Original title: Oddelenie restrikčnej a translokačnej funkcie rastrikčne modifikačného enzyme Typu 1 EcoR1241
Translated title: Uncoupling of DNA restriction and DNA translocation functions of the Type I restriction modification enzyme EcoR124I
Authors: Šišáková, Eva ; Weiserová, Marie (advisor) ; Janeček, Jiří (referee) ; Janščák, Pavel (referee)
Document type: Doctoral theses
Year: 2009
Language: eng
Abstract: [eng] [cze]
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...

Institution: Charles University Faculties (theses) (web)
Document availability information: Available in the Charles University Digital Repository.
Original record: http://hdl.handle.net/20.500.11956/93770

Permalink: http://www.nusl.cz/ntk/nusl-461669


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Universities and colleges > Public universities > Charles University > Charles University Faculties (theses)
Academic theses (ETDs) > Doctoral theses