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1、CHAPTER 8:The replication of DNAMeselsonandStahlsExperimentM. Meselson and F.W. Stahl. 1958. The replication of DNA in Escherichia coli Proc. Natl. Acad. Sci. U.S.A. 44: 671-682. M. Meselson and F. W. Stahl were interested in trying to devise a way to prove or disprove Watson and Cricks model of sem
2、i-conservative replication. In 1957, they successfully obtained the experimental proof for the semi-conservative replication of DNA. They did this by inventing a new technique called Density Gradient Centrifugation. Their paper was published in 1958 and ever since, the experiment has often been refe
3、rred to as: one of the most beautiful experiments in biology. How It Began:CsCl密度梯度离心分离密度梯度离心分离DNAA. A. The The MeselsonMeselson- -Stall experimentStall experimentB. the B. the interpretationinterpretation( (CsClCsCl gradient centrifuge) gradient centrifuge) N15N14N15 DNADNASemi-ConservationReplicat
4、ionComplications of DNA replicationComplications of DNA replicationlEnzymeslTheTopologicalProblemlDirectionproblem:semi-discontinuouslylPriminglThe Chemistry of DNA SynthesisThe Chemistry of DNA SynthesislDNA PolymeraseDNA PolymeraselThe Replication ForkThe Replication ForklThe Replication ProcessTh
5、e Replication ProcessCHAPTER 8 The replication of DNAl lRawmaterialsl lReactiondirectionl lChemicalreactionlThedrivingforceforDNAsynthesisThe Chemistry of DNA SynthesisThe Chemistry of DNA SynthesisSubstraterequiredforDNAsynthesisl lDiagram of the mechanism of DNA synthesisDiagram of the mechanism o
6、f DNA synthesislThe Chemistry of DNA SynthesisThe Chemistry of DNA SynthesislDNA PolymeraseDNA PolymeraselThe Replication ForkThe Replication ForklThe Replication ProcessThe Replication ProcessCHAPTER 8 The replication of DNADNA PolymeraseDNA PolymeraselThe specialization of DNA polymerasesl The mec
7、hanism of DNA Polymerasekinds of DNA polymerases The role of the subunits of DNA polymerases FunctionmechanismDNA polymerases of bacteriaDNA polymerases of bacteriaThecompositionoftheDNAPolIIIholoenzymeSliding clampslEncirclethenewlysynthesizeddouble-strandedDNAandthepolymeraseassociatedwiththeprime
8、r:templatejunctionlEnsurestherapidrebindingofDNAPoltothesameprimer:templatejunction,andthusincreasestheprocessivityofPol.lEukaryoticslidingDNAclampisPCNASlidingDNAClampSliding DNA clamps are found across all organism and share a similar structureSliding DNA Clamp Increases ProcessivityDNApolymerases
9、ofeukaryotesPolymeraseswitchingthe process of replacing DNA Pola/primase with DNA Pold or DNA Pole. DNA PolymeraseDNA PolymeraselThe specialization of DNA polymerasesl The mechanism of DNA PolymeraseMechanismFunctionlThe specialization of DNA polymeraseskinds of DNA polymerases The role of the subun
10、its of DNA polymerasesThumbFingersPalmlCatalytic sites for addition and removal of dNTPs. lBinds to two metal ions that alter the chemical environment around the catalytic site. lStabilization of the pyrophosphateDNAPolymerase-palmdomainlBinds to the incoming dNTP, encloses the correct paired dNTP t
11、o the position for catalysislBends the template to expose the only nucleotide at the template that ready for forming base pair with the incoming nucleotideDNAPolymerase-fingerdomainlNot directly involved in catalysislInteracts with the synthesized DNA to maintain correct position of the primer and t
12、he active site, and to maintain a strong association between DNA Pol and its substrate.DNAPolymerase-thumbdomainDNA Polymerase-palm domainDNAPolymerase-fingerdomainThumbFingersPalmDNA PolymeraseDNA PolymeraselThe specialization of DNA polymerasesl The mechanism of DNA PolymeraseSubunits of DNA polym
13、erases The role of the subunitsMechanismlThe specialization of DNA polymerasesSubunits of DNA polymerases The role of the subunitsFunctionlDNArapidprocessivesynthesislDNAaccuratesynthesisDNA Pol are processive enzymes lProcessivity is a characteristic of enzymes that operate on polymeric substrates.
14、lThe processivity of DNA Pol is the average number of nucleotides added each time the enzyme binds a primer:template junction (a few50,000). lAsinglesitetocatalyzetheadditionofanyofthefourdNTPs.lRecognitionofdifferentdNTPbymonitoringtheabilityofincomingdNTPinformingA-TandG-Cbasepairs;incorrectbasepa
15、irdramaticallylowerstherateofcatalysislDistinguishbetweenrNTPanddNTPbystericexclusionofrNTPsfromtheactivesite.DNA accurate synthesisDistinguishbetweenrNTPanddNTPbystericexclusionofrNTPsfromtheactivesiteExonucleasesproofreadnewlysynthesizedDNAThe occasional flicking of the bases into “wrong” tautomer
16、ic form results in incorrect base pair and mis-incorporation of dNTP. (10-5 mistake) The mismatched dNMP is removed by proofreading exonuclease.Post-replication mismatch repair processlThe Chemistry of DNA SynthesisThe Chemistry of DNA SynthesislDNA PolymeraseDNA PolymeraselThe Replication ForkThe R
17、eplication ForklThe Replication ProcessThe Replication ProcessThe replication forkThe junction between the newly separated template strands and the unreplicated duplex DNA 3.PrimingReason:polymeraseproofreadingactivityprimingprocess:1.UnwindthedoublehelixDNAhelicasesseparatethetwobase-pairedstrandso
18、fdupiexDNATopoisomeraseremovessupercoils 2.Semi-discontinuouslyLeadingstrand:Laggingstrand:Okazakifragment:DNAhelicasesunwindthedoublehelixinadvanceofthereplicationforkHexameric proteinTopoisomerase removes supercoils produced by DNA unwinding at the replication forkSingle-stranded binding proteins
19、(SSBs) stabilize single-stranded DNACooperative bindingSequence-independent manner (electrostatic interactions) 3.PrimingReason:polymeraseproofreadingactivityprimingprocess:1.UnwindthedoublehelixDNAhelicasesseparatethetwobase-pairedstrandsofdupiexDNATopoisomeraseremovessupercoils 2.Semi-discontinuou
20、slyLeadingstrand:Laggingstrand:Okazakifragment:Mechanism Of DNA Chain Growth, II. Accumulation Of Newly Synthesized Short Chains In E. Coli Infected With Ligase Defective T4 Phages* Kazunori Sugimoto, Kazunori Sugimoto, TsunekoTsuneko Okazaki, and Reiji Okazaki, Okazaki, and Reiji Okazaki, Proc. Nat
21、l. Acad. Sci. U.S.AProc. Natl. Acad. Sci. U.S.A 1968 60: 1356-1362.1968 60: 1356-1362.Evidence for the Semi-Discontinuous replication model was provided by the Okazakis (1968)Reiji Okazaki was born near Hiroshima, Japan, in 1930. He was a teenager there at the time of the explosion of the first of t
22、wo nuclear bombs that the US dropped at the end of World War II. His scientific career was cut short by his untimely death from cancer in 1975 at the age of 44, perhaps related to his exposure to the fallout of that blast.Tsuneko Okazaki, until recently, was a professor atThe University of Nagoya wh
23、ere she was the first woman at that institution to be named a professor.Currently she is on the faculty of Medicine in Fujita, and does research on centromeres.Okazakis ExperimentvThisexperimentclearlyshowedtheexistenceof1000-2000-nucleotidefragmentscalled“Okazakifragments”.vThesefragmentslaterbecam
24、eincorporatedintonormalDNAstrands,atthecompletionofDNAreplication.vAccordingtoOkazakisresearchDNAreplicationfollowsa“backandfill”mechanism.E.colit-2,7,15,60pulse-labelingindT-H3stop in KCN 0stop in KCN 0D.S.DNAS.S.DNADensitygradientofsucroseMeasureH3-Tpulse-chase20indT-H330transfer to transfer to dT
25、dT then then continuecontinueH3-Tpulse-chase pulse-labeling 120 60 15 7 2 10S(1kb)70S(Prok.400Nt/sec)DNA replication in Okazaki fragment 1kb3.PrimingReason:polymeraseproofreadingactivityprimingprocess:1.UnwindthedoublehelixDNAhelicasesseparatethetwobase-pairedstrandsofdupiexDNATopoisomeraseremovessu
26、percoils 2.Semi-discontinuouslyLeadingstrand:Laggingstrand:Okazakifragment:The initiation of a new strand of DNA require an RNA primerPrimase is a specialized RNA polymerase dedicated to making short RNA primers on an ssDNA template. Do not require specific DNA sequence.DNA Pol can extend both RNA a
27、nd DNA primers annealed to DNA template Priming of DNA synthesisPriming of DNA synthesisRNA primers must be removed to complete DNA replicationA joint efforts of RNase H, DNA polymerase & DNA ligase 3.PrimingReason:polymeraseproofreadingactivityprimingprocess:1.UnwindthedoublehelixDNAhelicasessepara
28、tethetwobase-pairedstrandsofdupiexDNATopoisomeraseremovessupercoils 2.Semi-discontinuouslyLeadingstrand:Laggingstrand:Okazakifragment:lThe Chemistry of DNA SynthesisThe Chemistry of DNA SynthesislDNA PolymeraseDNA PolymeraselThe Replication ForkThe Replication ForklThe Replication ProcessThe Replica
29、tion ProcessCHAPTER 8 The replication of DNAThe Replication ProcesslInitiationlElongation lTerminationlOrigins of replicationlthesitesatwhichDNAunwindingandinitiationofreplicationoccur.lTherepliconmodelofreplicationinitiationreplicatorinitiatorlInitiationofDNAreplicationlinE.colilInEukaryoteTherepli
30、conmodelofreplicationinitiation3/18/05ProposedbyJacobandBrennerin1963AlltheDNAreplicatedfromaparticularoriginisarepliconTwocomponents,replicatorandinitiator,controltheinitiationofreplicationInitiatorprotein:specificallyrecognizesaDNAelementinthereplicatorandactivatestheinitiationofreplicationReplica
31、tor:theentiresiteofcis-actingDNAsequencessufficienttodirecttheinitiationofDNAreplicationReplicator sequences3/18/05OriC in E. coli chromosomal DNAThreedifferentfunctionsofinitiatorprotein:1.bindstoreplicator,2.distorts/unwindsaregionofDNA,3.interactswithandrecruitsadditionalreplicationfactorsDnaAinE
32、. coli (all 3 functions),originrecognitioncomplex(ORC)ineukaryotes(functions 1 & 3)Initiation of DNA replication in E. coliInitiation in E. colilOrigins of replicationlthe sites at which DNA unwinding and initiation of replication occur.lTherepliconmodelofreplicationinitiationreplicatorinitiatorlIni
33、tiationofDNAreplicationlinE.colilInEukaryotePrereplicative Complexpre-RC activation & assembly of the replication fork in eukaryotespre-RCs are activated by two protein kinases (Cdk and Ddk) that are active only when the cells enter S phase.Origin activation:Pre-RC formation and activation is regula
34、ted to allow only a single round of replication during each cell cycle.Only one opportunity for pre-RCs to form, and only one opportunity for pre-RC activation.The regulation of initiation of replicationFigure 8-31 Effect of Cdk activity on pre-RC formation and activationCell cycle regulation of Cdk
35、 activity and pre-RC formatinThe Replication ProcesslInitiationlElongation lTerminationAtthereplication,theleadingstrandandlaggingstrandaresynthesizedsimultaneously.Tocoordinatethereplicationofbothstrands,multipleDNAPolsfunctionatthereplicationfork.DNAPolIIIholoenzymeissuchanexample.The composition
36、of the DNA Pol III holoenzymeTrombone modelThe Replication ProcesslInitiationlElongation lTerminationType II topoisomerases are required to separate daughter DNA moleculesTelomeres cannot be maintained by semi-conservative DNA replication35533553Leading strand synthesisLagging strand synthesis355353
37、53DNA polDNA polRegion of unreplicated DNALeading strand synthesisLagging strand synthesisTelomeres cannot be maintained by semi-conservative DNA replication35535353Region of unreplicated DNALeading strand synthesisLagging strand synthesisTelomeres cannot be maintained by semi-conservative DNA replicationTelomerase ReplicatesTelomeresTelomerase Extends 3 EndlThe Chemistry of DNA SynthesisThe Chemistry of DNA SynthesislDNA PolymeraseDNA PolymeraselThe Replication ForkThe Replication ForklThe Replication ProcessThe Replication Process
