Adenosine Triphosphate Stimulates Aquifex aeolicus MutL Endonuclease Activity 英文参考文献.docVIP

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Adenosine Triphosphate Stimulates Aquifex aeolicus MutL Endonuclease Activity 英文参考文献.doc

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Adenosine Triphosphate Stimulates Aquifex aeolicus MutL Endonuclease Activity 英文参考文献

AdenosineTriphosphateStimulatesAquifexaeolicus MutLEndonucleaseActivity JeromeMauris,ThomasC.Evans,Jr* NewEnglandBiolabs,Inc.,Ipswich,Massachusetts,UnitedStatesofAmerica Abstract Background: Human PMS2 (hPMS2) homologues act to nick 59 and 39 to misincorporated nucleotides during mismatch repairinorganismsthatlackMutH.Mn waspreviouslyfoundtostimulatetheendonucleaseactivityofthesehomologues. ++ ATPwasrequiredforthenickingactivityofhPMS2andyPMS1,butwasreportedtoinhibitbacterialMutLproteinsfrom Thermus thermophilus and Aquifex aeolicus that displayed homology to hPMS2. Mutational analysis has identified the DQHA(X)2E(X)4EmotifpresentintheC-terminusofPMS2homologuesasimportantforendonucleaseactivity. Methodologies/PrincipalFindings:WeexaminedtheeffectATPhadontheMn++inducednickingofsupercoiledpBR322 byfull-lengthandmutantA.aeolicusMutL(AaeMutL)proteins.Assaysweresingletimepoint,enzymetitrationexperiments or reaction time courses. The maximum velocity for MutL nicking was determined to be 1.660.0861025s21 and intheabsenceandpresenceofATP,respectively.AMPPNPstimulatedthenickingactivitytoasimilar 4.260.3610 25 21 s extentasATP.AtruncatedAaeMutLproteincomposedofonlytheC-terminal123aminoacidresidueswasfoundtonick supercoiledDNA.Furthermore,mutationsintheconservedC-terminalDQHA(X)2E(X)4EandCPHGRPmotifswereshownto abolishAaeMutLendonucleaseactivity. Conclusions:ATPstimulatedtheMn++inducedendonucleaseactivityofAaeMutL.ExperimentsutilizingAMPPNPimplied thatthestimulationdidnotrequireATPhydrolysis.AmutationintheDQHA(X)2E(X)4EmotifofAaeMutLfurthersupported the role of this region in endonclease activity. For the first time, to our knowledge, we demonstrate that changing the histidineresidueintheconservedCPHGRPmotifabolishesendonucleolyticactivityofahPMS2homologue.Finally,theC- ++ terminal123aminoacidresiduesofAaeMutLweresufficienttodisplayMn inducednickingactivity. Citation: Mauris J, Evans TC Jr (2009) Adenosine Triphosphate Stimulates Aquifex aeolicus MutL Endonucleas