基因的分子生物学DNAReplication

基因的分子生物学DNAReplication第1页/共29页2007-04-2724.14.1IntroductionThereplisome

isthemultiproteinstructurethatassemblesatthebacterialreplicatingforktoundertakesynthesisofDNA.ItcontainsDNApolymeraseandotherenzymes.Adnamutant

ofbacteriaistemperature-sensitive;itcannotsynthesizeDNAat42°C,butcandosoat37°C.Aquick-stopmutant

isatypeofDNAreplicationtemperature-sensitivemutant(dna)inE.colithatimmediatelystopsDNAreplicationwhenthetemperatureisincreasedto42°C.Aslow-stopmutant

isatypeofDNAreplicationtemperature-sensitivemutantinE.colithatcanfinisharoundofreplicationattheunpermissivetemperature,butcannotstartanother.Invitrocomplementation

isafunctionalassayusedtoidentifycomponentsofaprocess.Thereactionisreconstructedusingextractsfromamutantcell.Fractionsfromwild-typecellsarethentestedforrestorationofactivity.第2页/共29页2007-04-273ReplicationofduplexDNAisacomplexendeavorinvolvingaconglomerateofenzymeactivities.Differentactivitiesareinvolvedinthestagesofinitiation,elongation,andtermination.·Initiationinvolvesrecognitionofanoriginbyacomplexofproteins.BeforeDNAsynthesisbegins,theparentalstrandsmustbeseparatedand(transiently)stabilizedinthesingle-strandedstate.Thensynthesisofdaughterstrandscanbeinitiatedatthereplicationfork.·Elongationisundertakenbyanothercomplexofproteins.Thereplisome

existsonlyasaproteincomplexassociatedwiththeparticularstructurethatDNAtakesatthereplicationfork.Itdoesnotexistasanindependentunit(forexample,analogoustotheribosome).AsthereplisomemovesalongDNA,theparentalstrandsunwindanddaughterstrandsaresynthesized.·Attheendofthereplicon,joiningand/orterminationreactionsarenecessary.Followingtermination,theduplicatechromosomesmustbeseparatedfromoneanother,whichrequiresmanipulationofhigher-orderDNAstructure.第3页/共29页2007-04-274Replication

ofduplexDNAtakesplacebysynthesisoftwonewstrandsthatarecomplementarytotheparentalstrands.Theparentalduplexisreplacedbytwoidenticaldaughterduplexes,eachofwhichhasoneparentalstrandandonenewlysynthesizedstrand.Replicationiscalledsemiconservativebecausetheconservedunitsarethesinglestrandsoftheparentalduplex.Repair

ofdamagedDNAcantakeplacebyrepairsynthesis,whenastrandthathasbeendamagedisexcisedandreplacedbythesynthesisofanewstretch.Itcanalsotakeplacebyrecombinationreactions,whentheduplexregioncontainingthedamagedisreplacedbyanundamagedregionfromanothercopyofthegenome.ADNApolymerase

isanenzymethatsynthesizesadaughterstrand(s)ofDNA(underdirectionfromaDNAtemplate).Anyparticularenzymemaybeinvolvedinrepairorreplication(orboth).ADNAreplicase

isaDNA-synthesizingenzymerequiredspecificallyforreplication.Figure14.1

SemiconservativereplicationsynthesizestwonewstrandsofDNA.Figure14.2

RepairsynthesisreplacesashortstretchofonestrandofDNAcontainingadamagedbase.4.14.2DNApolymerasesaretheenzymesthatmakeDNA第4页/共29页2007-04-275Figure14.3

DNAsynthesisoccursbyaddingnucleotidestothe3’-OHendofthegrowingchain,sothatthenewchaingrowsinthe5’→3’direction.TheprecursorforDNAsynthesisisanucleosidetriphosphate,whichlosestheterminaltwophosphategroupsinthereaction.Figure14.4

OnlyoneDNApolymeraseisthereplicase.TheothersparticipateinrepairofdamagedDNA,restartingstalledreplicationforks,orbypassingdamage(tanslesion)inDNA.第5页/共29页2007-04-276Nicktranslation

describestheabilityofE.coliDNApolymeraseItouseanickasastartingpointfromwhichonestrandofaduplexDNAcanbedegradedandreplacedbyresynthesisofnewmaterial;isusedtointroduceradioactivelylabelednucleotidesintoDNAinvitro.·DNApolymeraseIhasaunique5’–3’exonucleaseactivitythatcanbecombinedwithDNAsynthesistoperformnicktranslation.Figure14.5

Nicktranslationreplacespartofapre-existingstrandofduplexDNAwithnewlysynthesizedmaterial.4.14.3DNApolymeraseshavevariousnucleaseactivities第6页/共29页2007-04-277Processivity

describestheabilityofanenzymetoperformmultiplecatalyticcycleswithasingletemplateinsteadofdissociatingaftereachcycle.Proofreading

referstoanymechanismforcorrectingerrorsinproteinornucleicacidsynthesisthatinvolvesscrutinyofindividualunitsaftertheyhavebeenaddedtothechain.·DNApolymerasesoftenhavea3’–5’exonucleaseactivitythatisusedtoexciseincorrectlypairedbases.·Thefidelityofreplicationisimprovedbyproofreadingbyafactorof~100.WecandividetheerrorsthatDNApolymerasemakesduringreplicationintotwoclasses.·Frameshifts·SubstitutionsFigure14.6

BacterialDNApolymerasesscrutinizethebasepairattheendofthegrowingchainandexcisethenucleotideaddedinthecaseofamisfit.4.14.4DNApolymerasescontrolthefidelityofreplication第7页/共29页2007-04-278·ManyDNApolymeraseshavealargecleftcomposedofthreedomainsthatresembleahand.·DNAliesacrossthe"palm"inagroovecreatedbythe"fingers"and"thumb".Figure14.7

ThecommonorganizationofDNApolymeraseshasapalmthatcontainsthecatalyticsite,fingersthatpositionthetemplate,athumbthatbindsDNAandisimportantinprocessivity,anexonucleasedomainwithitsownactivesite,andanN-terminaldomain.Figure14.8

ThecrystalstructureofphageT7DNApolymeraseshowsthatthetemplatestrandtakesasharpturninordertobeexposedtotheincomingnucleotide.4.14.5DNApolymeraseshaveacommonstructure第8页/共29页2007-04-279Theleadingstrand

ofDNAissynthesizedcontinuouslyinthe5’-3’direction.Thelaggingstrand

ofDNAmustgrowoverallinthe3’-5’directionandissynthesizeddiscontinuouslyintheformofshortfragments(5’-3’)thatarelaterconnectedcovalently.Okazakifragments

aretheshortstretchesof1000-2000basesproducedduringdiscontinuousreplication;theyarelaterjoinedintoacovalentlyintactstrand.Semidiscontinuousreplication

ismodeinwhichonenewstrandissynthesizedcontinuouslywhiletheotherissynthesizeddiscontinuously.·TheDNAreplicaseadvancescontinuouslywhenitsynthesizestheleadingstrand(5’–3’),butsynthesizesthelaggingstrandbymakingshortfragmentsthataresubsequentlyjoinedtogether.·Ontheleadingstrand

DNAsynthesiscanproceedcontinuouslyinthe5’to3’directionastheparentalduplexisunwound.·Onthelaggingstrand

astretchofsingle-strandedparentalDNAmustbeexposed,andthenasegmentissynthesizedinthereversedirection(relativetoforkmovement).Aseriesofthesefragmentsaresynthesized,each5’–3’;thentheyarejoinedtogethertocreateanintactlaggingstrand.Figure14.9

Theleadingstrandissynthesizedcontinuouslywhilethelaggingstrandissynthesizeddiscontinuously.sedimentingintherangeof7-11S,correspondingto~1000-2000bases.4.14.6DNAsynthesisissemidiscontinuous第9页/共29页2007-04-2710DNApolymeraseI:asinglepolypeptideof103kD.Thechaincanbecleavedintotwopartsbyproteolytictreatment:1,TheC-terminaliscalledtheKlenowfragment(68kD).Itisusedinsyntheticreactionsinvitro.Itcontainsthepolymeraseandthe3’–5’exonucleaseactivities.Theactivesitesare~30Åapartintheprotein,indicatingthatthereisspatialseparationbetweenaddingabaseandremovingone.2,Thesmallfragment(35kD)possessesa5’–3’exonucleolyticactivity,whichexcisessmallgroupsofnucleotides,upto~10basesatatime.Thisactivityiscoordinatedwiththesynthetic/proofreadingactivity.第10页/共29页2007-04-2711Ahelicase

isanenzymethatusesenergyprovidedbyATPhydrolysistoseparatethestrandsofanucleicacidduplex.Thesingle-strandbindingprotein(SSB)attachestosingle-strandedDNA,therebypreventingtheDNAfromformingaduplex.Figure14.10

AhexamerichelicasemovesalongonestrandofDNA.Itprobablychangesconformationwhenitbindstotheduplex,usesATPhydrolysistoseparatethestrands,andthenreturnstotheconformationithaswhenboundonlytoasinglestrand.Figure14.11

ФX174DNAcanbeseparatedintosinglestrandsbythecombinedeffectsof3functions:nickingwithAprotein,unwindingbyRep,andsingle-strandstabilizationbySSB.4.14.7TheФX

modelsystemshowshowsingle-strandedDNAisgeneratedforreplication第11页/共29页2007-04-2712Aprimer

isashortsequence(oftenofRNA)thatispairedwithonestrandofDNAandprovidesafree3’-OHendatwhichaDNApolymerasestartssynthesisofadeoxyribonucleotidechain.Theprimase

isatypeofRNApolymerasethatsynthesizesshortsegmentsofRNAthatwillbeusedasprimersforDNAreplication.Figure14.12

ADNApolymeraserequiresa3’-OHprimingendtoinitiatereplication.4.14.8PrimingisrequiredtostartDNAsynthesis第12页/共29页2007-04-2713Figure14.13

Thereareseveralmethodsforprovidingthefree3’-OHendthatDNApolymerasesrequiretoinitiateDNAsynthesis.Figure14.14

Initiationrequiresseveralenzymaticactivities,includinghelicases,single-strandbindingproteins,andsynthesisoftheprimer.第13页/共29页2007-04-2714Theclamploader

isa5subunitproteincomplexwhichisresponsibleforloadingthebclampontoDNAatthereplicationfork.·TheE.colireplicaseDNApolymeraseIIIisa900kDcomplexwithadimericstructure.·Eachmonomericunithasacatalyticcore,adimerizationsubunit,andaprocessivitycomponent.Figure14.17

DNApolymeraseIIIholoenzymeassemblesinstages,generatinganenzymecomplexthatsynthesizestheDNAofbothnewstrands.4.14.10DNApolymeraseholoenzymehas3subcomplexes第14页/共29页2007-04-27154.14.11TheclampcontrolsassociationofcoreenzymewithDNAFigure14.18

TheβsubunitofDNApolymeraseIIIholoenzymeconsistsofaheadtotaildimer(thetwosubunitsareshowninredandorange)thatformsaringcompletelysurroundingaDNAduplex(showninthecenter).Figure14.19

ThehelicasecreatingthereplicationforkisconnectedtotwoDNApolymerasecatalyticsubunits,eachofwhichisheldontoDNAbyaslidingclamp.Thepolymerasethatsynthesizestheleadingstrandmovescontinuously.ThepolymerasethatsynthesizesthelaggingstranddissociatesattheendofanOkazakifragmentandthenreassociateswithaprimerinthesingle-strandedtemplatelooptosynthesizethenextfragment.第15页/共29页2007-04-2716Figure14.20

EachcatalyticcoreofPolIIIsynthesizesadaughterstrand.DnaB(helicase)isresponsibleforforwardmovementatthereplicationfork.Figure14.21

CorepolymeraseandtheβclampdissociateatcompletionofOkazakifragmentsynthesisandreassociateatthebeginning.第16页/共29页2007-04-2717DNAligase

makesabondbetweenanadjacent3’-OHand5’-phosphateendwherethereisanickinonestrandofduplexDNA.Figure14.22

SynthesisofOkazakifragmentsrequirespriming,extension,removalofRNA,gapfilling,andnickligation.Figure14.23

DNAligasesealsnicksbetweenadjacentnucleotidesbyemployinganenzyme-AMPintermediate.4.14.12Okazakifragmentsarelinkedbyligase第17页/共29页2007-04-2718·Areplicationforkhas1complexofDNApolymeraseα/primaseand2complexesofDNApolymeraseδand/orε.Figure14.24

EukaryoticcellshavemanyDNApolymerases.Thereplicativeenzymesoperatewithhighfidelity.Exceptforthebenzyme,therepairenzymesallhavelowfidelity.Replicativeenzymeshavelargestructures,withseparatesubunitsfordifferentactivities.Repairenzymeshavemuchsimplerstructures.EachofthethreenuclearDNAreplicaseshasadifferentfunction:·DNApolymeraseαinitiatesthesynthesisofnewstrands.·DNApolymeraseδelongatestheleadingstrand.·DNApolymeraseε

maybeinvolvedinlaggingstrandsynthesis,butalsohasotherroles.4.14.13SeparateeukaryoticDNApolymerasesundertakeinitiationandelongation第18页/共29页2007-04-2719·PhageT4providesitsownreplicationapparatus,whichconsistsofDNApolymerase,thegene32SSB,ahelicase,aprimase,andaccessoryproteinsthatincreasespeedandprocessivity.Figure14.25

Similarfunctionsarerequiredatallreplicationforks.4.14.14PhageT4providesitsownreplicationapparatus第19页/共29页2007-04-27204.14.15CreatingthereplicationforksatanoriginFigure14.26

Theminimaloriginisdefinedbythedistancebetweentheoutsidemembersofthe13-merand9-merrepeats.Figure14.27

Prepriminginvolvesformationofacomplexbysequentialassociationofproteins,leadingtotheseparationofDNAstrands.第20页/共29页2007-04-2721·ThegeneralprincipleofbacterialinitiationisthattheoriginisinitiallyrecognizedbyaproteinthatformsalargecomplexwithDNA.·AshortregionofA·T-richDNAismelted.·DnaBisboundtothecomplexandcreatesthereplicationfork.Figure14.29

TranscriptioninitiatingatPRisrequiredtoactivatetheoriginoflambdaDNA.Figure14.30

Thelambdaoriginforreplicationcomprisestworegions.EarlyeventsarecatalyzedbyOprotein,whichbindstoaseriesof4sites;thenDNAismeltedintheadjacentA-T-richregion.AlthoughtheDNAisdrawnasastraightduplex,itisactuallybentattheorigin.4.14.16Commoneventsinprimingreplicationattheorigin第21页/共29页2007-04-2722Theprimosome

describesthecomplexofproteinsinvolvedintheprimingactionthatinitiatesreplicationonФX-typeorigins.Itisalsoinvolvedinrestartingstalledreplicationforks.Figure14.31

ReplicationishaltedbyadamagedbaseornickinDNA.4.14.17Theprimosomeisneededtorestartreplication第22页/共29页2007-04-2723Figure14.33

TheprimosomeisrequiredtorestartastalledreplicationforkaftertheDNAhasbeenrepaired.Figure14.34

Tusbindstoterasymmetricallyandblocksreplicationinonlyonedirection.第23页/共29页2007-04-2724Hemimethylated

DNAismethylatedononestrandofatargetsequencethathasacytosineoneachstrand.·oriCcontains11repeatsthataremethylatedonadenineonbothstrands.·ReplicationgenerateshemimethylatedDNA,whichcannotinitiatereplication.·Thereisa13mindelaybeforetherepeatsareremethylated.Figure14.35

ReplicationofmethylatedDNAgiveshemimethylatedDNA,whichmaintainsitsstateatGATCsitesuntiltheDammethylaserestoresthefullymethylatedcondition.Figure14.36

Onlyfullymethylatedoriginscaninitiatereplication;hemimethylateddaughteroriginscannotbeusedagainuntiltheyhavebeenrestoredtothefullymethylatedstate.4.14.18Doesmethylationattheoriginregulateinitiation?第24页/共29页2007-04-2725·SeqAbindstohemimethylatedDNAandisrequiredfordelayingrereplication.·SeqAmayinteractwithDnaA.·Whiletheoriginsarehemimethylated,theybindtothecellmembrane,andmaybeunavailabletomethylases.·Thenatureoftheconnectionbetweentheoriginandthemembraneisstillunclear.Figure14.37

Amembrane-boundinhibitorbindstohemim