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.

• Klíčová slova
• ArdA, ArdB, RMI, antirestriction, conjugative plasmid,
• Publikační typ
• časopisecké články MeSH

Although EcoR124 is one of the better-studied Type I restriction-modification enzymes, it still presents many challenges to detailed analyses because of its structural and functional complexity and missing structural information. In all available structures of its motor subunit HsdR, responsible for DNA translocation and cleavage, a large part of the HsdR C terminus remains unresolved. The crystal structure of the C terminus of HsdR, obtained with a crystallization chaperone in the form of pHluorin fusion and refined to 2.45 Å, revealed that this part of the protein forms an independent domain with its own hydrophobic core and displays a unique α-helical fold. The full-length HsdR model, based on the WT structure and the C-terminal domain determined here, disclosed a proposed DNA-binding groove lined by positively charged residues. In vivo and in vitro assays with a C-terminal deletion mutant of HsdR supported the idea that this domain is involved in complex assembly and DNA binding. Conserved residues identified through sequence analysis of the C-terminal domain may play a key role in protein-protein and protein-DNA interactions. We conclude that the motor subunit of EcoR124 comprises five structural and functional domains, with the fifth, the C-terminal domain, revealing a unique fold characterized by four conserved motifs in the IC subfamily of Type I restriction-modification systems. In summary, the structural and biochemical results reported here support a model in which the C-terminal domain of the motor subunit HsdR of the endonuclease EcoR124 is involved in complex assembly and DNA binding.

• Klíčová slova
• C-terminal domain, DNA binding protein, DNA endonuclease, EcoR124, Escherichia coli (E. coli), GFP fusion, HsdR, X-ray crystallography, crystal structure, restriction-modification,
• MeSH
• biofyzikální jevy MeSH
• DNA vazebné proteiny chemie   genetika   MeSH
• Escherichia coli chemie   genetika   MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• multiproteinové komplexy chemie   genetika   MeSH
• podjednotky proteinů chemie   genetika   MeSH
• proteinové domény genetika   MeSH
• proteiny z Escherichia coli chemie   genetika   MeSH
• restrikční endonukleasy typu I chemie   genetika   MeSH
• sekvence aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• endodeoxyribonuclease EcoR124I MeSH Prohlížeč
• HsdR protein, E coli MeSH Prohlížeč
• multiproteinové komplexy MeSH
• podjednotky proteinů MeSH
• proteiny z Escherichia coli MeSH
• restrikční endonukleasy typu I MeSH

Type I restriction-modification enzymes are multisubunit, multifunctional molecular machines that recognize specific DNA target sequences, and their multisubunit organization underlies their multifunctionality. EcoR124I is the archetype of Type I restriction-modification family IC and is composed of three subunit types: HsdS, HsdM, and HsdR. DNA cleavage and ATP-dependent DNA translocation activities are housed in the distinct domains of the endonuclease/motor subunit HsdR. Because the multiple functions are integrated in this large subunit of 1,038 residues, a large number of interdomain contacts might be expected. The crystal structure of EcoR124I HsdR reveals a surprisingly sparse number of contacts between helicase domain 2 and the C-terminal helical domain that is thought to be involved in assembly with HsdM. Only two potential hydrogen-bonding contacts are found in a very small contact region. In the present work, the relevance of these two potential hydrogen-bonding interactions for the multiple activities of EcoR124I is evaluated by analysing mutant enzymes using in vivo and in vitro experiments. Molecular dynamics simulations are employed to provide structural interpretation of the functional data. The results indicate that the helical C-terminal domain is involved in the DNA translocation, cleavage, and ATPase activities of HsdR, and a role in controlling those activities is suggested.

• Klíčová slova
• DNA restriction enzymes, Domain interactions, E. coli, Molecular modeling, Multisubunit enzyme complex,
• Publikační typ
• časopisecké články MeSH

The HsdR subunit of the type I restriction-modification system EcoR124I is responsible for the translocation as well as the restriction activity of the whole complex consisting of the HsdR, HsdM and HsdS subunits, and while crystal structures are available for the wild type and several mutants, the C-terminal domain comprising approximately 150 residues was not resolved in any of these structures. Here, three fusion constructs with the GFP variant pHluorin developed to overexpress, purify and crystallize the C-terminal domain of HsdR are reported. The shortest of the three encompassed HsdR residues 887-1038 and yielded crystals that belonged to the orthorhombic space group C2221, with unit-cell parameters a = 83.42, b = 176.58, c = 126.03 Å, α = β = γ = 90.00° and two molecules in the asymmetric unit (VM = 2.55 Å(3) Da(-1), solvent content 50.47%). X-ray diffraction data were collected to a resolution of 2.45 Å.

• Klíčová slova
• EcoR124I, Escherichia coli, GFP, HsdR, pHluorin, restriction-modification system,
• MeSH
• difrakce rentgenového záření MeSH
• Escherichia coli chemie   enzymologie   genetika   MeSH
• exprese genu MeSH
• klonování DNA MeSH
• krystalizace MeSH
• krystalografie rentgenová MeSH
• plazmidy chemie   metabolismus   MeSH
• podjednotky proteinů chemie   genetika   metabolismus   MeSH
• proteiny z Escherichia coli chemie   genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny chemie   genetika   metabolismus   MeSH
• restrikční endonukleasy typu I chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• zelené fluorescenční proteiny chemie   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• endodeoxyribonuclease EcoR124I MeSH Prohlížeč
• HsdR protein, E coli MeSH Prohlížeč
• PHluorin MeSH Prohlížeč
• podjednotky proteinů MeSH
• proteiny z Escherichia coli MeSH
• rekombinantní fúzní proteiny MeSH
• restrikční endonukleasy typu I MeSH
• zelené fluorescenční proteiny MeSH