CHAPTER3DNAReplicationNTU3章DNA复制台大.ppt

台大農藝系遺傳學60120000 Chapter3slide1 CHAPTER3DNAReplication PeterJ Russell editedbyYue WenWangPh D Dept ofAgronomy NTU AmolecularApproach2ndEdition 台大農藝系遺傳學60120000 Chapter3slide2 SemiconservativeDNAReplication 1 WatsonandCrickDNAmodelimpliesamechanismforreplication a UnwindtheDNAmolecule b Separatethetwostrands c Makeacomplementarycopyforeachstrand 台大農藝系遺傳學60120000 Chapter3slide3 2 ThreepossiblemodelswereproposedforDNAreplication a ConservativemodelproposedbothstrandsofonecopywouldbeentirelyoldDNA whiletheothercopywouldhavebothstrandsofnewDNA b DispersivemodelwasthatdsDNAmightfragment replicatedsDNA andthenreassemble creatingamosaicofoldandnewdsDNAregionsineachnewchromosome c SemiconservativemodelisthatDNAstrandsseparate andacomplementarystrandissynthesizedforeach sothatsiblingchromatidshaveoneoldandonenewstrand ThismodelwasthewinnerintheMeselsonandStahlexperiment 台大農藝系遺傳學60120000 Chapter3slide4 PeterJ Russell iGenetics Copyright PearsonEducation Inc publishingasBenjaminCummings Fig 3 1ThreemodelsforthereplicationofDNA 台大農藝系遺傳學60120000 Chapter3slide5 TheMeselson StahlExperiment Animation TheMeselson StahlExperiment1 MeselsonandStahl 1958 grewE coliinaheavy notradioactive isotopeofnitrogen 15Nintheformof15NH4Cl Becauseitisheavier DNAcontaining15NismoredensethanDNAwithnormal14N andsocanbeseparatedbyCsCldensitygradientcentrifugation Box3 1 2 OncetheE coliwerelabeledwithheavy15N theresearchersshiftedthecellstomediumcontainingnormal14N andtooksamplesattimepoints DNAwasextractedfromeachsampleandanalyzedinCsCldensitygradients Figure3 2 台大農藝系遺傳學60120000 Chapter3slide6 PeterJ Russell iGenetics Copyright PearsonEducation Inc publishingasBenjaminCummings Fig 3 2TheMeselson Stahlexperiment whichshowedthatDNAreplicatessemiconservatively 台大農藝系遺傳學60120000 Chapter3slide7 PeterJ Russell iGenetics Copyright PearsonEducation Inc publishingasBenjaminCummings BoxFig 3 1EquilibriumcentrifugationofDNAofdifferentdensitiesinacesiumchloridedensitygradient 台大農藝系遺傳學60120000 Chapter3slide8 3 Afteronereplicationcycleinnormal14Nmedium allDNAhaddensityintermediatebetweenheavyandnormal Aftertworeplicationcycles thereweretwobandsinthedensitygradient oneattheintermediateposition andoneatthepositionforDNAcontainingentirely14N 4 Resultscomparedwiththethreeproposedmodels a Doesnotfitconservativemodel becauseafteronegenerationthereisasingleintermediateband ratherthanonewithentirely15NDNAandanotherwithentirely14NDNA b ThedispersivemodelpredictedthatasinglebandofDNAofintermediatedensitywouldbepresentineachgeneration graduallybecominglessdenseasincreasingamountsof14Nwereincorporatedwitheachroundofreplication Instead MeselsonandStahlobservedtwobandsofDNA withtheintermediateformdecreasingovertime c Thesemiconservativemodelfitsthedataverywell 台大農藝系遺傳學60120000 Chapter3slide9 SemiconservativeDNAReplicationinEukaryotes 1 TovisualizeDNAofeukaryoticchromosomesreplicating CHO Chinesehamsterovary cellsaregrownin5 bromodeoxyuridine BUdR abaseanalogforthymine Aftertworoundsofreplication mitoticchromosomesarestainedwithfluorescentdyeandGiemsastain Figure3 3 台大農藝系遺傳學60120000 Chapter3slide10 2 DNAcontainingTstainsdarkly whileDNAcontainingtwoBUdRstrandsstainslightly Observedthatafteronegeneration bothchromatidsstainthesame eachwithoneBUdRstrandandoneTstrand Aftertwogenerations theystaindifferentlyandarecalledharlequinchromosomes onelight bothstrandshaveBUdR andonedark onestrandhasBUdRandotherstrandhasT 3 ShowedthateukaryotesalsousesemiconservativeDNAreplication 台大農藝系遺傳學60120000 Chapter3slide11 DNAPolymerases theDNAReplicatingEnzymes 1 FirstisolationofanenzymeinvolvedinDNAreplicationwasin1955 ArthurKornbergwonthe1959NobelPrizeinPhysiologyorMedicineforthiswork 台大農藝系遺傳學60120000 Chapter3slide12 DNAPolymeraseI 1 AccomplishedinvitrosynthesisofE coliDNA Hisreactionmixtureincluded a DNAfragments template b RadioactivelylabeleddNTPs dATP dGTP dTTPanddCTP c E colilysate 2 EnzymeoriginallycalledtheKornbergenzyme nowknownasDNAPolymeraseI Onceisolated couldcharacterizeitsactivity showingthattheabovecomponentsarerequired alongwithMg2 ionsformaximumactivity 台大農藝系遺傳學60120000 Chapter3slide13 RolesofDNAPolymerases Animation DNABiosynthesis HowaNewDNAStrandisMade1 AllDNApolymeraseslinkdNTPsintoDNAchains Figure3 4 Mainfeaturesofthereaction a Anincomingnucleotideisattachedbyits5 phosphategrouptothe3 OHofthegrowingDNAchain EnergycomesfromthedNTPreleasingtwophosphates TheDNAchainactsasaprimerforthereaction b Theincomingnucleotideisselectedbyitsabilitytohydrogenbondwiththecomplementarybaseinthetemplatestrand Theprocessisfastandaccurate c DNApolymerasessynthesizeonlyfrom5 to3 2 TheenzymeKornbergisolatedwasbelievedtobetheonlyDNApolymeraseinE coli However mutationsinthisgene polA1 werenotlethal indicatingthatotherDNApolymerasesmustexistinE coli 台大農藝系遺傳學60120000 Chapter3slide14 PeterJ Russell iGenetics Copyright PearsonEducation Inc publishingasBenjaminCummings Fig 3 4aDNAchainelongationcatalyzedbyDNApolymerase 台大農藝系遺傳學60120000 Chapter3slide15 PeterJ Russell iGenetics Copyright PearsonEducation Inc publishingasBenjaminCummings Fig 3 4bDNAchainelongationcatalyzedbyDNApolymerase 台大農藝系遺傳學60120000 Chapter3slide16 3 Temperature sensitivemutantsareusedtostudyessentialgenes At37 CpolAex1strainsarenormal andtheproteinhasnormalactivityinvitro At42 C however theproteinlacks5 3 exonucleaseactivity andbacterialcellswiththismutationaredead 4 AdditionalDNApolymeraseshavebeenisolated includingDNApolymeraseII 1970 DNApolymeraseIII 1971 DNApolymeraseIV andDNApolymeraseV 台大農藝系遺傳學60120000 Chapter3slide17 ThepropertiesofDNApolymerases 5 ThepropertiesofknownE coliDNApolymerasesare a DNApolymeraseIisasinglepeptideencodedbypolAandusedforDNAreplication ReplicatesDNAinthe5 3 direction Has5 3 exonucleaseactivitytoremovenucleotidesfrom5 endofDNAorfromanRNAprimer b DNApolymeraseIIisasinglepeptideencodedbypolB UsedforDNArepair c DNApolymeraseIIIhasthreepolypeptidesubunitsinthecatalyticcoreoftheenzyme encodedbythednaEgene dnaQ and holE Holoenzymehasanadditionalsixdifferentpolypeptides ReplicatesDNAinthe5 3 direction d DNApolymeraseIVisencodedbythedinBgene andisusedinDNArepair e DNApolymeraseVisencodedbyumuDC andisusedinDNArepair 6 E coliDNApolymerasesusedforDNAreplication DNApolymeraseIandDNApolymeraseIII have3 5 exonuclease proofreading activity 台大農藝系遺傳學60120000 Chapter3slide18 MolecularModelofDNAReplication 1 Table3 1showskeygenesandDNAsequencesinvolvedinreplication 台大農藝系遺傳學60120000 Chapter3slide19 InitiationofReplication 1 ReplicationstartswhenDNAattheoriginofreplicationdenaturestoexposethebases creatingareplicationfork Replicationisusuallybidirectionalfromtheorigin E colihasoneorigin oriC whichhas a Aminimalsequenceofabout245bprequiredforinitiation b Threecopiesofa13 bpAT richsequence c Fourcopiesofa9 bpsequence 2 EventsinE coliinitiatingDNAsynthesis derivedfrominvitrostudies Figure3 5 a Initiatorproteinsattach E coli sinitiatorproteinisDnaA fromthednaAgene b DNAhelicase fromdnaB bindsinitiatorproteinsontheDNA anddenaturestheAT richregionusingATPasanenergysource c DNAprimase fromdnaG bindshelicasetoformaprimosome whichsynthesizesashort 5 10nt RNAprimer 台大農藝系遺傳學60120000 Chapter3slide20 PeterJ Russell iGenetics Copyright PearsonEducation Inc publishingasBenjaminCummings Fig 3 5ModelfortheformationofareplicationbubbleatareplicationorigininE coliandtheinitiationofthenewDNAstrand 台大農藝系遺傳學60120000 Chapter3slide21 SemidiscontinuousDNAReplication Animation MolecularModelofDNAReplication 台大農藝系遺傳學60120000 Chapter3slide22 PeterJ Russell iGenetics Copyright PearsonEducation Inc publishingasBenjaminCummings Fig 3 6a bModelfortheeventsoccurringaroundasinglereplicationforkoftheE colichromosome 台大農藝系遺傳學60120000 Chapter3slide23 PeterJ Russell iGenetics Copyright PearsonEducation Inc publishingasBenjaminCummings Fig 3 6c eModelfortheeventsoccurringaroundasinglereplicationforkoftheE colichromosome 台大農藝系遺傳學60120000 Chapter3slide24 1 WhenDNAdenaturesattheoriC replicationforksareformed DNAreplicationisusuallybi directional butwillconsidereventsatjustonereplicationfork Figure3 6 a Single strandDNA bindingproteins SSBs bindthessDNAformedbyhelicase preventingreannealing b Primasesynthesizesaprimeroneachtemplatestrand c DNApolymeraseIIIaddsnucleotidestothe3 endoftheprimer synthesizinganewstrandcomplementarytothetemplate anddisplacingtheSSBs DNAismadeinoppositedirectionsonthetwotemplatestrands d Newstrandmade5 3 insamedirectionasmovementofthereplicationforkisleadingstrand whilenewstrandmadeinoppositedirectionislaggingstrand Leadingstrandneedsonlyoneprimer whilelaggingneedsaseriesofprimers 台大農藝系遺傳學60120000 Chapter3slide25 2 HelicasedenaturingDNAcausestighterwindinginotherpartsofthecircularchromosome Gyraserelievesthistension 3 Leadingstrandissynthesizedcontinuously whilelaggingstrandissynthesizeddiscontinuously intheformofOkazakifragments DNAreplicationisthereforesemidiscontinuous 4 Eachfragmentrequiresaprimertobegin andisextendedbyDNApolymeraseIII 5 Okazakidatashowthatthesefragmentsaregraduallyjoinedtogethertomakeafull lengthdsDNAchromosome DNApolymeraseIusesthe3 OHoftheadjacentDNAfragmentasaprimer andsimultaneouslyremovestheRNAprimerwhileresynthesizingtheprimerregionintheformofDNA ThenickremainingbetweenthetwofragmentsissealedwithDNAligase Fig 3 7 台大農藝系遺傳學60120000 Chapter3slide26 PeterJ Russell iGenetics Copyright PearsonEducation Inc publishingasBenjaminCummings Fig 3 7ActionofDNAligaseinsealingthegapbetweenadjacentDNAfragmentstoformalonger covalentlycontinuouschain 台大農藝系遺傳學60120000 Chapter3slide27 6 Keyproteinsareassociatedtoformareplisome TemplateDNAprobablybendstoallowsynthesisofbothleadingandlaggingstrandsatthereplicationfork Fig 3 8 7 EarlystagesofbidirectionalreplicationaresummarizedinFigure3 9 台大農藝系遺傳學60120000 Chapter3slide28 PeterJ Russell iGenetics Copyright PearsonEducation Inc publishingasBenjaminCummings Fig 3 8Modelforthe replicationmachine orreplisome thecomplexofkeyreplicationproteins withtheDNAatthereplicationfork 台大農藝系遺傳學60120000 Chapter3slide29 iActivity UnravelingDNAReplication 台大農藝系遺傳學60120000 Chapter3slide30 PeterJ Russell iGenetics Copyright PearsonEducation Inc publishingasBenjaminCummings Fig 3 9BidirectionalreplicationofcircularDNAmolecules 台大農藝系遺傳學60120000 Chapter3slide31 ReplicationofcircularDNAandthesupercoilingproblem 1 Somecircularchromosomes e g E coli arecircularthroughoutreplication creatingatheta like shape Asthestrandsseparateononesideofthecircle positivesupercoilsformelsewhereinthemolecule Replicationforkmovesabout500nt second soat10bp turn replicationforkrotatesat3 000rpm 2 Topoisomerasesrelievethesupercoils allowingtheDNAstrandstocontinueseparatingasthereplicationforksadvance 台大農藝系遺傳學60120000 Chapter3slide32 PeterJ Russell iGenetics Copyright PearsonEducation Inc publishingasBenjaminCummings Fig 3 11Diagramshowingtheunreplicated supercoiledparentstrandsandtheportionsalreadyreplicated 台大農藝系遺傳學60120000 Chapter3slide33 RollingCircleReplication 1 Anothermodelforreplicationisrollingcircle Figure3 10 whichisusedbyseveralbacteriophages including X174 afteracomplementismadeforthegenomicssDNA and aftercircularizationbybasepairingbetweenthe sticky ssDNAcosends 2 Rollingcirclereplicationbeginswithanick single strandedbreak attheoriginofreplication The5 endisdisplacedfromthestrand andthe3 endactsasaprimerforDNApolymeraseIII whichsynthesizesacontinuousstrandusingtheintactDNAmoleculeasatemplate 3 The5 endcontinuestobedisplacedasthecircle rolls andisprotectedbySSBsuntildiscontinuousDNAsynthesismakesitadsDNAagain 台大農藝系遺傳學60120000 Chapter3slide34 PeterJ Russell iGenetics Copyright PearsonEducation Inc publishingasBenjaminCummings Fig 3 10Thereplicationprocessofdouble strandedcircularDNAmoleculesthroughtherollingcirclemechanism 台大農藝系遺傳學60120000 Chapter3slide35 4 ADNAmoleculemanygenomesinlengthcanbemadebyrollingcirclereplication Duringviralassemblyitiscutintoindividualviralchromosomesandpackagedintophagehead 5 Bacteriophage regardlessofwhetherenteringthelyticorlysogenicpathway circularizesitschromosomeimmediatelyafterinfection a Inalysogenicinfection thecircularDNAintegratedintoaspecificsiteintheE colichromosomebyacrossoverevent b Inalyticinfection rollingcirclereplicationproducesalongconcatamerof DNA andtheaviralendonuclease productofthetergene recognizethecossitesandmakesthestaggeredcutsthatusedtoassemblenewvirusparticles 台大農藝系遺傳學60120000 Chapter3slide36 PeterJ Russell iGenetics Copyright PearsonEducation Inc publishingasBenjaminCummings Fig 3 11 chromosomestructurevariesatstagesoflyticinfectionofE coli 台大農藝系遺傳學60120000 Chapter3slide37 DNAReplicationinEukaryotes 1 DNAreplicationisverysimilarinbothprokaryotesandeukaryotes exceptthateukaryoteshavemorethanonechromosome 台大農藝系遺傳學60120000 Chapter3slide38 Replicons 1 EukaryoticchromosomesgenerallycontainmuchmoreDNAthanthoseofprokaryotes andtheirreplicationforksmovemuchmoreslowly Iftheywereliketypicalprokaryotes withonlyoneoriginofreplicationperchromosome DNAreplicationwouldtakemanydays 2 Instead eukaryoticchromosomescontainmultipleorigins atwhichDNAdenaturesandreplicationthenproceedsbidirectionallyuntilanadjacentreplicationforkisencountered TheDNAreplicatedfromasingleoriginiscalledareplicon orreplicationunit Figure3 12 台大農藝系遺傳學60120000 Chapter3slide39 PeterJ Russell iGenetics Copyright PearsonEducation Inc publishingasBenjaminCummings Fig 3 12ReplicatingDNAofDrosophilamelanogaster 台大農藝系遺傳學60120000 Chapter3slide40 3 Ineukaryotes repliconsizeissmallerthanitisinprokaryotes replicationisslower andeachchromosomecontainsmanyreplicons Numberandsizeofrepliconsvarywithcelltype 4 NotalloriginswithinagenomeinitiateDNAsynthesissimultaneously Cell specificpatternsoforiginactivationareobserved sothatchromosomalregionsarereplicatedinapredictableorderineachcellcycle Figure3 13 台大農藝系遺傳學60120000 Chapter3slide41 PeterJ Russell iGenetics Copyright PearsonEducation Inc publishingasBenjaminCummings Fig 3 13TemporalorderingofDNAreplicationinitiationeventsinreplicationunitsofeukaryoticchromosomes 台大農藝系遺傳學60120000 Chapter3slide42 InitiationofReplication 1 Eukaryoticoriginsaregenerallynotwellcharacterized thoseoftheyeastSaccharomycescerevisiaeareamongthebestunderstood 2 ChromosomalDNAfragments about100bp thatareabletoreplicateautonomouslywhenintroducedintoyeastasextracellular circularDNAareknownasARSs autonomouslyreplicatingsequences 3 ARSsareyeastreplicators ThethreesequenceelementstypicallyfoundinARSsareA B1 andB2 4 Initiatorproteininyeastsisthemultiproteinoriginrecognitioncomplex ORC whichbindstoAandB1 Otherreplicationproteinsjoin includingonethatunwindsDNAatB2 TheyeastoriginofreplicationisbetweenregionsB1andB2 5 DNAandhistonesmustbedoubledineachcellcycle G1preparesthecellforDNAreplication chromosomeduplicationoccursduringSphase G2preparesforcelldivision andsegregationofprogenychromosomesoccursduringMphase allowingthecelltodivide 台大農藝系遺傳學60120000 Chapter3slide43 6 Cellcyclecontroliscomplex andonlyoutlinedhere Yeasts inwhichchromosomalreplicationiswellstudied serveasaeukaryoticmodelorganism 7 Initiationofreplicationhastwoseparatesteps controlledbycyclin dependentkinases Cdks thatarepresentthroughoutthecellcycle exceptduringG1 a IntheabsenceofCdskduringG1 replicatorselectionoccurs ORCandotherproteinsassembleoneachreplicatortofrompre replicativecomplexes pre RC b WhencellentersSphase Cdksarepresent andactivatepre RCstoinitiatereplication c Cdkactivityinhibitsanotherroundofpre RCformationuntilthecellagainentersG1 whenCdksareabsent 台大農藝系遺傳學60120000 Chapter3slide44 PeterJ Russell iGenetics Copyright PearsonEducation Inc publishingasBenjaminCummings Fig 3 xSomeofthemoleculareventsthatcontrolprogressionthroughthecellcycleinyeasts 台大農藝系遺傳學60120000 Chapter3slide45 EukaryoticReplicationEnzymes 1 EnzymesofeukaryoticDNAreplicationaren taswellcharacterizedastheirprokaryoticcounterparts Thereplicationprocessissimilarinbothgroups DNAdenatures replicationissemiconservativeandsemidiscontinuousandprimersarerequired 2 FifteenDNApolymerasesareknowninmammaliancells a ThreeDNApolymerasesareusedtoreplicatenuclearDNA Pol alpha extendsthe10 ntRNAprimerbyabout30nt Pol delta andPol epsilon extendtheRNA DNAprimers oneontheleadingstrandandtheotheronthelagging itisnotclearwhichsynthesizeswhich b OtherDNApolsreplicatemitochondrialorchloroplastDNA orareusedinDNArepair 台大農藝系遺傳學60120000 Chapter3slide46 ReplicatingtheEndsofChromosomes 1 Whentheendsofchromosomesarereplicatedandtheprimersareremovedfromthe5 ends thereisnoadjacentDNAstrandtoserveasaprimer andsoasingle strandedregionisleftatthe5 endofthenewstrand Ifthegapisnotaddressed chromosomeswouldbecomeshorterwitheachroundofreplication Figure3 14 台大農藝系遺傳學60120000 Chapter3slide47 PeterJ Russell iGenetics Copyright PearsonEducation Inc publishingasBenjaminCummings Fig 3 14Theproblemofreplicatingcompletelyalinearchromosomeineukaryotes 台大農藝系遺傳學60120000 Chapter3slide48 2 Mosteukaryoticchromosomeshaveshort species specificsequencestandemlyrepeatedattheirtelomeres BlackburnandGreiderhaveshownthatchromosomelengthsaremaintainedbytelomerase whichaddstelomererepeatswithoutusingthecell sregularreplicationmachinery 3 IntheciliateTetrahymena thetelomererepeatsequenceis5 TTGGGG 3 Figure3 15 a Telomerase anenzymecontainingbothproteinandRNA bindstotheterminaltelomererepeatwhenitissinglestranded synthesizinga3 ntsequence TTG b The3 endofthetelomeraseRNAcontainsthesequenceAAC whichbindstheTTGpositioningtelomerasetocompleteitssynthesisoftheTTGGGGtelomererepeat c Additionalroundsoftelomeraseactivitylengthenthechromosomebyaddingtelomererepeats 台大農藝系遺傳學60120000 Chapter3slide49 4 Aftertelomeraseaddstelomeresequences chromosomalreplicationproceedsintheusualway Anyshorteningofthechromosomeendsiscompensatedbytheadditionofthetelomererepeats 5 IfthesequenceofthetelomeraseRNAismutated telomereswillcorrespondtothemutantsequence ratherthantheorganism snormaltelomeresequence UsinganRNAtemplatetomakeDNA telomerasefunctionsasareversetranscriptasecalledTERT telomerasereversetranscriptase 6 Telomerelengthmayvary butorganismsandcelltypeshavecharacteristictelomerelengths Mutantsaffectingtelomerelengthhavebeenidentified anddataindicatethattelomerelengthisgeneticallycontrolled Shorteningoftelomereseventuallyleadstocelldeath andthismaybeafactorintheregulationofnormalcelldeath 台大農藝系遺傳學60120000 Chapter3slide50 PeterJ Russell iGenetics Copyright PearsonEducation Inc publishingasBenjaminCummings Fig 3 15SynthesisoftelomericDNAbytelomerase 台大農藝系遺傳學60120000 Chapter3slide51 AssemblingNewDNAintoNucleosomes 1 WheneukaryoticDNAisreplicated itcomplexeswithhistones Thisrequiressynthesisofhistoneproteinsandassemblyofnewnucleosomes 2 TranscriptionofhistonegenesisinitiatedneartheendofG1phase andtranslationofhistoneproteinsoccursthroughoutSphase 3 AssemblyofnewlyreplicatedDNAintonucleosomesisshowninFigure3 16 a ParentalhistonecoresseparateintoanH3 H4tetramer andtwoH2A H2Bdimers b H3 H4tetramer preexistingornewlymade bindstoreplicateddsDNAandbeginsnucleosomeassembly c H2A H2Bdimers preexistingornewlymade areaddedinanassemblyprocessthatrequireshistonechaperoneproteinstodirectit 4 Self assemblyofnucleosomeshasbeenobservedonlyinvitro 台大農藝系遺傳學60120000 Chapter3slide52