Clamp loader of Escherichia coli DNA Polymerase III : kinetics of the ATP-dependent steps in the sliding-clamp loading reaction PDF
Preview Clamp loader of Escherichia coli DNA Polymerase III : kinetics of the ATP-dependent steps in the sliding-clamp loading reaction
THECLAMPLOADEROFEscherichiacoliDNAPOLYMERASEIII: KINETICSOFTHEATP-DEPENDENTSTEPSINTHESLIDING-CLAMP LOADINGREACTION By CHRISTOPHERR. WILLIAMS ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT OFTHEREQUIREMENTSFORTHEDEGREEOF DOCTOROFPHILOSOPHY UNIVERSITYOFFLORIDA 2003 Copyright2003 by ChristopherR.Williams Thisdocumentisdedicatedtomyparents,family,andHeatherRunyan. ACKNOWLEDGMENTS ForemostIwouldliketothankmyadvisor,LindaBloom,Ph.D.,foroutstanding guidanceandprovidinganexceptionalworkingenvironmentinastate-of-the-artresearch laboratory. IthankmycommitteeatUF,Drs.DanielL. Purich,ArthurS.Edison,J.Bert Flanagen,andAlfredS.Lewin,fortheirassistancenotonlyinguidingmyproject,butin guidingmetobecomemorewiseandperceptiveasascientist. Imustalsoacknowledge mycommitteeatArizonaStateUniversity,Drs.NealWoodbury,YuriL. Lyubchenko, andKennethJ.Hoober,fortheirassistanceintheearlystagesofthisdissertationproject. IacknowledgeManjuHingorani,Ph.D.,forhelpfuldiscussionsattheKeystone Symposiameetingsandproteinpreparation,MikeO'Donnell,Ph.D.forthegenerous giftsofclamploaders,MartinWebb,Ph.D.,MyronF. Goodman,Ph.D.,andJeffery Bertramforthegiftofthephosphatebindingproteinplasmid,andhelpin characterizationanduseofMDCC-PBP,andPetrKuzmic,Ph.D.,forcontributionofthe customversionofDynaFitandassistancewithkineticmodeling. Iwouldliketothank myfollowingcolleaguesfortheirfriendshipandinvaluablediscussionsregardingthis projectandscienceingeneral:BrandonAson,RyanShaw,JohnC.Lopez,Gabriel Montaho,Ph.D.,GregoryUyeda,JoseClemente,Ph.D.,JoyceFeller,Ph.D. Finally,I wouldliketothankmyparentsfortheirunwaveringsupportofmyendevours,andfor purchasingmyfirstmicroscopeandchemistrysetsyearsago. TABLEOFCONTENTS Page ACKNOWLEDGMENTS' iv ., LISTOFTABLES * LISTOFFIGURES » ABSTRACT xiv : CHAPTER 1 INTRODUCTION-STATEMENTOFPROBLEM 1 TheProcessiveEscherichiacoliDNAPolymeraseIIIHoloenzyme 1 DNAPolymeraseIIIHoloenzyme 1 ThepSlidingClamp 2 TheDnaXClampLoader-StoichiometryandOrganizationofSubunits 3 ClampLoaderSubunitFunctions 4 TheMechanismofpClampLoading 5 ImportanceoftheE.coliModelReplicationSystem 7 TheGeneralProblemUnderStudyandResearchQuestionsAddressed 8 ConformationalDynamicsoftheClampLoadingMachine 10 EnhancementoftheClampLoadingMachinebyitsClamp 13 The%andySubunitsareRequiredforOptimumActivityoftheClampLoaderl4 ApplicationoftheAnalysesoftheClampLoadingMachinetoOtherComplex MolecularMachines 17 NovelHybridDevicesBasedontheClampLoaderandSlidingClamp 19 "Off-the-WaH"ExampleoftheClampandClampLoaderinaNovelDevice...20 DesignofResearchProject 21 2 LITERATUREREVIEW 24 DNAReplicationinEscherichiacoli 24 DNAPolymeraseIIIHoloenzyme 25 Thet.SubunitistheCoordinatorofPolIIIHoloenzymeFunctionand Processivity 29 Structureofthe|3SlidingClampProcessivityProtein 31 TheDnaXClampLoadingMachine 35 TheAAA+SuperfamilyofMotorProteins 40 X-rayCrystalStructureoftheClampLoadingMachine 45 StructureoftheNucleotideBindingSiteandtheProposedConformational ChangeoftheySubunit 48 X-RayCrystalStructureofthe8SubunitBoundtoa|3Monomerandthe ' MechanismforOpeningthePSlidingClamp 52 DNAstructuralrequirementsforpclamploadingbyycomplex 56 MechanismofthepClampLoadingReactionCyclebyyComplex 60 MutationsofthePClamp,andyComplex8'andySubunits:Effectsonthe ClampLoadingMechanism 65 TheClampLoadingMachineWithinPolymeraseIIIHoloenzyme 68 ClampsandClampLoadersofBacteriophage,Eukaryotic,and ArchaealOrganisms 71 BacteriophageT4ClampandClamploader 73 EukaryoticPCNAClampandReplicationFactor-CClampLoader 75 ArchaealPCNAClampandReplicationFactor-CClampLoader 80 MATERIALSANDMETHODS 84 Proteins,Reagents,andOligonucleotideSubstrates 84 DNAPolymeraseIIIProteins 84 Reagents 85 OligonucleotideSubstrates 85 PurificationofEscherichiacoliPhosphateBindingProtein 86 LabelingofPhosphateBindingProteinwithMDCC 89 CharacterizationofMDCC-LabeledPhosphateBindingProtein 90 RemovalofFree-InorganicPhosphate(P,)Contaminationwiththe"Pi-mop"..91 ConcentrationandEfficiencyofLabelingofMDCC-PBP 91 Characterizationofthefluorescence-molarresponseofMDCC-PBPtoPi 92 ActiveSiteTitrationofMDCC-PBP 93 FluorescenceAnisotropyBindingAssays 94 CalculationofAnisotropy 94 Steady-stateMeasurementofClampLoader-RhX-DNABindingKinetics 95 Pre-Steady-StateMeasurementofClampLoader-RhX-DNABinding Kinetics 99 Fluorescence-basedMDCC-PBPATPHydrolysis(ATPase)Assay 101 Steady-StateKineticsofATPhydrolysis 101 Pre-Steady-StateKineticsofATPHydrolysis 104 ComputerModelingofATPHydrolysisKineticData 109 CorrelatedPre-Steady-StateMDCC-PBPATPaseAssaysandFluorescence AnisotropyBindingAssays 110 Stopped-FlowDeadTimeDeterminations 112 DeterminationoftheDeadTimefortheBiologicSFM-4Stopped-Flow 112 DeterminationoftheAppliedPhotophysicsSX.18MVStopped-Flow ReactionAnalyzerSequential-andSingle-mixDeadTimes 114 8 4 ATP-DEPENDENTCONFORMATIONALCHANGEINTHECLAMPLOADER11 Introduction 118 Steady-StateCharacterizationofyComplexATPHydrolysis,DNABindingand ClampLoadingActivities 120 EnhancementofSteady-StateATPHydrolysisKineticsofyComplexbyp clamp 120 Steady-StateDNABindingandClampLoadingActivitiesofyComplex 122 Pre-Steady-StateKineticsofDNA-DependentATPHydrolysisbyyComplex 127 Pre-Steady-StateMDCC-PBPATPaseAssaysforyComplexintheAbsence andPresenceofpClamp 127 Pre-Steady-StateMDCC-PBPATPaseAssaysatDifferentConcentrationsofy ComplexinthePresenceofp 129 ATPyS-ChaseofPre-Steady-StateATPHydrolysisActivitybyyComplex...131 Pre-Steady-StateKineticsofpClampLoadingbyyComplexInitiatedat DifferentStepsoftheReactionCycle 135 KineticsofClampLoadingwhenyComplexisEquilibratedwithATP 135 KineticsofClampLoadingwhenyComplexisEquilibratedwithATPandP 137 KineticsofClampLoadingwhenyComplexisAddedDirectlytoaSolution ofATP,p,andDNA 141 KineticsofATPHydrolysisDuringClampLoadingwhenyComplexis EquilibratedwithATP 142 KineticsofATPHydrolysiswhenyComplexisnotEquilibratedwithATP 144 KineticsofFormationoftheTwoPopulationsofyComplex 146 ComputerModelingofATPHydrolysisReactionKinetics 149 Discussion 153 5 CHARACTERIZATIONOFTHEMINIMALCLAMPLOADERCOMPLEX ANDCOMPARISONTOGAMMACOMPLEX 163 Introduction 163 y388'istheMinimalClampLoaderComplexWhichCanBindDNA 165 AnalysisofDNABindingActivityoftheIndividualSubunitsor Sub-complexesoftheClampLoader 165 AnalysisoftheDNABindingActivityofy388'MinimalComplexinthe AbsenceandPresenceofp 167 ComparisonofpClampBindingAffinityoftheMinimalComplexandyComplexl69 EquilibriumPpyreneBindingActivityoftheMinimalComplexandyComplex169 ApparentDissociationConstantforATPBindingtheMinimalComplexory Complex 171 KineticsofATPHydrolysisbytheMinimalComplexMeasuredUsingtheMDCC- PBPATPaseAssay 173 Steady-StateATPHydrolysisKineticsofy388'MinimalComplexinthe AbsenceandPresenceofpClamp 173 Pre-Steady-StateKineticsofATPHydrolysisbyy388' MinimalComplexin theAbsenceandPresenceofpClamp 176 KineticsofATPHydrolysiswhentheMinimalComplexisnotequilibrated withATP 180 ATPyS-ChaseofPre-Steady-StateATPHydrolysisActivitybythey388' MinimalComplex I...183 ATPyS-ChaseofSteady-StateATPHydrolysisActivitybythe7386'Minimal Complexor7Complex 186 ClampLoadingActivityoftheMinimalComplexisMoreSensitivetoADP than7Complex 188 Pre-Steady-StateKineticsofClampLoadingbytheMinimalComplexInitiatedat DifferentStepsoftheReactionCycle 190 KineticsofClampLoadingwhentheMinimalComplexisEquilibratedwith ATPandP 191 KineticsofClampLoadingwhentheMinimalComplexisEquilibratedwith ATP 194 KineticsofClampLoadingWhentheMinimalComplexisMixedDirectly withaSolutionofATP,P,andDNA 195 DirectRealTimeCorrelationoftheMinimalComplexDNABindingandATP HydrolysisKineticsinthePresenceandAbsenceofpClamp 196 Discussion 200 UnderstandingyComplexKineticsbyCharacterizationofandComparison withy388'MinimalComplex 200 7388' istheMinimalComplexwithDNABindingAbility,andBindsATPand PwithAffinitySimilartoyComplex 202 Pre-Steady-StateATPHydrolysisandDNABindingKinetics:Analysesofthe ActiveandInactiveClampLoaderStates 202 ThexandySubunits,MissingfromtheMinimalComplex,MayFacilitatethe ConformationalDynamicsofyComplex 210 PClampEnhancestheSwitchfromInactivetoActiveClampLoader Populations 211 ExperimentswhentheMinimalComplexwasnotEquilibratedwithATP RevealSlowerConformationalChangeKineticsthanyComplex 213 TheNatureofNucleotideBindingtotheMinimalComplexandyComplex..214 6 CONCLUSIONSANDRECOMMENDATIONS 221 Introduction 221 Steady-StateKineticsoftheClampLoaderintheAbsenceorPresenceofP 224 KineticsofATP-DependentConformationalChangeswithintheClampLoader...226 APossibleMechanismforpClampEnhancementofClampLoaderActivity 233 TheMissing%andv|/SubunitsareResponsiblefortheKineticDifferences BetweentheMinimalComplexandyComplex 236 The%and>)/SubunitsareE. coliClampLoaderAAA+AdaptorProteins 237 ProgrammaticRecommendations 239 Pre-Steady-StateKineticsofpClampBinding 239 FluorescenceLifetimeMeasurementofMDCC-PBPTitratedwithInorganic Phosphate 241 FurtherInvestigationofATPBindingandNucleotideExchangebytheClamp Loader 242 AnalysisofClampLoaderConformationalDynamicsbyCircularDichroism Spectroscopy 244 IdentificationofthePutativeClampLoaderDNABindingSurface 245 TheiandyAAA+adaptorhypothesis 246 APPENDIX COMPUTERMODELINGOFEXPERIMENTALKINETICDATA 248 DynaFitScriptForFittingShowninFigure4-9 248 DynaFitScriptForFittinginShowninFigure4-10C 249 DynaFitScriptForFittinginShowninFigure5-6 251 SimulationMechanismsforEquilibrationSteps:KinTekSimProgram 252 DynaFitOutputIndices 255 FittingforyComplexDatainFigure4-9 255 FittingforyComplexDatainFigure4-IOC 258 FittingforMinimalComplexDatainFigure5-6 260 FittingforMinimalComplexDataForEstimationofConformationalRate ConstantsFromaSingleDataset 262 AlternateDynafitModelwitha"Branch"StepatAfterHydrolysisofSecondATP265 AlternateDynafitModelAppliedtoyComplexinFigure4-9 265 AlternateDynafitModelAppliedtoyComplexinFigure4-10C 267 AlternateDynafitModelAppliedtotheMinimalComplexinFigure5-7 269 AlternateDynafitModelAppliedtoyComplexinaDNABindingAssayinthe AbsenceofPClamp 271 LISTOFREFERENCES 274 BIOGRAPHICALSKETCH 287 LISTOFTABLES Table page 2-1. Clampsandclamploadersthroughevolution 72 3-1. Assayandproteinbuffers 85 3-2. TG-plusmediacontents 87 4-1. Steady-stateATPhydrolysiskineticsofycomplexintheabsenceand presenceofpa 121 5-1. Steady-stateATPhydrolysiskineticparametersfortheminimalcomplexandy complexintheabsenceandpresenceofp" 174 5-2. Steady-stateATPyS-chaseassayresults:comparisonoftheminimalcomplexandy complexinthepresenceorabsenceofP 187
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