1、Happy Birthday,Double Helix DNA Replication Background Information Watson&Crick General Features 1)Many enzymes and proteins are required 2)Template&dNTPs/Mg 2+are required 3)Semi-conservative A key experiment designed by M.Meselson and W.F.Stahl（1958）4)DNA Unwinding is necessary 5)A Primer with a f
2、ree 3-OH group is required 6)Only in the 53direction 7)Specific Origin of Replication-Ori C and ARS(Autonomously Replicating Sequence）Three Common Features of Replication Origins 8)Bi-directional(With some exceptions)9)Semi-discontinuous Replication fork，Leading strand,Lagging strand and Okazaki fra
3、gments 10)Highly processive,Highly ordered and Extremely accurate Molecular Structure of Nucleic Acids:Molecular Structure of Nucleic Acids:A Structure for Deoxyribose Nucleic Acid A Structure for Deoxyribose Nucleic Acid(Nature,April 25,1953.volume 171:737-738.)uThe novel feature of the structure i
4、s the manner in which the two chains are held together by the purine and pyrimidine bases.The(bases)are joined together in pairs,a single base from one chain being hydrogen-bonded to a single base from the other chain,so that the two lie side by side.One of the pair must be a purine and the other a
5、pyrimidine for bonding to occur.Only specific pairs of bases can bond together.These pairs are:adenine(purine)with thymine(pyrimidine),and guanine(purine)with cytosine(pyrimidine).u.in other words,if an adenine forms one member of a pair,on either chain,then on these assumptions the other member mus
6、t be thymine;similarly for guanine and cytosine.The sequence of bases on a single chain does not appear to be restricted in any way.However,if only specific pairs of bases can be formed,it follows that if the sequence of bases on one chain is given,then the sequence on the other chain is automatical
7、ly determined.u.It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.The structure itself suggested that each strand could separate and act as a template for a new strand,therefore doubling the amount of
8、 DNA,yet keeping the genetic information,in the form of the original sequence,intact.Testing Models for DNA replicationMatthew Meselson and Franklin Stahl(1958)Matthew Meselson and Franklin Stahl more recentlyFaculty member at HarvardMechanisms of Molecular EvolutionFaculty Chair for CBW StudiesFacu
9、lty member at U.of OregonMeiotic RecombinationTesting Models for DNA replicationMeselson and Stahl(1958)Density labeling experiment on E.coli(bacterial)DNABacterial culture15NH4Cl(Sole N source)Grow for several generationsBacterial culturewith dense DNAThis is the starting material for the experimen
10、tMeselson and Stahl(continued)Harvest cells and resuspend in media with14NH4Cl as the sole N sourceBacterial culturewith dense DNAGrow for 1 generationHarvest some cells“1st generation”Grow for another generationHarvest some cells“2nd generation”Grow for another generationetcFor each generation isol
11、ate the DNA and spin through a density(CsCl)gradient).Detect DNA in the gradient(eg by UV absorption)Monitor how many DNA bands there are after each generationBacterial culture“0 generation”14NH4ClMeselson and Stahl Original DataDNA Replication Since DNA replication is semiconservative,therefore the
12、 helix must be unwound.John Cairns(1963)showed that initial unwinding is localized to a region of the bacterial circular genome,called an“origin”or“ori”for short.E.colichromosomeLocalizedunwindingoriginORDNA replicationunidirectionalbidirectionalReplication forksJohn CairnsGrow cells for several gen
13、erationsSmall amounts of 3H thymidineare incorporated into new DNAGrow for brief period of timeAdd a high concentration of 3H-thymidinein media with lowconcentration of 3H-thymidineBacterial culture*T*T*T*TDense label at the replication forkwhere new DNA is being made*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T*T
14、*T*T*T*T*T*T*T*T*T*T*TAll DNA is lightlylabeled with radioactivity*T*T*TCairns then isolated the chromosomes by lysing the cells very very gently and placed them on an electron micrograph(EM)grid which he exposed to X-ray film for two months.Evidence points to bidirectional replicationLabel at both
15、replication forksDNA Replication is Semi-discontinuousConsider one replication fork:5353Direction ofunwindingContinuous replication53PrimerPrimer53Primer53Discontinuous replicationEvidence for the Semi-Discontinuous replication model was provided by the Okazakis(1968)Reiji Okazaki was born near Hiro
16、shima,Japan,in 1930.He was a teenager there at the time of the explosion of the first of two 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 t
17、hat blast.Evidence for Semi-Discontinuous Replication(pulse-chase experiment)Bacteria arereplicatingBacterial cultureAdd 3H ThymidineFor a SHORT time(i.e.seconds)Flood with non-radioactive TAllow replicationTo continue Harvest the bacteriaat different timesafter the chaseIsolate their DNASeparate th
18、e strands(using alkali conditions)Run on a sizing gradientsmallestlargestRadioactivity will onlybe in the DNA that was made during the pulsesmallestlargestResults of pulse-chase experiment Pulse5353Direction ofunwinding35PrimerPrimer53Primer53*ChaseContinuous synthesisDiscontinuous synthesisDNA repl
19、ication is semi-discontinuousEnzymes and Proteins Involved in DNA Replication DNA dependent DNA polymerase(DNA pol,DNA聚合酶）-incorporation of nucleotides DNA Helicase（DNA解链酶）-promotes strand separation,requires ATP and unwinds ds DNA at replication fork Single-stranded DNA binding proteins（SSB，单链结合蛋白）
20、-keep strands apart,coat DNA and prevent re-association of strands and stimulate DNA polymerase Primase（引发酶引发酶）-formation of RNA primers DNA ligase（DNA 连接酶）-joining of Okazaki fragments Topoisomerase（拓扑异构酶）-release stress of unwinding:relieves stress by breaking and sealing-otherwise DNA becomes too
21、 tightly coiled and stops the replicating fork The Enzymes responsible for removing RNA primers Uracil-DNA N-glycosylase（尿嘧啶-DNA-N-糖苷酶）Telomerase（端聚酶）-maintain telomeric DNA integrityDNA-dependent DNA polymerases Common Reaction Equation:Prokaryotic DNA pol Eukaryotic DNA pol E.coli DNA polymerasesE
22、.coli DNA polymerases Identification Structure and Function of DNA pol I DNA pol II and DNA pol III DNA pol IV and DNA pol V Conclusion Protein extracts from E.coli+Template DNAIs new DNA synthesized?-dNTPs(substrates)all 4 at once-Mg2+(cofactor)-ATP(energy source)-free 3OH end(primer)In vitro assay
23、 for DNA synthesisUsed the assay to purify a DNA polymerizing enzymeDNA polymerase ICurrently a faculty member at Stanford School of Medicine How Amazing!l a 3 to 5 exonuclease activityl a 5 to 3 exonuclease activityl a 5 to 3 DNA polymerizing activityMore on Pol I Why the exonuclease activity?The 3
24、-5 exonuclease activity serves a proofreading function It removes incorrectly matched bases,so that the polymerase can try again Conceptual model for proofreading based on Conceptual model for proofreading based on kinetic considerationskinetic considerationsstalling transient melting exonuclease si
25、te occupancyProof reading activityof the 3 to 5 exonuclease.Proof reading activity is slowcompared to polymerizingactivity,but the stalling ofDNAP I after insertion of an incorrect base allows the proofreading activity to catch up with the polymerizingactivity and remove theincorrect base.Notice how
26、 the newly-formed strand oscillates between the polymerase and 3-exonuclease sites,adding a base and then checking itMore on Pol I 3 to 5 exonuclease activity Structure of the Klenow fragmentEven More on Pol I 5-exonuclease activity,working together with the polymerase,accomplishes nick translation
27、DNA Polymerase I is great,but.In 1969 John Cairns and Paula deLucia-isolated a mutant bacterial strain with only 1%DNAP I activity(polA)-mutant was super sensitive to UV radiation-but otherwise the mutant was fine-it could divideConclusion:DNAP I is NOT the principal replication enzyme in E.coliOthe
28、r clues.-DNAP I is too slow(600 dNTPs added/minute)-DNAP I is only moderately processive(processivity refers to the number of dNTPs added to a growing DNA chain before the enzyme dissociates from the template)Conclusion:There must be additional DNA polymerases.Biochemists purified them from the polA
29、 mutantWhat does DNAP I do?-functions in multiple processes that require only short lengths of DNA synthesis-has a major role in DNA repair(Cairns-deLucia mutant was UV-sensitive)-its role in DNA replication is to remove primers and fill in the gaps left behind-for this it needs the nick-translation
30、 activityThe DNA Polymerase Family A total of 5 different DNAPs have been reported in E.coli DNAP I:does 90%of polymerizing activity DNAP II:functions in DNA repair(proven in 1999)DNAP III:principal DNA replication enzyme DNAP IV:functions in DNA repair(discovered in 1999)DNAP V:functions in DNA rep
31、air(discovered in 1999)DNA Polymerase III The real replicative polymerase in E.coli Its fast:up to 1,000 dNTPs added/sec/enzyme Its highly processive:500,000 dNTPs added before dissociatingIts accurate:makes 1 error in 107 dNTPs added,with proofreading,this gives a final error rate of 1 in 1010 over
32、all.Genetic mutant(Ts)ITSCOMPLICATED!The subunits of E.coli DNA polymerase IIISubunit Functiona ae eq qt tb bg gd dd dc cy y5 to 3 polymerizing activity3 to 5 exonuclease activitya a and e e assembly(scaffold)Assembly of holoenzyme on DNASliding clamp=processivity factorClamp-loading complexClamp-lo
33、ading complexClamp-loading complexClamp-loading complexClamp-loading complexCoreEnzymedimerHoloenzymeThe structure formed by two beta subunits of the E.coli DNA polymerase III.This structure can clamp a DNA molecule and slide with the core polymerase along the DNA molecule.DNA Polymerase IIIholoenzy
34、me CoreCoreb bb bb bb bb b clampst t2 2t t subunits hold2 cores in a dimerg g complex(clamp loader)g gReplicationForkLeading Strand synthesisLagging Strand synthesisComparison of E.Coli Comparison of E.Coli DNA pol I,II,and IIIDNA pol I,II,and IIIEukaryotic DNA polymeraseEukaryotic DNA polymeraseOth
35、er Enzymes and Proteins Involved Other Enzymes and Proteins Involved in DNA Replicationin DNA Replication Helicase:I and II；ATPase Helicase II is involved in DNA replication E.coli:dna B蛋白 and Rep蛋白 Werner syndrome(WS)and Helicase mutation SSB：without any enzymatic activity Prokaryotic:Act in a coop
36、erative fashion Eukaryotic:Replication Factor A(RFA)Primase:A kind of DNA-dependent RNA polymerase The Enzyme removing primers Prokaryotic:DNA pol I;Enkaryotic:RNase H(5-3 exonuclease activity active only on RNA-DNA hybrids)or MF1(5-3 exonuclease)DNA ligase Prokaryotic:NAD+;Eukaryotic and Viral:ATP
37、Topoisomerase:I,II(E.coli-Gyrase),III,and IV II and IV are involved in DNA replication Uracil-DNA N-glycosylase Removing the mis-incorporated dUMP during DNA replication Telomease Specific to eukaryotes;A kind of retro-transcriptaseAction of Topoisomerase II Action of DNA LigaseThe“End-Replication P
38、roblem”The leading strand is made as a continuous molecule that can replicate all the way to the end of a chromosome.The lagging strand is made as short Okazaki fragments,each requiring a new primer to be laid down on the template,that are then ligated to make a continuous strand.The lagging strand
39、cannot replicate all the way to the end of linear chromosome,since there is no DNA beyond the end for apriming event to fill in the gap between the last Okazaki fragment and the terminus.This leaves a 3 overhang.Act as protective“caps”on the ends of chromosomes.They are composed of short,tandem repe
40、ats.In humans:5-TTAGGG-3 repeated at the ends ofeach chromosome for a total length of 15 kilobases.Telomeres are non-coding DNATherefore,if telomeres gradually get eroded by DNAreplication,there is less harm to the organismTelomeresTelomerase=a protein componentwith reverse transcriptase activity pl
41、us an RNA component containing 1.5 copies of the telomere repeat sequence.Reverse transcriptase is a DNA polymerasethat uses RNA as a template(not DNA)Just like other DNA polymerases it requires a primerTelomere Repeats are Added by the enzyme,TelomeraseThe RNA component of telomerase base-pairs wit
42、h the last telomere repeat.The lest of the telomere RNA“hangs off”the end of the chromosome.This makes the end of the chromosome into a primer that can be extended by telomerase.Telomerase makes a DNA copy of its RNA,which is just like adding a telomere repeat.Then the enzyme translocates again to t
43、he new end of the chromosome and repeats the process.How telomerase works:Details of DNA ReplicationDetails of DNA Replication Initiation（）Elongation（）Termination and Separation（）()Protein NameFunctionDNA GyraseUnwinding DNASSBSingle-stranded DNA bindingDnaAInitiation factorHUHistone-like(DNA bindin
44、g and bending)PriAPrimosome assemblyPriBPrimosome assemblyPriCPrimosome assemblyDnaBDNA unwinding(helicase)DnaCDnaB chaperoneDnaTAssists DnaC in delivery of DnaBPrimaseSynthesis of an RNA primerDNAP III holoenzymeElongation(DNA synthesis)DNAP IExcises RNA primer,fills in with DNALigaseCovalently lin
45、ks Okazaki fragmentsTusTerminationDNA Replication is an Ordered Series of StepslFind the origin:DnaA(origin recognition protein)+HUlUnwind the helix:DnaB(helicase),DnaC+DnaT(deliver DnaB to the origin),SSB(keeps helix unwound),DNA Gyrase facilitates efficient unwindinglSynthesize primers:DnaG(primas
46、e)+PriA,PriB,PriC (assembly and function of the primosome)lElongate(new strand synthesis):DNAP III holoenzymelRemove the primers and ligate Okazaki fragments:(DNAP I+Ligase)lTerminate replication:Ter(termination sequence)+Tus(termination utilization substance)l Separate Daughter DNAs:DNA Topo IVPrim
47、osome-引发体Gyrase-旋转酶Finding and unwinding the origin of replication13 base pair repeat=5-GATCNTNTTNTT-34 DnaA tetramersfirst bind to the repeats.Binding is cooperative.Each DnaA binds ATP.They recruit additional DnaA monomers to bind to adjacent DNA generating a nucleosome-like structureDnaA powers t
48、he unwindingof adjacent A-T-rich repeatsby hydrolyzing ATP.A proteincalled HU also helps.DnaB(a helicase,is now delivered tothe unwound region with the help ofDnaC and DnaT.You need one helicaseat each replication fork to do theunwinding.Delivery and assembly ofDnaB onto DNA requires ATP.SSB coats t
49、he unwound DNA strandsto prevent them from reassociating.Unwinding starts in both directions,andshoves off(displaces)the DnaA proteins.This a prepriming complex.Primase is now recruited to each forkso that a primer can be laid down for DNAsynthesis on each strand at each fork.Primase is associated w
50、ith helicase.Primase lays down an on the leading strand.Primase lays down a primer on the laggingstrand.This a primosome.Addition of DNA polymerase III holoenzyme forms a replisomePrimers must be occasionally laid down on the lagging strand to prime Okazaki fragment synthesis.This is done by the Dna
