Zhang J. Y., Bondy-Denomy J. (2021). Mobile element warfare via CRISPR and anti-CRISPR in Pseudomonas aeruginosa. Nucleic Acids Res. 49, 2114–2125. 10.1093/nar/gkab006 PubMed DOI PMC

Łobocka M. B., Rose D. J., Plunkett G., Rusin M., Samojedny A., Lehnherr H., et al. . (2004). Genome of bacteriophage P1. J. Bacteriol. 186, 7032–7068. 10.1128/JB.186.21.7032-7068.2004 PubMed DOI PMC

McMahon S. A., Roberts G. A., Johnson K. A., Cooper L. P., Liu H., White J. H., et al. . (2009). Extensive DNA mimicry by the ArdA anti-restriction protein and its role in the spread of antibiotic resistance. Nucleic Acids Res. 37, 4887–4897. 10.1093/nar/gkp478 PubMed DOI PMC

Melkina O. E., Goryanin I. I., Zavilgelsky G. B. (2016). The DNA–mimic antirestriction proteins ArdA ColIB-P9, Arn T4, and Ocr T7 as activators of H-NS-dependent gene transcription. Microbiol. Res. 192, 283–291. 10.1016/j.micres.2016.07.008 PubMed DOI

Murray N. E. (2000). Type I restriction systems: sophisticated molecular machines (a legacy of Bertani and Weigle). Microbiol. Mol. Biol. Rev. 64, 412–434. 10.1128/MMBR.64.2.412-434.2000 PubMed DOI PMC

Patel J., Taylor I., Dutta C. F., Kneale G., Firman K. (1992). High-level expression of the cloned genes encoding the subunits of and intact DNA methyltransferase, M·EcoR124. Gene 112, 21–27. 10.1016/0378-1119(92)90298-4 PubMed DOI

Price C., Lingner J., Bickle T. A., Firman K., Glover S. W. (1989). Basis for changes in DNA recognition by the EcoR124 and EcoR 124 3 Type I DNA restriction and modification enzymes. J. Mol. Biol. 205, 115–125. 10.1016/0022-2836(89)90369-0 PubMed DOI

Rao D. N., Dryden D. T. F., Bheemanaik S. (2014). Type III restriction-modification enzymes: a historical perspective. Nucleic Acids Res. 42, 45–55. 10.1093/nar/gkt616 PubMed DOI PMC

Serfiotis-Mitsa D., Herbert A. P., Roberts G. A., Soares D. C., White J. H., Blakely G. W., et al. . (2009). 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. Nucleic Acids Res. 38, 1723–1737. 10.1093/nar/gkp1144 PubMed DOI PMC

Taylor I., Patel J., Firman K., Kneale G. (1992). Purification and biochemical characterisation of the EcoR124 type I modification methylase. Nucleic Acids Res. 20, 179–186. 10.1093/nar/20.2.179 PubMed DOI PMC

Weiserova M., Janscak P., Benada O., Hubácek J., Zinkevich V. E., Glover S. W., et al. . (1993). Cloning, production and characterisation of wild type and mutant forms of the R·EcoK endonucleases. Nucleic Acids Res. 21, 373–379. 10.1093/nar/21.3.373 PubMed DOI PMC

Wood E. (1983). Molecular cloning. A laboratory manual. Biochem. Educ. 11, 82. 10.1016/0307-4412(83)90068-7 DOI

Zavilgelsky G. B., Kotova V. Y., Rastorguev S. M. (2011). Antimodification activity of the ArdA and Ocr proteins. Russ. J. Genet. 47, 159–167. 10.1134/S1022795410081034 PubMed DOI

An Initial Genome Editing Toolset for Caldimonas thermodepolymerans, the First Model of Thermophilic Polyhydroxyalkanoates Producer