ArdA Protein Specificity to Type I Restriction–Modification Systems

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Abstract

ArdA are DNA-mimic proteins which inhibit type I restriction-modification (RMI) systems by binding to them instead of DNA. The question of specificity to DNA methylation sites recognized by RMI complexes remains to be answered: is ArdA able to mimic a specific DNA site? In this work, we cloned ardA genes from three Gram-positive bacteria Agrobacterium tumefaciens, Pseudomonas monteilii and Xanthomonas sp. Antirestriction abilities of these genes were tested against three RMI systems of Escherichia coli, differing in DNA recognition/methylation sites. It was shown that despite the similarity of predicted structures of the studied ArdA proteins, they have significant specificity for three RMI systems. The results obtained may indicate the ability of DNA-mimetics to imitate specific DNA sites.

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A. A. Kudryavtseva

Moscow Institute of Physics and Technology (National Research University)

Email: manukhovi@mail.ru
Russian Federation, Dolgoprudny, Moscow Region, 141707

A. V. Vlasov

Moscow Institute of Physics and Technology (National Research University); RussianBiotechnological University (ROSBIOTECH); Joint Institute for Nuclear Research

Email: manukhovi@mail.ru
Russian Federation, Dolgoprudny, Moscow Region, 141707; Moscow, 125080; Dubna, 141980

E. V. Zinovev

Moscow Institute of Physics and Technology (National Research University)

Email: manukhovi@mail.ru
Russian Federation, Dolgoprudny, Moscow Region, 141707

D. D. Yanovskaya

Skolkovo Institute of Science and Technology

Email: manukhovi@mail.ru
Russian Federation, Moscow, 143028

A. A. Utkina

Moscow Institute of Physics and Technology (National Research University)

Email: manukhovi@mail.ru
Russian Federation, Dolgoprudny, Moscow Region, 141707

S. M. Rastorguev

Pirogov Russian National Research Medical University

Email: manukhovi@mail.ru
Russian Federation, Moscow, 117997

I. V. Manukhov

Moscow Institute of Physics and Technology (National Research University); RussianBiotechnological University (ROSBIOTECH)

Author for correspondence.
Email: manukhovi@mail.ru
Russian Federation, Dolgoprudny, Moscow Region, 141707; Moscow, 125080

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2. Figure S1. Supplementary material to the article
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3. Fig. 1. Alignment of amino acid sequences encoded by the ardA genes of the conjugative plasmid pKM101, transposon Tn916, A. tumefaciens, P. monteilii and Xanthomonas sp. (-) – absence of homologous amino acid, Cov value shows the percentage coverage of each sequence, pid – content (%) of identical amino acids.

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4. Fig. 2. Alignment of ArdA protein structures with the known structure of ArdA Tn916 (PDB2W82) using PMOL v.1.9. a – Alignment of ArdA_At with ArdA_Tn916. b – Alignment of ArdA_Pm with ArdA_Tn916. c – Alignment of ArdA_Xs with ArdA_Tn916.

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5. Fig. 3. Efficiency of seeding of unmodified λо phage on E. coli TG1 cells containing plasmids carrying restriction genes and restriction and antirestriction genes: EcoKI-pACYCEcoKI; EcoKI + ardA_At – pACYCEcoKI, pArdA_At; EcoKI + ardA_Pm – pACYCEcoKI, pArdA_Pm; EcoKI + ardA_Xs – pACYCEcoKI, pArdA_Xs; EcoAI – pAM35; EcoAI + ardA_At – pAM35, pArdA_At; EcoAI + ardA_Pm – pAM35, pArdA_Pm; EcoAI + ardA_Xs – pAM35, pArdA_Xs; EcoR124II – pKF650; EcoR124II + ardA_At – pKF650 + pArdA_At; EcoR124II + ardA_Pm – pKF650 + pArdA_Pm; EcoR124II + ardA_Xs – pKF650 + pArdA_Xs.

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6. Fig. 4. Efficiency of seeding of unmodified λо phage on E. coli TG1 cells containing plasmids carrying restriction genes and restriction and antirestriction genes: EcoR124I – pEcoR124I; EcoR124I + ardA_At – pEcoR124I + pArdA_At; EcoR124I + ardA_Pm – pEcoR124I + pArdA_Pm; EcoR124I + ardA_Xs – pEcoR124I + pArdA_Xs EcoR124II – pKF650; EcoR124II + ardA_At – pKF650 + pArdA_At; EcoR124II + ardA_Pm – pKF650 + pArdA_Pm; EcoR124II + ardA_Xs – pKF650 + pArdA_Xs.

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7. Fig. 5. Alignment of the structure of the EcoKI S-subunit complex with the ArdA_Xs dimer with the PDB structure (5YBB): the EcoKI S-subunit complex bound to a DNA fragment. a – General view of the alignment (ArdA_Xs are shown in green; S-subunit of EcoKI obtained in AlphaFold is shown in cream; S-subunit of EcoKI from PDB (5YBB) is shown in gray). b – Possible scheme of protein-protein contacts of ArdA_Xs and the S-subunit of the EcoKI complex in comparison with the PDB structure of the protein-DNA complex. c – The same contacts of ArdA_Xs and the S-subunit of EcoKI (modeled in AlphaFold) without superposition of the PDB structure of the protein-DNA complex. d – Putative scheme of protein-protein contacts of ArdA_Xs and the S-subunit of the EcoKI complex on another, symmetrical, region of the S-subunit.

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