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.
- 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
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.
- 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
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
Restriction-modification systems protect bacteria from foreign DNA. Type I restriction-modification enzymes are multifunctional heteromeric complexes with DNA-cleavage and ATP-dependent DNA translocation activities located on endonuclease/motor subunit HsdR. The recent structure of the first intact motor subunit of the type I restriction enzyme from plasmid EcoR124I suggested a mechanism by which stalled translocation triggers DNA cleavage via a lysine residue on the endonuclease domain that contacts ATP bound between the two helicase domains. In the present work, molecular dynamics simulations are used to explore this proposal. Molecular dynamics simulations suggest that the Lys-ATP contact alternates with a contact with a nearby loop housing the conserved QxxxY motif that had been implicated in DNA cleavage. This model is tested here using in vivo and in vitro experiments. The results indicate how local interactions are transduced to domain motions within the endonuclease/motor subunit.
- MeSH
- adenosintrifosfát chemie metabolismus MeSH
- aminokyselinové motivy MeSH
- DNA chemie metabolismus MeSH
- fenotyp MeSH
- genotyp MeSH
- hydrolýza MeSH
- katalýza MeSH
- kinetika MeSH
- konzervovaná sekvence MeSH
- kvantová teorie MeSH
- lysin MeSH
- mutace MeSH
- mutageneze cílená MeSH
- restrikční endonukleasy typu I chemie genetika metabolismus MeSH
- simulace molekulární dynamiky MeSH
- terciární struktura proteinů MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. 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 chemie 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
- práce podpořená grantem MeSH
The Type I restriction-modification enzyme EcoR124I is an ATP-dependent endonuclease that uses dsDNA translocation to locate and cleave distant non-specific DNA sites. Bioinformatic analysis of the HsdR subunits of EcoR124I and related Type I enzymes showed that in addition to the principal PD-(E/D)xK Motifs, I, II and III, a QxxxY motif is also present that is characteristic of RecB-family nucleases. The QxxxY motif resides immediately C-terminal to Motif III within a region of predicted alpha-helix. Using mutagenesis, we examined the role of the Q and Y residues in DNA binding, translocation and cleavage. Roles for the QxxxY motif in coordinating the catalytic residues or in stabilizing the nuclease domain on the DNA are discussed.
- MeSH
- aminokyselinové motivy MeSH
- DNA metabolismus MeSH
- exodeoxyribonukleasa V chemie MeSH
- financování organizované MeSH
- kinetika MeSH
- molekulární sekvence - údaje MeSH
- mutageneze MeSH
- podjednotky proteinů chemie MeSH
- restrikční endonukleasy typu I genetika chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- substituce aminokyselin MeSH
- transport proteinů MeSH
BACKGROUND: Type I restriction-modification (R-M) systems are the most complex restriction enzymes discovered to date. Recent years have witnessed a renaissance of interest in R-M enzymes Type I. The massive ongoing sequencing programmes leading to discovery of, so far, more than 1 000 putative enzymes in a broad range of microorganisms including pathogenic bacteria, revealed that these enzymes are widely represented in nature. The aim of this study was characterisation of a putative R-M system EcoA0ORF42P identified in the commensal Escherichia coli A0 34/86 (O83: K24: H31) strain, which is efficiently used at Czech paediatric clinics for prophylaxis and treatment of nosocomial infections and diarrhoea of preterm and newborn infants. RESULTS: We have characterised a restriction-modification system EcoA0ORF42P of the commensal Escherichia coli strain A0 34/86 (O83: K24: H31). This system, designated as EcoAO83I, is a new functional member of the Type IB family, whose specificity differs from those of known Type IB enzymes, as was demonstrated by an immunological cross-reactivity and a complementation assay. Using the plasmid transformation method and the RM search computer program, we identified the DNA recognition sequence of the EcoAO83I as GGA(8N)ATGC. In consistence with the amino acids alignment data, the 3' TRD component of the recognition sequence is identical to the sequence recognized by the EcoEI enzyme. The A-T (modified adenine) distance is identical to that in the EcoAI and EcoEI recognition sites, which also indicates that this system is a Type IB member. Interestingly, the recognition sequence we determined here is identical to the previously reported prototype sequence for Eco377I and its isoschizomers. CONCLUSION: Putative restriction-modification system EcoA0ORF42P in the commensal Escherichia coli strain A0 34/86 (O83: K24: H31) was found to be a member of the Type IB family and was designated as EcoAO83I. Combination of the classical biochemical and bacterial genetics approaches with comparative genomics might contribute effectively to further classification of many other putative Type-I enzymes, especially in clinical samples.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- DNA restrikčně-modifikační enzymy genetika metabolismus MeSH
- Escherichia coli enzymologie genetika MeSH
- financování organizované MeSH
- genomika MeSH
- proteiny z Escherichia coli genetika metabolismus MeSH
- protilátky bakteriální metabolismus MeSH
- restrikční endonukleasy typu I genetika metabolismus MeSH
- sekvence nukleotidů MeSH
- sekvenční homologie nukleových kyselin MeSH
- sekvenční seřazení MeSH
- testy genetické komplementace MeSH
Phosphorylation of Type I restriction-modification (R-M) enzymes EcoKI, EcoAI, and EcoR124I - representatives of IA, IB, and IC families, respectively - was analysed in vivo by immunoblotting of endogenous phosphoproteins isolated from Escherichia coli strains harbouring the corresponding hsd genes, and in vitro by a phosphorylation assay using protein kinase present in subcellular fractions of E. coli. From all three R-M enzymes, the HsdR subunit of EcoKI system was the only subunit that was phosphorylated. Further, evidence is presented that HsdR is phosphorylated in vivo only when coproduced with HsdM and HsdS subunits - as part of assembled EcoKI restriction endonuclease, while the individually produced HsdR subunit is not phosphorylated. In vitro phosphorylation of the HsdR subunit of purified EcoKI endonuclease occurs on Thr, and is strictly dependent on the addition of a catalytic amount of cytoplasmic fraction isolated from E. coli. So far this is the first case of phosphorylation of a Type I R-M enzyme reported.
- MeSH
- chromatografie na tenké vrstvě MeSH
- elektroforéza MeSH
- Escherichia coli enzymologie metabolismus MeSH
- financování organizované MeSH
- fosfoaminokyseliny metabolismus MeSH
- fosforylace MeSH
- imunoprecipitace MeSH
- podjednotky proteinů metabolismus MeSH
- proteiny z Escherichia coli metabolismus MeSH
- restrikční endonukleasy typu I metabolismus MeSH
- restrikční enzymy metabolismus MeSH
- western blotting MeSH
