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

Abstract

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..