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Uncoupling of DNA restriction and DNA translocation functions of the Type I restriction modification enzyme EcoR124I
Šišáková, Eva ; Weiserová, Marie (advisor) ; Janeček, Jiří (referee) ; Janščák, Pavel (referee)
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...
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Restrikčně-modifikační enzymy Typu I - identifikace pomocí dvourozměrné elektroforesy a studium fosforylace podjednotek Hsd.
Cajthamlová, Kamila ; Weiserová, Marie (advisor) ; Janeček, Jiří (referee) ; Pospíšek, Martin (referee)
107 7. Závěr 1. Vytvořili jsme podmínky pro dělení a identifikaci všech tří podjednotek Hsd R-M enzymů Typu I EcoKI a EcoR124I na dvourozměrném gelu. Optimalizovali jsme podmínky dělení těchto enzymů v nerovnovážném gradientu pH (NEPHGE) v prvním rozměru 2D-PAGE na pozadí celkových proteinů buněčného extraktu. Tento systém nám v budoucnu umožní provést detailnější studie exprese a postranslační modifikace R-M enzymů Typu I v závislosti na různých fyziologických podmínkách a v souvislosti celkové buněčné proteinové exprese. 2. Využití tohoto systému v primárních pokusech odhalilo existenci dvou isoforem podjednotky HsdR systému EcoKI lišících se velikostí náboje. Usoudili jsme, že jedna z těchto isoforem by mohla být posttranslačně modifikována. 3. Sledování fosforylace zástupců tří skupin R-M enzymů Typu I EcoKI (skupina IA), EcoAI (skupina IB) a EcoR124I (skupina IC), na základě imunoprecipitačních analys kmenů E. coli produkujících tyto enzymy, prokázalo fosforylaci enzymu EcoKI na podjednotce HsdR. Podjednotky HsdM ani HsdS enzymu EcoKI fosforylovány nejsou. Žádná z podjednotek Hsd systémů EcoAI a EcoR124I také fosforylována nebyla. 4. Podjednotka HsdR enzymu EcoKI je fosforylována in vivo pokud je součástí komplexní REasy EcoKI. Samotná HsdR, bez současné exprese HsdM a HsdS, kdy se netvoří funkční...
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Uncoupling of DNA restriction and DNA translocation functions of the Type I restriction modification enzyme EcoR124I
Šišáková, Eva ; Weiserová, Marie (advisor) ; Janeček, Jiří (referee) ; Janščák, Pavel (referee)
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...
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SILVER NANOPARTICLES AND THEIR BACTERICIDAL EFFECT
Piksová, K. ; Weiserová, Marie ; Jedličková, A. ; Fojtík, A.
Rapid development of bio-nanotechnology lead to the new way in the combating of bacteria and to searching specific properties of nanomaterials The study of bactericidal nanomaterials is particularly timely considering the recent increase of new resistant strains of bacteria to the most potent antibiotics The present work studies the bactericidal effect of silver nanoparticles in the range of 10-30 nm on Gram-negative bacteria and Gram-positive bacteria
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Charakterizace restrikčně-modifikačního systému komensálního kmene
Weiserová, Marie ; Ryu, J.
We have characterised a putative restriction-modification system EcoA0ORF42P in commensal Escherichia coli strain A0 34/86 (O83: K24: H31). This system is a functional member of the Type IB family, whose specificity differs from those of known Type IB enzymes, as proved by immunological cross-reactivity and complementation assay. Using a plasmid transformation method and RM search computer program, we have identified the DNA recognition sequence of EcoA0ORF42P as GGA(8N)ATGC
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Kontinuální analýzy DNA translokace enzymem Typu I EcoR124I
Šišáková, Eva ; Seidel, R. ; Szczelkun, M. ; Weiserová, Marie
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 translocation
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