Clamp loader of Escherichia coli DNA Polymerase III : kinetics of the ATP-dependent steps in the sliding-clamp loading reaction

306 Pages·2003·8 MB·English

by  Williams, Christopher R.

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