Structural model for the multisubunit Type IC restriction–modification DNA methyltransferase M.EcoR124I in complex with DNA

Recent publication of crystal structures for the putative DNA-binding subunits (HsdS) of the functionally uncharacterized Type I restriction–modification (R-M) enzymes MjaXIP and MgeORF438 have provided a convenient structural template for analysis of the more extensively characterized members of this interesting family of multisubunit molecular motors. Here, we present a structural model of the Type IC M.EcoR124I DNA methyltransferase (MTase), comprising the HsdS subunit, two HsdM subunits, the cofactor AdoMet and the substrate DNA molecule. The structure was obtained by docking models of individual subunits generated by fold-recognition and comparative modelling, followed by optimization of inter-subunit contacts by energy minimization. The model of M.EcoR124I has allowed identification of a number of functionally important residues that appear to be involved in DNA-binding. In addition, we have mapped onto the model the location of several new mutations of the hsdS gene of M.EcoR124I that were produced by misincorporation mutagenesis within the central conserved region of hsdS, we have mapped all previously identified DNA-binding mutants of TRD2 and produced a detailed analysis of the location of surface-modifiable lysines. The model structure, together with location of the mutant residues, provides a better background on which to study protein–protein and protein–DNA interactions in Type I R-M systems

Structural model for the multisubunit Type IC restriction–modification DNA methyltransferase M.EcoR124I in complex with DNA

Recent publication of crystal structures for the putative DNA-binding subunits (HsdS) of the functionally uncharacterized Type I restriction–modification (R-M) enzymes MjaXIP and MgeORF438 have provided a convenient structural template for analysis of the more extensively characterized members of this interesting family of multisubunit molecular motors. Here, we present a structural model of the Type IC M.EcoR124I DNA methyltransferase (MTase), comprising the HsdS subunit, two HsdM subunits, the cofactor AdoMet and the substrate DNA molecule. The structure was obtained by docking models of individual subunits generated by fold-recognition and comparative modelling, followed by optimization of inter-subunit contacts by energy minimization. The model of M.EcoR124I has allowed identification of a number of functionally important residues that appear to be involved in DNA-binding. In addition, we have mapped onto the model the location of several new mutations of the hsdS gene of M.EcoR124I that were produced by misincorporation mutagenesis within the central conserved region of hsdS, we have mapped all previously identified DNA-binding mutants of TRD2 and produced a detailed analysis of the location of surface-modifiable lysines. The model structure, together with location of the mutant residues, provides a better background on which to study protein–protein and protein–DNA interactions in Type I R-M systems

A RecB-family nuclease motif in the Type I restriction endonuclease EcoR124I

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 α-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

Uncoupling of DNA restriction and DNA translocation functions of the Type I restriction modification enzyme EcoR124I

v vuuuerJ Type I restriďion-modificationenzymeEcoRl24I is a multifunctional,hetero.oligomeric enzyme complex that cleaves DNA after extensiveATP hydrolysis coupled to processive DNA translocation.ATP hydrolysis and DNA translocationare conferredby superfamily2 (SF2) helicase motifs in the central domain of its HsdR subunit.The N-terminal domain carries a conservedregion with catalytic residuesreminiscentof the PD-@/D)xK catalytic motif of Type II restrictionenzymes. Single amino acid substitutionsin the motifs II and III reducedor removedDNA cleavage activiý of the enzymecomplexwithoutďfecting an assemblyof the complex and its DNA- binding properties.Using a combinationof bulk solution and single-moleculeassays,we investigatedthe influence of these mutationson the DNA translocationpropertiesof the enzyme,conferredby the helicase domain. Reduced ATPase activiý of the mutantswas detectedby steady-statestopped flow measurementswith the use of phosphate-binding protein.These results do not show a clear relationshipbetweenthe translocationratesand ATPase rates.Probably the broaderand bimodal distributionof hanslocationratesand the stalling eventsduring initiation revealedin single molecule experimentsall lead to a lower apparentATPase rates.We suggestanexistenceof possibleinterdomďninteractionsbetween the..

Kontinuální analýzy DNA translokace enzymem Typu I EcoR124I

Using combination of bulk solution and single-molecule assays we have investigated the influence of several mutations in the PD-(D/E)-X-K catalytic motif of HsdR on the DNA translocation and ATPase properties of EcoR124I, the restriction-modification enzyme Type I. The gained results have given us the insight into the possible interdomain interactions between helicase and nuclease domains of the HsdR subunit and the effect of their interactions on DNA translocatio

Uncoupling DNA restriction and DNA translocation functions of type I restriction modification enzyme ECOR124RI - a potential molecular motor

Type I restiction enzymes are large, oligometric, multifunctional enzymes containing both restriction endonuclease and modification methyltransferase activities in a single molecule

Oddelenie restrikčnej a translokačnej funkcie rastrikčne modifikačného enzyme Typu 1 EcoR1241

v vuuuerJ Type I restriďion-modificationenzymeEcoRl24I is a multifunctional,hetero.oligomeric enzyme complex that cleaves DNA after extensiveATP hydrolysis coupled to processive DNA translocation.ATP hydrolysis and DNA translocationare conferredby superfamily2 (SF2) helicase motifs in the central domain of its HsdR subunit.The N-terminal domain carries a conservedregion with catalytic residuesreminiscentof the PD-@/D)xK catalytic motif of Type II restrictionenzymes. Single amino acid substitutionsin the motifs II and III reducedor removedDNA cleavage activiý of the enzymecomplexwithoutďfecting an assemblyof the complex and its DNA- binding properties.Using a combinationof bulk solution and single-moleculeassays,we investigatedthe influence of these mutationson the DNA translocationpropertiesof the enzyme,conferredby the helicase domain. Reduced ATPase activiý of the mutantswas detectedby steady-statestopped flow measurementswith the use of phosphate-binding protein.These results do not show a clear relationshipbetweenthe translocationratesand ATPase rates.Probably the broaderand bimodal distributionof hanslocationratesand the stalling eventsduring initiation revealedin single molecule experimentsall lead to a lower apparentATPase rates.We suggestanexistenceof possibleinterdomďninteractionsbetween the...4 Lívery l. Miestne cielenou mutagerÉzousme pripravili kolekciu Ínutantovsjednou substitrlciou konzervovanýchaminokyselinových zvyškov v motívochII a III v N.ÍerminálnejónÉne podjednotkyHsdRenzymuEcoRl24I:Dl5lA, El65A, El65D, EI65IIadK167A. r Pozitívnym a negativnym komplementďným tostonrilr ylvo sme zistili' Že všet$ mutantyprejavili fenoýp ťď. r Test restrikcie DNA ',?vjrro potvrdil výsledky in vivo testov,Že Žiadnyz mutantov nebolschopníštiepiťplazmidovúDNA nalineá'muforrnu. r TestviizbovostiDNA (EMSA) neodhalilžiadnevýznamrÉrozďely medzidivolcým a mutantnýmienzýmamiv schopnostiviazaťDNA. r Metóda na meranieATPázovej aktivity,zaloŽeruínapoužitíproteínuviaŽúcehgfosfáL ukázalu Že substituciev motíveII a Itr u váčšinymutantovspÓsobila virc než2. nrásobnúredukciu hydrolýzy ATP. Mutant Kl67A bol jeďný' ktoý prejavil aktivifu porovnatelhús divolcýmenzýmom. r Translokačnýprocesbol analyzovanýdisociácioutriplexu na stopped.fbw fluorimetri a pornocoumagnetickejpinzeý. obe techniky ukázali zntženétranslokďnérýchloati mutantov,rozdiely medzi mutantamia divohým enzýmom boli ešteýraznejšie pri merani na magrretickej pinzete. Pozorovali sme takztiez zmenerrú procesivih1iniciáciua bimoďílnudistribúciuRl ýchlostí. 2. Aminokyselinovézvyšlry Q a Y v motíveQxxxY podjednotkyHsdR boli substituované...Department of Genetics and MicrobiologyKatedra genetiky a mikrobiologieFaculty of SciencePřírodovědecká fakult

Uncoupling of DNA restriction and DNA translocation functions of the Type I restriction modification enzyme EcoR124I

v vuuuerJ Type I restriďion-modificationenzymeEcoRl24I is a multifunctional,hetero.oligomeric enzyme complex that cleaves DNA after extensiveATP hydrolysis coupled to processive DNA translocation.ATP hydrolysis and DNA translocationare conferredby superfamily2 (SF2) helicase motifs in the central domain of its HsdR subunit.The N-terminal domain carries a conservedregion with catalytic residuesreminiscentof the PD-@/D)xK catalytic motif of Type II restrictionenzymes. Single amino acid substitutionsin the motifs II and III reducedor removedDNA cleavage activiý of the enzymecomplexwithoutďfecting an assemblyof the complex and its DNA- binding properties.Using a combinationof bulk solution and single-moleculeassays,we investigatedthe influence of these mutationson the DNA translocationpropertiesof the enzyme,conferredby the helicase domain. Reduced ATPase activiý of the mutantswas detectedby steady-statestopped flow measurementswith the use of phosphate-binding protein.These results do not show a clear relationshipbetweenthe translocationratesand ATPase rates.Probably the broaderand bimodal distributionof hanslocationratesand the stalling eventsduring initiation revealedin single molecule experimentsall lead to a lower apparentATPase rates.We suggestanexistenceof possibleinterdomďninteractionsbetween the..

DNA cleavage by Type ISP Restriction-Modification enzymes is initially targeted to the 3'-5' strand

The mechanism by which a double-stranded DNA break is produced following collision of two translocating Type I Restriction–Modification enzymes is not fully understood. Here, we demonstrate that the related Type ISP Restriction–Modification enzymes LlaGI and LlaBIII can cooperate to cleave DNA following convergent translocation and collision. When one of these enzymes is a mutant protein that lacks endonuclease activity, DNA cleavage of the 3′-5′ strand relative to the wild-type enzyme still occurs, with the same kinetics and at the same collision loci as for a reaction between two wild-type enzymes. The DNA nicking activity of the wild-type enzyme is still activated by a protein variant entirely lacking the Mrr nuclease domain and by a helicase mutant that cannot translocate. However, the helicase mutant cannot cleave the DNA despite the presence of an intact nuclease domain. Cleavage by the wild-type enzyme is not activated by unrelated protein roadblocks. We suggest that the nuclease activity of the Type ISP enzymes is activated following collision with another Type ISP enzyme and requires adenosine triphosphate binding/hydrolysis but, surprisingly, does not require interaction between the nuclease domains. Following the initial rapid endonuclease activity, additional DNA cleavage events then occur more slowly, leading to further processing of the initial double-stranded DNA break

The Type ISP Restriction-Modification enzymes LlaBIII and LlaGI use a translocation-collision mechanism to cleave non-specific DNA distant from their recognition sites

The Type ISP Restriction–Modification (RM) enzyme LlaBIII is encoded on plasmid pJW566 and can protect Lactococcus lactis strains against bacteriophage infections in milk fermentations. It is a single polypeptide RM enzyme comprising Mrr endonuclease, DNA helicase, adenine methyltransferase and target-recognition domains. LlaBIII shares >95% amino acid sequence homology across its first three protein domains with the Type ISP enzyme LlaGI. Here, we determine the recognition sequence of LlaBIII (5′-TnAGCC-3′, where the adenine complementary to the underlined base is methylated), and characterize its enzyme activities. LlaBIII shares key enzymatic features with LlaGI; namely, adenosine triphosphate-dependent DNA translocation (∼309 bp/s at 25°C) and a requirement for DNA cleavage of two recognition sites in an inverted head-to-head repeat. However, LlaBIII requires K(+) ions to prevent non-specific DNA cleavage, conditions which affect the translocation and cleavage properties of LlaGI. By identifying the locations of the non-specific dsDNA breaks introduced by LlaGI or LlaBIII under different buffer conditions, we validate that the Type ISP RM enzymes use a common translocation–collision mechanism to trigger endonuclease activity. In their favoured in vitro buffer, both LlaGI and LlaBIII produce a normal distribution of random cleavage loci centred midway between the sites. In contrast, LlaGI in K(+) ions produces a far more distributive cleavage profile