Type I restriction-modification enzymes differ significantly from the type II enzymes commonly used as molecular biology reagents. On hemi-methylated DNAs type I enzymes like the EcoR124I restriction-modification complex act as conventional adenine methylases at their specific target sequences, but unmethylated targets induce them to translocate thousands of base pairs through the stationary enzyme before cleaving distant sites nonspecifically. EcoR124I is a superfamily 2 DEAD-box helicase like eukaryotic double-strand DNA translocase Rad54, with two RecA-like helicase domains and seven characteristic sequence motifs that are implicated in translocation. In Rad54 a so-called extended region adjacent to motif III is involved in ATPase activity. Although the EcoR124I extended region bears sequence and structural similarities with Rad54, it does not influence ATPase or restriction activity as shown in this work, but mutagenesis of the conserved glycine residue of its motif III does alter ATPase and DNA cleavage activity. Through the lens of molecular dynamics, a full model of HsdR of EcoR124I based on available crystal structures allowed interpretation of functional effects of mutants in motif III and its extended region. The results indicate that the conserved glycine residue of motif III has a role in positioning the two helicase domains.

• Klíčová slova
• DNA restriction enzymes, Domain interactions, Molecular mechanics, Molecular modeling, Multisubunit enzyme complex, Principal components analysis,
• MeSH
• adenosintrifosfát chemie   MeSH
• aktivace enzymů MeSH
• analýza hlavních komponent MeSH
• DNA-helikasy chemie   genetika   metabolismus   MeSH
• hydrolýza MeSH
• interakční proteinové domény a motivy * MeSH
• konformace proteinů MeSH
• multienzymové komplexy chemie   MeSH
• mutace MeSH
• podjednotky proteinů chemie   genetika   metabolismus   MeSH
• restrikční endonukleasy typu I chemie   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• simulace molekulární dynamiky MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosintrifosfát MeSH
• DNA-helikasy MeSH
• multienzymové komplexy MeSH
• podjednotky proteinů 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

Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA cleavage and ATP-dependent DNA translocation activities located on motor subunit HsdR. Functional coupling of DNA cleavage and translocation is a hallmark of the Type I restriction systems that is consistent with their proposed role in horizontal gene transfer. DNA cleavage occurs at nonspecific sites distant from the cognate recognition sequence, apparently triggered by stalled translocation. The X-ray crystal structure of the complete HsdR subunit from E. coli plasmid R124 suggested that the triggering mechanism involves interdomain contacts mediated by ATP. In the present work, in vivo and in vitro activity assays and crystal structures of three mutants of EcoR124I HsdR designed to probe this mechanism are reported. The results indicate that interdomain engagement via ATP is indeed responsible for signal transmission between the endonuclease and helicase domains of the motor subunit. A previously identified sequence motif that is shared by the RecB nucleases and some Type I endonucleases is implicated in signaling.

• MeSH
• adenosintrifosfát chemie   metabolismus   MeSH
• DNA bakterií MeSH
• Escherichia coli genetika   metabolismus   MeSH
• exodeoxyribonukleasa V chemie   genetika   metabolismus   MeSH
• exprese genu MeSH
• konformace nukleové kyseliny MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• mutace MeSH
• plazmidy chemie   metabolismus   MeSH
• podjednotky proteinů chemie   genetika   metabolismus   MeSH
• proteiny - lokalizační signály MeSH
• proteiny z Escherichia coli chemie   genetika   metabolismus   MeSH
• restrikční endonukleasy typu I chemie   genetika   metabolismus   MeSH
• signální transdukce MeSH
• štěpení DNA MeSH
• terciární struktura proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• DNA bakterií MeSH
• exodeoxyribonuclease V, E coli MeSH Prohlížeč
• exodeoxyribonukleasa V MeSH
• HsdR protein, E coli MeSH Prohlížeč
• podjednotky proteinů MeSH
• proteiny - lokalizační signály MeSH
• proteiny z Escherichia coli MeSH
• restrikční endonukleasy typu I MeSH

The type I restriction-modification enzyme EcoR124I comprises three subunits with the stoichiometry HsdR2/HsdM2/HsdS1. The HsdR subunits are archetypical examples of the fusion between nuclease and helicase domains into a single polypeptide, a linkage that is found in a great many other DNA processing enzymes. To explore the interrelationship between these physically linked domains, we examined the DNA translocation properties of EcoR124I complexes in which the HsdR subunits had been mutated in the RecB-like nuclease motif II or III. We found that nuclease mutations can have multiple effects on DNA translocation despite being distinct from the helicase domain. In addition to reductions in DNA cleavage activity, we also observed decreased translocation and ATPase rates, different enzyme populations with different characteristic translocation rates, a tendency to stall during initiation and altered HsdR turnover dynamics. The significance of these observations to our understanding of domain interactions in molecular machines is discussed.

• MeSH
• adenosintrifosfatasy metabolismus   MeSH
• aminokyselinové motivy MeSH
• biologický transport MeSH
• biotest MeSH
• DNA-helikasy chemie   MeSH
• DNA metabolismus   MeSH
• endonukleasy chemie   MeSH
• Escherichia coli enzymologie   MeSH
• kinetika MeSH
• molekulární motory chemie   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• mutageneze MeSH
• mutantní proteiny chemie   metabolismus   MeSH
• optická pinzeta MeSH
• podjednotky proteinů chemie   metabolismus   MeSH
• restrikční endonukleasy typu I chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• terciární struktura proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfatasy MeSH
• DNA-helikasy MeSH
• DNA MeSH
• endodeoxyribonuclease EcoR124I MeSH Prohlížeč
• endonukleasy MeSH
• molekulární motory MeSH
• mutantní proteiny MeSH
• podjednotky proteinů MeSH
• restrikční endonukleasy typu I MeSH