DNA-replication@生物化学讲义课件

1、Happy Birthday,Double Helix DNA ReplicationBackground Information Watson & CrickGeneral 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 Pr

2、imer with a free 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 , Lagg

3、ing strand and Okazaki fragments 10) Highly processive , Highly ordered and Extremely accurate Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid (Nature, April 25, 1953. volume 171:737-738.) The novel feature of the structure is the manner in which the two chains are hel

4、d 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 pyrimidine for bonding to occur. .Only

5、specific pairs of bases can bond together. These pairs are: adenine (purine) with thymine (pyrimidine), and guanine (purine) with cytosine (pyrimidine). .in other words, if an adenine forms one member of a pair, on either chain, then on these assumptions the other member must be thymine; similarly f

6、or 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 automatically determined. .It

7、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 DNA, yet keeping

8、 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 StudiesFaculty member a

9、t 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 experimentMesels

10、on 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 isolate th

11、e 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 h

12、elix 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 sever

13、al generationsSmall 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

14、*T*T*T*T*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 replicationLabe

15、l at both 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 bor

16、n near Hiroshima, 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

17、the fallout of that 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 th

18、eir DNASeparate the 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

19、 synthesisDNA replication is semi-discontinuousEnzymes and Proteins Involved in DNA ReplicationDNA dependent DNA polymerase (DNA pol, DNA聚合酶）- incorporation of nucleotidesDNA Helicase（DNA解链酶）- promotes strand separation, requires ATP and unwinds ds DNA at replication fork Single-stranded DNA binding

20、 proteins（ SSB，单链结合蛋白）-keep strands apart, coat DNA and prevent re-association of strands and stimulate DNA polymerase Primase（引发酶）- formation of RNA primersDNA ligase （DNA 连接酶）-joining of Okazaki fragmentsTopoisomerase（拓扑异构酶）- release stress of unwinding: relieves stress by breaking and sealing-oth

21、erwise DNA becomes too tightly coiled and stops the replicating fork The Enzymes responsible for removing RNA primersUracil-DNA N-glycosylase （尿嘧啶-DNA-N-糖苷酶）Telomerase（端聚酶）-maintain telomeric DNA integrityDNA-dependent DNA polymerasesCommon Reaction Equation: Mg2+ DNA + Primer-OH + dNTP DNA/Primer-d

22、NMP + PPi 5 3 Subsequent hydrolysis of PPi drives the reaction forwardProkaryotic DNA pol DNA pol I,II,III,IV and VEukaryotic DNA pol DNA pol ,and E. coli DNA polymerasesIdentification Kornberg and DNA pol I (Kornberg enzyme)Structure and Function of DNA pol I A multi-functional enzymeDNA pol II and

23、 DNA pol IIIDNA pol IV and DNA pol VConclusion DNA pol III is a major polymerase involved in E. coli chromosome DNA replicationArthur Kornberg (1957)Protein extracts from E. coli+Template DNAIs new DNA synthesized?- dNTPs (substrates) all 4 at once- Mg2+ (cofactor)- ATP (energy source)- free 3OH end

24、 (primer)In vitro assay for DNA synthesisUsed the assay to purify a DNA polymerizing enzymeDNA polymerase ICurrently a faculty member at Stanford School of Medicine How Amazing! a 3 to 5 exonuclease activity a 5 to 3 exonuclease activity a 5 to 3 DNA polymerizing activityDNA Pol I from E. coli is 92

25、8 aa (109 kD) monomer A single polypeptide with at least three different Enzymatic activities!The protein is folded into discrete domainsHans Klenow used proteases (subtilisin or trypsin) to cleave between residues 323 and 324, separating 5-exonuclease (on the small fragment) and the other two activ

26、ities (on the large fragment, the so-called Klenow fragment”) Tom Steitz has determined the structure of the Klenow fragment More on Pol I Why the exonuclease activity? The 3-5 exonuclease activity serves a proofreading function It removes incorrectly matched bases, so that the polymerase can try ag

27、ain Conceptual model for proofreading based on kinetic considerationsstalling transient melting exonuclease site 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 all

28、ows the proofreading activity to catch up with the polymerizingactivity and remove theincorrect base.Notice how 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 fragme

29、ntEven More on Pol I 5-exonuclease activity, working together with the polymerase, accomplishes nick translation 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-

30、but otherwise the mutant was fine- it could divideConclusion: DNAP I is NOT the principal replication enzyme in E. coliOther 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 e

31、nzyme dissociates from the template)Conclusion: There must be additional DNA polymerases.Biochemists purified them from the polA 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 U

32、V-sensitive)- its role in DNA replication is to remove primers and fill in the gaps left behind- for this it needs the nick-translation 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 rep

33、air (proven in 1999)DNAP III: principal DNA replication enzyme DNAP IV: functions in DNA repair (discovered in 1999)DNAP V: functions in DNA repair (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: 5

34、00,000 dNTPs added before dissociatingIts accurate: makes 1 error in 107 dNTPs added, with proofreading, this gives a final error rate of 1 in 1010 overall. Genetic mutant(Ts)ITS COMPLICATED!The subunits of E. coli DNA polymerase IIISubunitFunctionaeqtbgddcy5 to 3 polymerizing activity3 to 5 exonucl

35、ease activitya and e assembly (scaffold)Assembly of holoenzyme on DNASliding clamp = processivity factorClamp-loading complexClamp-loading complexClamp-loading complexClamp-loading complexClamp-loading complexCoreEnzymedimerHoloenzymeThe structure formed by two beta subunits of the E. coli DNA polym

36、erase III . This structure can clamp a DNA molecule and slide with the core polymerase along the DNA molecule. DNA Polymerase IIIholoenzyme CoreCorebbbbb clampst2t subunits hold2 cores in a dimerg complex(clamp loader)gReplicationForkLeading Strand synthesisLagging Strand synthesisComparison of E. C

37、oli DNA pol I, II, and IIIEukaryotic DNA polymeraseOther Enzymes and Proteins Involved in DNA ReplicationHelicase: 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 mutationSSB：without any enzymatic activity Prokaryotic: Act in a c

38、ooperative fashion Eukaryotic: Replication Factor A (RFA)Primase: A kind of DNA-dependent RNA polymeraseThe 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

39、 and Viral: ATPTopoisomerase: I,II (E.coli- Gyrase),III, and IV II and IV are involved in DNA replicationUracil-DNA N-glycosylase Removing the mis-incorporated dUMP during DNA replicationTelomease Specific to eukaryotes; A kind of retro-transcriptaseAction of Topoisomerase II Action of DNA LigaseThe

40、 “End-Replication Problem”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 stra

41、nd. The lagging strand 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 c

42、omposed of short, tandem repeats.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

43、 reverse transcriptase activity plus 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 co

44、mponent of telomerase base-pairs with 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 repe

45、at. Then the enzyme translocates again to the new end of the chromosome and repeats the process.How telomerase works:Details of DNA ReplicationThree steps 1) Initiation（起始） 2) Elongation（延伸） 3) Termination and Separation（终止与分离）DNA replication in E.coli- “form”DNA replication in eukaryotesD-loop repl

46、ication and Rolling-circle replication (-form)Proteins Involved in DNA Replication in E. coliDNA Replication is an Ordered Series of StepsFind the origin: DnaA (origin recognition protein) + HUUnwind the helix: DnaB (helicase), DnaC + DnaT (deliver DnaB to the origin), SSB (keeps helix unwound), DNA

47、 Gyrase facilitates efficient unwindingSynthesize primers: DnaG (primase) + PriA, PriB,PriC (assembly and function of the primosome)Elongate (new strand synthesis): DNAP III holoenzymeRemove the primers and ligate Okazaki fragments: (DNAP I + Ligase)Terminate replication: Ter (termination sequence)+

48、 Tus (termination utilization substance) Separate Daughter DNAs: DNA Topo IVPrimosome- 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

49、monomers to bind to adjacent DNA generating a nucleosome-like structureDnaA powers the 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 replicati

50、on fork to do theunwinding. Delivery and assembly ofDnaB onto DNA requires ATP.SSB coats the 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

51、 a primer can be laid down for DNAsynthesis on each strand at each fork. Primase is associated with helicase.Primase lays down an RNA primer on the leading strand. Primase lays down a primer on the laggingstrand. This a primosome. Addition of DNA polymerase III holoenzyme forms a replisomePrimers mu

52、st be occasionally laid down on the lagging strand to prime Okazaki fragment synthesis. This is done by the DnaG primase which occasionally reassociates with the DnaB helicase to lay down a new primer on the lagging strand.Leading strandLeading strandA “snapshot” of DNA replicationPol III core dimer

53、 synthesizing leading & lagging strands.Tau subunit of Pol III binds to helicase.b Clamp loader g Complex of Pol III holoenzyme( g 2 , d, d, c, psi) Uses ATP to open dimer and position it at 3 -end of primer.“Loaded” clamp then binds Pol III core (and releases from ).Processive DNA synthesis. - load

54、s b subunit dimer onto primerOrder of eventsRecycling phaseOnce Okazaki fragment completed, b clamp releases from core. b binds to g . g unloads b clamp from DNA. b clamp recycles to next primer. Termination of ReplicationTermination occurs at ter region of E. coli chromosome. ter region rich in Gs

55、and Ts, signals the end of replication. Terminator utilization substance (Tus) binds to ter region.Tus prevents replication fork from passing by inhibiting helicase activity.Terminating DNA synthesis in prokaryotes.Fig. 21.27Each fork stops at the Ter regions, which are 22 bp, 3 copies, and bind the

56、 Tus protein.Eukaryotic DNA Replication Like E. coli, but more complex Chromatin and Nucleosome Multiple origins of replication DNA replication occurs just at S phase of the cell cycle and is controlled by many proteins Okazaki fragments are shorter than in ProkaryotesReplication forks run a slower

57、speed than in ProkaryotesTwo rounds of replication cannot occur at the same timeTelomerase is requiredLicensing: positive control of Eukaryotic DNA replication An Origin Recognition Complex of proteins (ORC). These remain on the DNA throughout the process. Accessory proteins called licensing factors. These accumulate in the nucleus during G1 of the cell cycle. They include: Cdc6 and Cdt1, which bind to