Gene-X-ppt(Gene10-基因十)--Chapter11

Chapter11TheReplicon11.1Introduction•replicon–AunitofthegenomeinwhichDNAisreplicated.Eachcontainsanoriginforinitiationofreplication.•origin–AsequenceofDNAatwhichreplicationisinitiated.•terminus–AsegmentofDNAatwhichreplicationends.11.1Introduction•single-copyreplicationcontrol–Acontrolsysteminwhichthereisonlyonecopyofarepliconperunitbacterium.–Thebacterialchromosomeandsomeplasmidshavethistypeofregulation.•multicopyreplicationcontrol–Occurswhenthecontrolsystemallowstheplasmidtoexistinmorethanonecopyperindividualbacterialcell.11.2RepliconsCanBeLinearorCircular•semiconservativereplication–Replicationaccomplishedbyseparationofthestrandsofaparentalduplex,witheachstrandthenactingasatemplateforsynthesisofacomplementarystrand.•AreplicatedregionappearsasabubblewithinnonreplicatedDNA.•AreplicationforkisinitiatedattheoriginandthenmovessequentiallyalongDNA.FIGURE02:ReplicatedDNAisseenasareplicationbubbleflankedbynonreplicatedDNA11.2RepliconsCanBeLinearorCircular•Replicationisunidirectionalwhenasinglereplicationforkiscreatedatanorigin.•Replicationisbidirectionalwhenanorigincreatestworeplicationforksthatmoveinoppositedirections.FIGURE03:Repliconsmaybeunidirectionalorbidirectional,dependingonwhetheroneortworeplicationforksareformedattheorigin11.3OriginsCanBeMappedbyAutoradiographyandElectrophoresis•ReplicationforkmovementcanbedetectedbylabelingofnewlysynthesizedDNAwithpulsesofeitherradioactiveorfluorescentnucleotides.FIGURE06:InitialreplicationisdetectedbyincorporationofgreenlabelPhotoreproducedfromK.Marheineke,etal.,VisualizationofbidirectionalinitiationofchromosomalDNAreplicationinahumancellfreesystem,NucleicAcidsRes.,vol.33(21),pp.6931-3941,bypermissionofOxfordUniversityPress.PhotocourtesyofKathrinMarheinekeandTorstenKrude.11.3OriginsCanBeMappedbyAutoradiographyandElectrophoresis•ReplicationforkscreateY-shapedstructuresthatchangetheelectrophoreticmigrationofDNAmolecules.FIGURE07:Thepositionoftheoriginandthenumberofreplicatingforksdeterminetheshapeofareplicatingrestrictionfragment11.4TheBacterialGenomeIs(Usually)aSingleCircularReplicon•Bacterialrepliconsareusuallycirclesthatreplicatebidirectionallyfromasingleorigin.•TheoriginofE.coli,oriC,is245bpinlength.FIGURE08:ReplicationrequiresDNAsynthesisandchromosomeseparation11.4TheBacterialGenomeIs(Usually)aSingleCircularReplicon•Thetworeplicationforksusuallymeethalfwayaroundthecircle,buttherearetersitesthatcauseterminationifthereplicationforksgotoofar.FIGURE09:Forksusuallymeetbeforeterminating11.5MethylationoftheBacterialOriginRegulatesInitiation•oriCcontainsbindingsitesforDnaA–dnaA-boxes.•oriCalsocontainselevenGATC/CTAGrepeatsthataremethylatedonadenineonbothstrands.FIGURE10:TheE.colioriginofreplication,oriC,containsmultiplebindingsitesfortheDnaAinitiatorprotein11.5MethylationoftheBacterialOriginRegulatesInitiation•ReplicationgenerateshemimethylatedDNA,whichcannotinitiatereplication.•Thereisa13-minutedelaybeforetheGATC/CTAGrepeatsareremethylated.FIGURE11:Onlyfullymethylatedoriginscaninitiatereplication11.5MethylationoftheBacterialOriginRegulatesInitiation•SeqAbindstohemimethylatedDNAandisrequiredfordelayingrereplication.•SeqAmayinteractwithDnaA.•Astheoriginsarehemimethylatedtheybindtothecellmembraneandmaybeunavailabletomethylases.•Thenatureoftheconnectionbetweentheoriginandthemembraneisstillunclear.11.6ArchaealChromosomesCanContainMultipleReplicons•SomeArchaeahavemultiplereplicationorigins.•Theseoriginsareboundbyhomologsofeukaryoticreplicationinitiationfactors.•OriginbindingintroducesunderwindingofDNA.StructurefromProteinDataBank2QBY.E.LDueber,etal.,Science317(2007):1210-1213.FIGURE12:ThecrystalstructureofaheterodimerofarchaealOrc1/Cdc6replicationinitiatorproteinsboundtoanoriginofreplication11.7EachEukaryoticChromosomeContainsManyReplicons•Achromosomeisdividedintomanyreplicons.•TheprogressionintoSphaseistightlycontrolled.•checkpoint–Abiochemicalcontrolmechanismthatpreventsthecellfromprogressingfromonestagetothenextunlessspecificgoalsandrequirementshavebeenmet.FIGURE13:Theeukaryotecellcycle11.7EachEukaryoticChromosomeContainsManyReplicons•Eukaryoticrepliconsare40to100kbinlength.•IndividualrepliconsareactivatedatcharacteristictimesduringSphase.•Regionalactivationpatternssuggestthatrepliconsnearoneanotherareactivatedatthesametime.FIGURE14:Aeukaryoticchromosomecontainsmultipleoriginsofreplicationthatultimatelymergeduringreplication11.8ReplicationOriginsCanBeIsolatedinYeast•OriginsinS.cerevisiaeareshortA-Tsequencesthathaveanessential11bpsequence.•TheORCisacomplexofsixproteinsthatbindstoanARS.FIGURE16:AnARSextendsfor~50bpandincludesaconsensussequence(A)andadditionalelements(B1–B3)11.8ReplicationOriginsCanBeIsolatedinYeast•Adomain–Theconserved11bpsequenceofA-TbasepairsintheyeastARSelementthatcomprisesthereplicationorigin.•RelatedORCcomplexesarefoundinmulticellulareukaryotes.11.9LicensingFactorControlsEukaryoticRereplication•Licensingfactorisnecessaryforinitiationofreplicationateachorigin.•Licensingfactorispresentinthenucleuspriortoreplication,butisremoved,inactivated,ordestroyedbyrepl