chapter-28-DNA-replication

PartIVInformationTransfer•DNAreplication•Transcription•TranslationWhatisthecentraldogmaofthemolecularbiology?Chapter28DNAreplication28.1HowisDNAreplicated?1.DNAreplicationissemiconservative2.DNAreplicationisbidirectional3.DNAreplicationissemidiscontinuousDNAreplicationissemiconservativeEachoriginalstrandiscopiedtoyieldacompletecomplementarystrand.HowisDNAreplicated?-IHowisDNAreplicated?-IIDNAreplicactionisbidirectionalIfreplicationisbidirectional,autoradiogramsofradioactivelylabeledreplicatingchromosomesshouldshowtworeplicationforksheavilylabeledwithradioactivethymidine.AnautoradiogramofthechromosomefromadividingE.colicellshowsbidirectionalreplication.HowisDNAreplicated?-IIIDNAreplicactionissemidiscontinuousDNApolymeraseonlypolymerizenucleotides5-3,bothstrandmustbesynthesizedin5-3.Thustheleadingstrandissynthesizedcontinuously;andthelaggingstrandissynthesizeddiscontinuouslyin1000to2000nucleotidesOkazakifragments,whicharethencovalentlyjoinedbyDNAligasetoformuninterruptedDNAstrand.AllDNApolymerasessharethefollowingproperties:1.TheincomingbaseisselectedwithintheDNApolymeraseactivesitethroughbase-pairingwiththecorrespondingbaseinthetemplatestrand2.Chaingrowthisinthe5-3directionantiparalleltothetemplatestrand3.DNApolymerasescannotinitiateDNAsynthesisdenovo—allrequireaprimerwithafree3-OHtobuildupon.28.2DNAreplicationinbacteriaNucleicacidsarelinearpolymersofnucleotideslinked3’to5’byphosphodiesterbridges.Sanger’schainterminationmethod(a)ADNAfragmentservesastemplateinapolymerizationreactionusingDNApolymeraseandaprimer.(b)2’,3’-dideoxynucleotidelack3’-OHgroups,theycannotserveasacceptorsfornext5’-nucleotideaddition,andthusthechainisterminatedatddNTP.(c)Fourparallelreactionsarerun,ineach,adifferentddNTPisincludedwithfourdNTPs.AnestedsetofDNAfragmentswasgenerated,withtheterminalnucleotide’sidentityrevealedbytheddNTP.E.coliDNApolymeraseIIIholoenzyme-replicateschromosomeE.coliDNApolymeraseIIIholoenzymeiscomposedofdifferentkindsofsubunitsandsitsateachreplicationforkAtthereplicationfork,theDNAduplexisunwoundbytheDNAgyrase促旋酶andhelicaseandthesinglestrandsarecoatedwithSSB(ssDNAbindingprotein).Primaseperiodicallyprimes启动synthesisonthelaggingstrand.Eachhalfofthedimericreplicativepolymeraseisacorepolymeraseboundtoitstemplatestrandbyabeta-subunitslidingclamp.DNApolymeraseIandDNAligaseactdownstreamonthelaggingstrandtoremoveRNAprimers,replacethemwithDNAandligatetheOkazakifragments.ThreeStepsinStartingaPrimerforaNewOkazakiFragmentPriortoprimerformation,thebasesoftheparentalDNAstrandarecoveredwithSSBproteins.A)First,thePriAproteindisplacestheSSBproteins.B)Second,aprimaseassociateswiththePriAprotein.C)Lastly,theprimasemakestheshortRNAprimerneededtoinitiatetheOkazakifragment.ThreeStepsinJoiningtheOkazakiFragmentsWhenfirstmade,thelaggingstrandiscomposedofalternatingOkazakifragmentsandRNAprimers.ThefirststepinreplacingtheRNAprimerwithDNAisthebindingofDNApolymeraseItotheprimerregion.AsPolImovesforwarditdegradestheRNAandreplacesitwithDNA.Lastly,DNAligasesealsthenickthatremains.ribonucleaseH(RNaseH)EnzymethatdegradestheRNAstrandofDNA:RNAhybriddoublehelixes.InbacteriaitremovesthemajorportionofRNAprimersusedtoinitiateDNAsynthesis.E.coliDNApolymeraseIisaproofreaderandeditorThe3-5exonulceaseactivityofDNApolymeraseIremovesnucleotidesfromthe3endofthegrowingDNAchains.ChromosomeReplicationInitiatesatoriCReplicationofabacterialchromosomestartsataspecificpoint,theoriginofreplication.SequencesattheOriginofDNAReplicationSequencerepeatsattheoriginofreplicationareoftwovarieties,bothbeingAT-rich.ChromosomeReplicationTerminatesatterCTerminationofreplicationbyTusandTerSitesThecircularbacterialchromosomehasaterminationregion,orterminus,withseveralsitesthatstopreplicationforksmovingclockwise(TerF,TerBandTerC)andcounterclockwise(TerE,TerDandTerA).TheConceptoftheRepliconArepliconisanyDNA(orRNA)moleculethatiscapableofsurvivingandreplicatingitselfinsideacell.Arepliconmustpossessanoriginofreplicationwherereplicationisinitiated.Arepliconmustalsobeanintact,“complete”moleculeofDNA(orRNA)withendsthatareprotectedfrombeingattackedbyacell‘sdefensesystem.Thusitmustbecircular(orhaveendsthatareprotected).Inprokaryotes,repliconsareusuallyclosedcirclesofDNAthathavenoends.Inmostbacteria,linearmoleculesofDNAaredegradedbyexonucleases.Consequently,linearsegmentsofDNAthatenterabacterialcellduringconjugationortransformationwilleventuallybedegraded.Plasmidsareanothergroupofreplicons.Theyareextraself-replicatingmoleculesofDNAthatarenotnecessaryforsurvivalofthehostcellCircularrepliconsareoccasionallyfoundineukaryoticcells,includingplasmidssuchasthe2umcircleofyeast.Mitochondriaandchloroplastsalsocontaintheirowngenomes,orreplicons,whicharecircularmoleculesofself-replicatingDNA.Repliconsotherthanchromosome28.3HowisDNAreplicatedinEukaryoticcells?EukaryoticDNAisorganizedintochromosomeswithinthenucleus.Thesechromosomesmustbereplicatedonce(andonlyonce!)eachcellcycle.Progressionthroughthecellcycleisregulatedthroughcheckpointsthatcontrolwhetherthecellcontinuesintothenextphase.EukaryoticChro