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Chapter13DNAreplicationChapter13DNAreplication113.1Introduction

13.2DNApolymerasesaretheenzymesthatmakeDNA

13.3DNAsynthesisissemidiscontinuous

13.4Coordinatingsynthesisofthelaggingandleading

strands

13.5ThereplicationapparatusofphageT4

13.6Creatingthereplicationforksatanorigin

13.7Commoneventsinprimingreplicationattheorigin

13.8Doesmethylationattheoriginregulateinitiation?

13.9Licensingfactorcontrolseukaryoticrereplication

13.1Introduction

13.2DNApo2ReplisomeisthemultiproteinstructurethatassemblesatthebacterialreplicatingforktoundertakesynthesisofDNA.ContainsDNApolymeraseandotherenzymes.

13.1IntroductionReplisomeisthemultiprotein3DNApolymerasesareenzymesthatsynthesizeadaughterstrand(s)ofDNA(underdirectionfromaDNAtemplate).Maybeinvolvedinrepairorreplication.

DNAreplicaseisaDNA-synthesizingenzymerequiredspecificallyforreplication.

RepairofdamagedDNAcantakeplacebyrepairsynthesis,whenastrandthathasbeendamagedisexcisedandreplacedbythesynthesisofanewstretch.Itcanalsotakeplacebyrecombinationreactions,whentheduplexregioncontainingthedamagedisreplacedbyanundamagedregionfromanothercopyofthegenome.

ReplicationofduplexDNAtakesplacebysynthesisoftwonewstrandsthatarecomplementarytotheparentalstrands.Theparentalduplexisreplacedbytwoidenticaldaughterduplexes,eachofwhichhasoneparentalstrandandonenewlysynthesizedstrand.Itiscalledsemiconservativebecausetheconservedunitsarethesinglestrandsoftheparentalduplex.13.2DNApolymerasesaretheenzymesthatmakeDNA

DNApolymerasesareenzymesth4Figure13.1SemiconservativereplicationsynthesizestwonewstrandsofDNA.13.2DNApolymerasesaretheenzymesthatmakeDNA

Figure13.1Semiconservativer5Figure13.2RepairsynthesisreplacesadamagedstrandofDNA.13.2DNApolymerasesaretheenzymesthatmakeDNAFigure13.2Repairsynthesisr6Figure13.3DNAsynthesisoccursbyaddingnucleotidestothe3-OHendofthegrowingchain,sothatthenewchainissynthesizedinthe5-3direction.TheprecursorforDNAsynthesisisanucleosidetriphosphate,whichlosestheterminaltwophosphategroupsinthereaction.13.2DNApolymerasesaretheenzymesthatmakeDNAFigure13.3DNAsynthesisoccu7Figure13.4OnlyoneDNApolymeraseisthereplicase.TheothersparticipateinrepairofdamagedDNA.13.2DNApolymerasesaretheenzymesthatmakeDNAFigure13.4OnlyoneDNApolym8

NicktranslationdescribestheabilityofE.coliDNApolymeraseItouseanickasastartingpointfromwhichonestrandofaduplexDNAcanbedegradedandreplacedbyresynthesisofnewmaterial;isusedtointroduceradioactivelylabelednucleotidesintoDNAinvitro.

13.3DNApolymeraseshavevariousnucleaseactivities

Nicktranslationdescribesth9Proofreadingreferstoanymechanismforcorrectingerrorsinproteinornucleicacidsynthesisthatinvolvesscrutinyofindividualunitsaftertheyhavebeenaddedtothechain.

13.4DNApolymerasescontrolthefidelityofreplicationProofreadingreferstoanymec10Figure13.6BacterialDNApolymerasesscrutinizethebasepairattheendofthegrowingchainandexcisethenucleotideaddedinthecaseofamisfit.13.4DNApolymerasescontrolthefidelityofreplicationFigure13.6BacterialDNApoly11Figure13.5Nicktranslationreplacespartofapre-existingstrandofduplexDNAwithnewlysynthesizedmaterial.13.3DNApolymeraseshavevariousnucleaseactivitiesFigure13.5Nicktranslationr1213.3DNApolymeraseshavevariousnucleaseactivities13.3DNApolymeraseshavevari1313.3DNApolymeraseshavevariousnucleaseactivities13.3DNApolymeraseshavevari14Figure13.12Thereareseveralmethodsforprovidingthefree3-OHendthatDNApolymerasesrequiretoinitiateDNAsynthesis.13.8PrimingisrequiredtostartDNAsynthesisFigure13.12Thereareseveral15Figure13.7CrystalstructureofphageT7DNApolymerasehasarighthandstructure.DNAliesacrossthepalmandisheldbythefingersandthumb.PhotographkindlyprovidedbyCharlesRichardsonandTomEllenberger.13.5SomeDNApolymeraseshaveacommonstructureFigure13.7Crystalstructure16Figure13.8ThecatalyticdomainofaDNApolymerasehasaDNA-bindingcleftcreatedbythreesubdomains.Theactivesiteisinthepalm.Proofreadingisprovidedbyaseparateactivesiteinanexonucleasedomain.13.5SomeDNApolymeraseshaveacommonstructureFigure13.8Thecatalyticdoma17LaggingstrandofDNAmustgrowoverallinthe3′-5′directionandissynthesizeddiscontinuouslyintheformofshortfragments(5′-3′)thatarelaterconnectedcovalently.

LeadingstrandofDNAissynthesizedcontinuouslyinthe5′-3′direction.

Okazakifragmentsaretheshortstretchesof1000-2000basesproducedduringdiscontinuousreplication;theyarelaterjoinedintoacovalentlyintactstrand.

Semidiscontinuousreplicationismodeinwhichonenewstrandissynthesizedcontinuouslywhiletheotherissynthesizeddiscontinuously.13.6DNAsynthesisissemidiscontinuous

LaggingstrandofDNAmustgro18Figure13.9Theleadingstrandissynthesizedcontinuouslywhilethelaggingstrandissynthesizeddiscontinuously.13.6DNAsynthesisissemidiscontinuousFigure13.9Theleadingstrand19SSB

isthesingle-strandproteinofE.coli,aproteinthatbindstosingle-strandedDNA.13.7Single-strandedDNAisneededforreplicationSSBisthesingle-strandprote20Figure13.22SynthesisofOkazakifragmentsrequirespriming,extension,removalofRNA,gapfilling,andnickligation.13.10CoordinatingsynthesisofthelaggingandleadingstrandsFigure13.22SynthesisofOkaz21Figure13.23DNAligasesealsnicksbetweenadjacentnucleotidesbyemployinganenzyme-AMPintermediate.13.10CoordinatingsynthesisofthelaggingandleadingstrandsFigure13.23DNAligaseseals22Figure13.10fX174DNAcanbeseparatedintosinglestrandsbythecombinedeffectsof3functions:nickingwithAprotein,unwindingbyRep,andsingle-strandstabilizationbySSB.

13.7Single-strandedDNAisneededforreplicationFigure13.10fX174DNAcanbe23Figure13.13Initiationrequiresseveralenzymaticactivities,includinghelicases,single-strandbindingproteins,andsynthesisoftheprimer.13.8PrimingisrequiredtostartDNAsynthesisFigure13.13Initiationrequir24Figure13.13Initiationrequiresseveralenzymaticactivities,includinghelicases,single-strandbindingproteins,andsynthesisoftheprimer.13.8PrimingisrequiredtostartDNAsynthesisFigure13.13Initiationrequir25Figure13.12Leadingandlaggingstrandpolymerasesmoveapart.13.8PrimingisrequiredtostartDNAsynthesisFigure13.12Leadingandlaggi26Figure13.18DNApolymeraseIIIholoenzymeassemblesinstages,generatinganenzymecomplexthatsynthesizestheDNAofbothnewstrands.13.10CoordinatingsynthesisofthelaggingandleadingstrandsFigure13.18DNApolymeraseII27Figure13.19ThebsubunitofDNApolymeraseIIIholoenzymeconsistsofaheadtotaildimer(thetwosubunitsareshowninredandorange)thatformsaringcompletelysurroundingaDNAduplex(showninthecenter).PhotographkindlyprovidedbyJohnKuriyan.13.10CoordinatingsynthesisofthelaggingandleadingstrandsFigure13.19Thebsubunitof28Figure13.20EachcatalyticcoreofPolIIIsynthesizesadaughterstrand.DnaBisresponsibleforforwardmovementatthereplicationfork.13.10CoordinatingsynthesisofthelaggingandleadingstrandsFigure13.20Eachcatalyticco29Figure13.21CorepolymeraseandthebclampdissociateatcompletionofOkazakifragmentsynthesisandreassociateatthebeginning.13.10CoordinatingsynthesisofthelaggingandleadingstrandsFigure13.21Corepolymerasea30Figure13.16Tusbindstoterasymmetricallyandblocksreplicationinonlyonedirection.13.9TheprimosomeisneededtorestartreplicationFigure13.16Tusbindstoter31Figure13.24Similarfunctionsarerequiredatallreplicationforks.13.11ThereplicationapparatusofphageT4

Figure13.24Similarfunctions32Figure13.24Similarfunctionsarerequiredatallreplicationforks.13.11ThereplicationapparatusofphageT4

Figure13.24Similarfunctions33Figure13.25Theminimaloriginisdefinedbythedistancebetweentheoutsidemembersofthe13-merand9-merrepeats.

13.12Creatingthereplicationforksatanorigin

Figure13.25Theminimalorigi34Figure13.26Prepriminginvolvesformationofacomplexbysequentialassociationofproteins,leadingtotheseparationofDNAstrands.13.12CreatingthereplicationforksatanoriginFigure13.26Prepriminginvolv35Figure13.27ThecomplexatoriCcanbedetectedbyelectronmicroscopy.BothcomplexeswerevisualizedwithantibodiesagainstDnaBprotein.PhotographskindlyprovidedbyBarbaraFunnell.13.12CreatingthereplicationforksatanoriginFigure13.27Thecomplexator3613.13CommoneventsinprimingreplicationattheoriginFigure13.28TranscriptioninitiatingatPRisrequiredtoactivatetheoriginoflambdaDNA.13.13Commoneventsinpriming37Figure13.29Thelambdaoriginforreplicationcomprisestworegions.EarlyeventsarecatalyzedbyOprotein,whichbindstoaseriesof4sites;thenDNAismeltedintheadjacentA-T-richregion.AlthoughtheDNAisdrawnasastraightduplex,itisactuallybentattheorigin.13.13CommoneventsinprimingreplicationattheoriginFigure13.29Thelambdaorigin3813.13Commoneventsinprimingreplicationattheorigin13.13Commoneventsinpriming3913.14Doesmethylationattheoriginregulateinitiation?

Figure13.30ReplicationofmethylatedDNAgiveshemimethylatedDNA,whichmaintainsitsstateatGATCsitesuntiltheDammethylaserestoresthefullymethylatedcondition.13.14Doesmethylationatthe4013.14Doesmethylationattheoriginregulateinitiation?Figure13.31

Onlyfullymethylatedoriginscaninitiatereplication;hemimethylateddaughteroriginscannotbeusedagainuntiltheyhavebeenrestoredtothefullymethylatedstate.13.14Doesmethylationatthe4113.14Doesmethylationattheoriginregulateinitiation?Figure13.32Amembrane-boundinhibitorbindstohemimethylatedDNAattheorigin,andmayfunctionbypreventingthebindingofDnaA.ItisreleasedwhentheDNAisremethylated.13.14Doesmethylationatthe4213.15Licensingfactorcontrolseukaryoticrereplication

Figure13.33AnucleusinjectedintoaXenopuseggcanreplicateonlyonceunlessthenuclearmembraneispermeabilizedtoallowsubsequentreplicationcycles.13.15Licensingfactorcontrol4313.15LicensingfactorcontrolseukaryoticrereplicationFigure13.34Licensingfactorinthenucleusisinactivatedafterreplication.Anewsupplyoflicensingfactorcanenteronlywhenthenuclearmembranebreaksdownatmitosis.13.15Licensingfactorcontrol4413.15LicensingfactorcontrolseukaryoticrereplicationFigure13.35Proteinsattheorigincontrolsusceptibilitytoinitiation.13.15Licensingfactorcontrol4513.16SummaryDNAsynthesisoccursbysemidiscontinuousreplication,inwhichtheleadingstrandofDNAgrowing53isextendedcontinuously,butthelaggingstrandthatgrowsoverallintheopposite35directionismadeasshortOkazakifragments,eachsynthesized53.TheleadingstrandandeachOkazakifragmentofthelaggingstrandinitiatewithanRNAprimerthatisextendedbyDNApolymerase.BacteriaandeukaryoteseachpossessmorethanoneDNApolymeraseactivity.DNApolymeraseIIIsynthesizesbothlaggingandleadingstrandsinE.coli.ManyproteinsarerequiredforDNApolymeraseIIIactionandseveralconstitutepartofthereplisomewithinwhichitfunctions.13.16SummaryDNAsynthesisocc46Figure12.16Therollingcirclegeneratesamultimericsingle-strandedtail.13.7Single-strandedDNAisneededforreplicationFigure12.16Therollingcircl47Primer

isashortsequence(oftenofRNA)thatispairedwithonestrandofDNAandprovidesafree3′-OHendatwhichaDNApolymerasestartssynthesisofadeoxyribonucleotidechain.13.8PrimingisrequiredtostartDNAsynthesisPrimerisashortsequence(of48Figure13.11ADNApolymeraserequiresa3-OHendtoinitiatereplication.13.8PrimingisrequiredtostartDNAsynthesisFigure13.11ADNApolymerase49Figure12.34ReplicationofColE1DNAisinitiatedbycleavingtheprimerRNAtogeneratea3-OHend.Theprimerformsapersistenthybridintheoriginregion.13.8PrimingisrequiredtostartDNAsynthesisFigure12.34ReplicationofCo50Figure12.16Therollingcirclegeneratesamultimericsingle-strandedtail.13.8PrimingisrequiredtostartDNAsynthesisFigure12.16Therollingcircl51Figure13.5Nicktranslationreplacespartofapre-existingstrandofduplexDNAwithnewlysynthesizedmaterial.13.8PrimingisrequiredtostartDNAsynthesisFigure13.5Nicktranslationr52Figure12.15Adenovirusterminalproteinbindstothe5endofDNAandprovidesaC-OHendtoprimesynthesisofanewDNAstrand.13.8PrimingisrequiredtostartDNAsynthesisFigure12.15Adenovirustermin53PrimosomedescribesthecomplexofproteinsinvolvedintheprimingactionthatinitiatesreplicationonfX-typeorigins.Itisalsoinvolvedinrestartingstalledreplicationforks.13.9TheprimosomeisneededtorestartreplicationPrimosomedescribesthecomple54Figure13.14ReplicationishaltedbyadamagedbaseornickinDNA.13.9TheprimosomeisneededtorestartreplicationFigure13.14Replicationisha55Figure14.35AnE.coliretrievalsystemusesanormalstrandofDNAtoreplacethegapleftinanewlysynthesizedstrandoppositeasiteofunrepaireddamage.

13.9TheprimosomeisneededtorestartreplicationFigure14.35AnE.coliretrie56Figure13.15TheprimosomeisrequiredtorestartastalledreplicationforkaftertheDNAhasbeenrepaired.13.9TheprimosomeisneededtorestartreplicationFigure13.15Theprimosomeis57Figure12.7ReplicationterminiinE.coliarelocatedbeyondthepointatwhichthereplicationforksactuallymeet.13.9TheprimosomeisneededtorestartreplicationFigure12.7Replicationtermin58Figure13.17Leadingandlaggingstrandpolymerasesmoveapart.13.10CoordinatingsynthesisofthelaggingandleadingstrandsFigure13.17Leadingandlaggi59Figure13.26Prepriminginvolvesformationofacomplexbysequentialassociationofproteins,leadingtotheseparationofDNAstrands.13.13Commoneventsinprimingreplicationattheorigin

Figure13.26Prepriminginvolv6013.14Doesmethylationattheoriginregulateinitiation?Figure12.26AttachmentofbacterialDNAtothemembranecouldprovideamechanismforsegregation.13.14Doesmethylationatthe6113.15LicensingfactorcontrolseukaryoticrereplicationFigure1.10ReplicationofDNAissemiconservative.13.15Licensingfactorcontrol6213.15LicensingfactorcontrolseukaryoticrereplicationFigure12.10AnARSextendsfor~50bpandincludesaconsensussequence(A)andadditionalelements(B1-B3).13.15Licensingfactorcontrol6313.16SummaryDNAsynthesisoccursbysemidiscontinuousreplication,inwhichtheleadingstrandofDNAgrowing53isextendedcontinuously,butthelaggingstrandthatgrowsoverallintheopposite35directionismadeasshortOkazakifragments,eachsynthesized53.TheleadingstrandandeachOkazakifragmentofthelaggingstrandinitiatewithanRNAprimerthatisextendedbyDNApolymerase.BacteriaandeukaryoteseachpossessmorethanoneDNApolymeraseactivity.DNApolymeraseIIIsynthesizesbothlaggingandleadingstrandsinE.coli.ManyproteinsarerequiredforDNApolymeraseIIIactionandseveralconstitutepartofthereplisomewithinwhichitfunctions.13.16SummaryDNAsynthesisocc64ThereplisomecontainsanasymmetricdimerofDNApolymeraseIII;eachnewDNAstrandissynthesizedbyadifferentcorecomplexcontainingacatalytic()subunit.Processivityofthecorecomplexismaintainedbytheclamp,whichformsaringroundDNA.Theloopingmodelforthereplicationforkproposesthat,asonehalfofthedimeradvancestosynthesizetheleadingstrand,theotherhalfofthedimerpullsDNAthroughasasingleloopthatprovidesthetemplateforthelaggingstrand.ThetransitionfromcompletionofoneOkazakifragmenttothestartofthenextrequiresthelaggingstrandcatalyticsubunittodissociatefromDNAandthentoreattachtoaclampattheprimingsiteforthenextOkazakifragment.Thereplisomecontainsanasym65DnaBprovidesthehelicaseactivityatareplicationfork;thisdependsonATPcleavage.DnaBmayfunctionbyitselfinoriCrepliconstoprovideprimosomeactivitybyinteractingperiodicallywithDnaG,whichprovidestheprimasethatsynthesizesRNA.DnaBprovidesthehelicaseact66PhageT4codesforasizeablereplicationapparatus,consistingof7proteins:DNApolymerase,helicase,single-strandbindingprotein,primingactivities,andaccessoryproteins.Similarfunctionsarerequiredinotherreplicationsystems,includingaHeLacellsystemthatreplicatesSV40DNA.Differentenzymes,DNApolymeraseandDNApolymerase,initiateandelongatethenewstrandsofDNA.PhageT4codesforasizeable67Thecommonmodeoforiginactivationinvolvesaninitiallimitedmeltingofthedoublehelix,followedbymoregeneralunwindingtocreatesinglestrands.SeveralproteinsactsequentiallyattheE.coliorigin.DnaAbindstoaseriesof9bprepeatsand13bprepeats,forminganaggregateof2040monomerswithDNAinwhichthe13bprepeatsaremelted.ThehelicaseactivityofDnaB,togetherwithDnaC,unwindsDNAfurther.Similareventsoccuratthelambdaorigin,wherephageproteinsOandParethecounterpartsofbacterialproteinsDnaAandDnaC,respectively.InSV40replication,severaloftheseactivitiesarecombinedinthefunctionsofTantigen.Thecommonmodeoforiginacti68TheXprimingeventalsorequiresDnaB,DnaC,andDnaT.PriAisthecomponentthatdefinestheprimosomeassemblysite(pas)forXreplicons;itdisplacesSSBfromDNAinanactionthatinvolvescleavageofATP.PriBandPriCareadditionalcomponentsoftheprimosome.TheXprimingeventalsorequi69SeveralsitesthataremethylatedbytheDammethylasearepresentintheE.coliorigin,includingthoseofthe13-merbindingsitesforDnaA.Theoriginremainshemimethylatedandisinasequesteredstatefor~10minutesfollowinginitiationofareplicationcycle.Duringthisperioditisassociatedwiththemembrane,andreinitiationofreplicationisrepressed.Severalsitesthataremethyla70Aftercelldivision,nucleiofeukaryoticcellshavealicensingfactorthatisneededtoinitiatereplication.Itsdestructionafterinitiationofreplicationpreventsfurtherreplicationcyclesfromoccurringinyeast.Licensingfactorcannotbeimportedintothenucleusfromthecytoplasm,andcanbereplacedonlywhenthenuclearmembranebreaksdownduringmitosis.Aftercelldivision,nucleiof71Chapter13DNAreplicationChapter13DNAreplication7213.1Introduction

13.2DNApolymerasesaretheenzymesthatmakeDNA

13.3DNAsynthesisissemidiscontinuous

13.4Coordinatingsynthesisofthelaggingandleading

strands

13.5ThereplicationapparatusofphageT4

13.6Creatingthereplicationforksatanorigin

13.7Commoneventsinprimingreplicationattheorigin

13.8Doesmethylationattheoriginregulateinitiation?

13.9Licensingfactorcontrolseukaryoticrereplication

13.1Introduction

13.2DNApo73ReplisomeisthemultiproteinstructurethatassemblesatthebacterialreplicatingforktoundertakesynthesisofDNA.ContainsDNApolymeraseandotherenzymes.

13.1IntroductionReplisomeisthemultiprotein74DNApolymerasesareenzymesthatsynthesizeadaughterstrand(s)ofDNA(underdirectionfromaDNAtemplate).Maybeinvolvedinrepairorreplication.

DNAreplicaseisaDNA-synthesizingenzymerequiredspecificallyforreplication.

RepairofdamagedDNAcantakeplacebyrepairsynthesis,whenastrandthathasbeendamagedisexcisedandreplacedbythesynthesisofanewstretch.Itcanalsotakeplacebyrecombinationreactions,whentheduplexregioncontainingthedamagedisreplacedbyanundamagedregionfromanothercopyofthegenome.

ReplicationofduplexDNAtakesplacebysynthesisoftwonewstrandsthatarecomplementarytotheparentalstrands.Theparentalduplexisreplacedbytwoidenticaldaughterduplexes,eachofwhichhasoneparentalstrandandonenewlysynthesizedstrand.Itiscalledsemiconservativebecausetheconservedunitsarethesinglestrandsoftheparentalduplex.13.2DNApolymerasesaretheenzymesthatmakeDNA

DNApolymerasesareenzymesth75Figure13.1SemiconservativereplicationsynthesizestwonewstrandsofDNA.13.2DNApolymerasesaretheenzymesthatmakeDNA

Figure13.1Semiconservativer76Figure13.2RepairsynthesisreplacesadamagedstrandofDNA.13.2DNApolymerasesaretheenzymesthatmakeDNAFigure13.2Repairsynthesisr77Figure13.3DNAsynthesisoccursbyaddingnucleotidestothe3-OHendofthegrowingchain,sothatthenewchainissynthesizedinthe5-3direction.TheprecursorforDNAsynthesisisanucleosidetriphosphate,whichlosestheterminaltwophosphategroupsinthereaction.13.2DNApolymerasesaretheenzymesthatmakeDNAFigure13.3DNAsynthesisoccu78Figure13.4OnlyoneDNApolymeraseisthereplicase.TheothersparticipateinrepairofdamagedDNA.13.2DNApolymerasesaretheenzymesthatmakeDNAFigure13.4OnlyoneDNApolym79

NicktranslationdescribestheabilityofE.coliDNApolymeraseItouseanickasastartingpointfromwhichonestrandofaduplexDNAcanbedegradedandreplacedbyresynthesisofnewmaterial;isusedtointroduceradioactivelylabelednucleotidesintoDNAinvitro.

13.3DNApolymeraseshavevariousnucleaseactivities

Nicktranslationdescribesth80Proofreadingreferstoanymechanismforcorrectingerrorsinproteinornucleicacidsynthesisthatinvolvesscrutinyofindividualunitsaftertheyhavebeenaddedtothechain.

13.4DNApolymerasescontrolthefidelityofreplicationProofreadingreferstoanymec81Figure13.6BacterialDNApolymerasesscrutinizethebasepairattheendofthegrowingchainandexcisethenucleotideaddedinthecaseofamisfit.13.4DNApolymerasescontrolthefidelityofreplicationFigure13.6BacterialDNApoly82Figure13.5Nicktranslationreplacespartofapre-existingstrandofduplexDNAwithnewlysynthesizedmaterial.13.3DNApolymeraseshavevariousnucleaseactivitiesFigure13.5Nicktranslationr8313.3DNApolymeraseshavevariousnucleaseactivities13.3DNApolymeraseshavevari8413.3DNApolymeraseshavevariousnucleaseactivities13.3DNApolymeraseshavevari85Figure13.12Thereareseveralmethodsforprovidingthefree3-OHendthatDNApolymerasesrequiretoinitiateDNAsynthesis.13.8PrimingisrequiredtostartDNAsynthesisFigure13.12Thereareseveral86Figure13.7CrystalstructureofphageT7DNApolymerasehasarighthandstructure.DNAliesacrossthepalmandisheldbythefingersandthumb.PhotographkindlyprovidedbyCharlesRichardsonandTomEllenberger.13.5SomeDNApolymeraseshaveacommonstructureFigure13.7Crystalstructure87Figure13.8ThecatalyticdomainofaDNApolymerasehasaDNA-bindingcleftcreatedbythreesubdomains.Theactivesiteisinthepalm.Proofreadingisprovidedbyaseparateactivesiteinanexonucleasedomain.13.5SomeDNApolymeraseshaveacommonstructureFigure13.8Thecatalyticdoma88LaggingstrandofDNAmustgrowoverallinthe3′-5′directionandissynthesizeddiscontinuouslyintheformofshortfragments(5′-3′)thatarelaterconnectedcovalently.

LeadingstrandofDNAissynthesizedcontinuouslyinthe5′-3′direction.

Okazakifragmentsaretheshortstretchesof1000-2000basesproducedduringdiscontinuousreplication;theyarelaterjoinedintoacovalentlyintactstrand.

Semidiscontinuousreplicationismodeinwhichonenewstrandissynthesizedcontinuouslywhiletheotherissynthesizeddiscontinuously.13.6DNAsynthesisissemidiscontinuous

LaggingstrandofDNAmustgro89Figure13.9Theleadingstrandissynthesizedcontinuouslywhilethelaggingstrandissynthesizeddiscontinuously.13.6DNAsynthesisissemidiscontinuousFigure13.9Theleadingstrand90SSB

isthesingle-strandproteinofE.coli,aproteinthatbindstosingle-strandedDNA.13.7Single-strandedDNAisneededforreplicationSSBisthesingle-strandprote91Figure13.22SynthesisofOkazakifragmentsrequirespriming,extension,removalofRNA,gapfilling,andnickligation.13.10CoordinatingsynthesisofthelaggingandleadingstrandsFigure13.22SynthesisofOkaz92Figure13.23DNAligasesealsnicksbetweenadjacentnucleotidesbyemployinganenzyme-AMPintermediate.13.10CoordinatingsynthesisofthelaggingandleadingstrandsFigure13.23DNAligaseseals93Figure13.10fX174DNAcanbeseparatedintosinglestrandsbythecombinedeffectsof3functions:nickingwithAprotein,unwindingbyRep,andsingle-strandstabilizationbySSB.

13.7Single-strandedDNAisneededforreplicationFigure13.10fX174DNAcanbe94Figure13.13Initiationrequiresseveralenzymaticactivities,includinghelicases,single-strandbindingproteins,andsynthesisoftheprimer.13.8PrimingisrequiredtostartDNAsynthesisFigure13.13Initiationrequir95Figure13.13Initiationrequiresseveralenzymaticactivities,includinghelicases,single-strandbindingproteins,andsynthesisoftheprimer.13.8PrimingisrequiredtostartDNAsynthesisFigure13.13Initiationrequir96Figure13.12Leadingandlaggingstrandpolymerasesmoveapart.13.8PrimingisrequiredtostartDNAsynthesisFigure13.12Leadingandlaggi97Figure13.18DNApolymeraseIIIholoenzymeassemblesinstages,generatinganenzymecomplexthatsynthesizestheDNAofbothnewstrands.13.10CoordinatingsynthesisofthelaggingandleadingstrandsFigure13.18DNApolymeraseII98Figure13.19ThebsubunitofDNApolymeraseIIIholoenzymeconsistsofaheadtotaildimer(thetwosubunitsareshowninredandorange)thatformsaringcompletelysurroundingaDNAduplex(showninthecenter).Photographkindlyprovidedby

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