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1、Happy Birthday,Double Helix,DNA Replication,Background 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 P

2、rimer 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 , Lag

3、ging 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 he

4、ld 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

6、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 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 keepin

8、g the genetic information, in the form of the original sequence, intact. ,Testing Models for DNA replication,Matthew Meselson and Franklin Stahl (1958),Matthew Meselson and Franklin Stahl more recently,Faculty member at HarvardMechanisms of Molecular EvolutionFaculty Chair for CBW Studies,Faculty me

9、mber at U. of OregonMeiotic Recombination,Density labeling experiment on E. coli (bacterial) DNA,Meselson and Stahl (continued),Harvest some cells“1st generation”,Harvest some cells“2nd generation”,For each generation isolate the DNA and spin through a density (CsCl) gradient).Detect DNA in the grad

10、ient (eg by UV absorption)Monitor how many DNA bands there are after each generation,Meselson and Stahl Original Data,DNA Replication,Since DNA replication is semiconservative, therefore the helix must be unwound.John Cairns (1963) showed that initial unwinding is localized to a region of the bacter

11、ial circular genome, called an “origin” or “ori” for short.,OR,John Cairns,Cairns 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 replication,DNA Rep

12、lication is Semi-discontinuous,Consider one replication fork:,Continuous replication,Discontinuous replication,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 e

13、xplosion 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 that blast.,Evidence for Semi-Discontinuous Replication(pulse-c

14、hase experiment),Harvest the bacteriaat different timesafter the chase,Isolate their DNASeparate the strands(using alkali conditions)Run on a sizing gradient,Radioactivity will onlybe in the DNA that was made during the pulse,Results of pulse-chase experiment,Continuous synthesis,Discontinuous synth

15、esis,DNA replication is semi-discontinuous,Enzymes and Proteins Involved in DNA Replication,DNA 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 pr

16、oteins（ 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-otherw

17、ise 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 integrity,DNA-dependent DNA polymerases,Common Reaction Equation: Mg2+ DNA + Primer-OH + dNTP DNA/Primer-dN

18、MP + 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 polymerases,Identification Kornberg and DNA pol I (Kornberg enzyme)Structure and Function of DNA pol I A multi-functional enzymeDNA pol II an

19、d DNA pol IIIDNA pol IV and DNA pol VConclusion DNA pol III is a major polymerase involved in E. coli chromosome DNA replication,Arthur 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

20、 end (primer)In vitro assay for DNA synthesis,Used the assay to purify a DNA polymerizing enzymeDNA polymerase I,Currently 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 activity,DNA Pol I from E. col

21、i is 928 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 t

22、wo activities (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 ca

23、n try again,Conceptual model for proofreading based on kinetic considerations,stalling transient melting exonuclease site occupancy,Proof reading activityof the 3 to 5 exonuclease.Proof reading activity is slowcompared to polymerizingactivity, but the stalling ofDNAP I after insertion of an incorrec

24、t base allows 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 it,More on Pol I 3 to 5 exonuclease activity,Structure of the K

25、lenow fragment,Even 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 U

26、V radiation- but otherwise the mutant was fine- it could divideConclusion: DNAP I is NOT the principal replication enzyme in E. coli,Other 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 chai

27、n before the enzyme dissociates from the template)Conclusion: There must be additional DNA polymerases.Biochemists purified them from the polA mutant,What does DNAP I do?,- functions in multiple processes that require only short lengths of DNA synthesis- has a major role in DNA repair (Cairns- deLuc

28、ia 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 activity,The DNA Polymerase Family,A total of 5 different DNAPs have been reported in E. coli DNAP I: does 90% of polymerizing activity DNAP II: func

29、tions 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 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 high

30、ly 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 overall. Genetic mutant(Ts),ITS COMPLICATED!,Subunit,Function,The structure formed by two beta subunits of the E. coli DNA polymerase II

31、I . This structure can clamp a DNA molecule and slide with the core polymerase along the DNA molecule.,DNA Polymerase IIIholoenzyme,ReplicationFork,Leading Strand synthesis,Lagging Strand synthesis,Comparison of E. Coli DNA pol I, II, and III,Eukaryotic DNA polymerase,Other Enzymes and Proteins Invo

32、lved in 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 mutationSSB：without any enzymatic activity Prokaryotic: Act in a cooperative fashion Eukaryotic: Replication Factor A (RFA)Primase: A kind of DNA-d

33、ependent 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 and Viral: ATPTopoisomerase: I,II (E.coli- Gyrase),III, and IV II and IV are inv

34、olved in DNA replicationUracil-DNA N-glycosylase Removing the mis-incorporated dUMP during DNA replicationTelomease Specific to eukaryotes; A kind of retro-transcriptase,Action of Topoisomerase II,Action of DNA Ligase,The “End-Replication Problem”,The leading strand is made as a continuous molecule

35、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 cannot replicate all the way to the end of linear chrom

36、osome, 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 repeats.In humans: 5-TTAGGG-3 repeated at the ends of

37、each 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 organism,Telomeres,Telomerase = a protein componentwith reverse transcriptase activity plus an RNA component containing 1.5 copies

38、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 primer,Telomere Repeats are Added by the enzyme, Telomerase,The RNA component of telomerase base-pairs with the last telomere repeat. The lest o

39、f 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 the new end of the chromosome an

40、d repeats the process.,How telomerase works:,Details of DNA Replication,Three 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 replication and Rolling-circle replication (-form),Proteins Involved in DNA

41、 Replication in E. coli,DNA Replication is an Ordered Series of Steps,Find the origin: DnaA (origin recognition protein) + HUUnwind the helix: DnaB (helicase), DnaC + DnaT (deliver DnaB to the origin), SSB (keeps helix unwound), DNA Gyrase facilitates efficient unwindingSynthesize primers: DnaG (pri

42、mase) + 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)+ Tus (termination utilization substance) Separate Daughter DNAs: DNA

43、 Topo IVPrimosome- 引发体Gyrase- 旋转酶,Finding and unwinding the origin of replication,13 base pair repeat = 5-GATCNTNTTNTT-3,4 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 st

44、ructure,DnaA 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 replication fork to do theunwinding. Delivery and assembly ofDnaB onto

45、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 a primer can be laid down for DNAsynthesis on each strand a

46、t 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.,Addition of DNA polymerase III holoenzyme forms a replisome,Primers must be occasionally laid down on the lagging strand to prime Okazaki fragment

47、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 strand,Leading strand,A “snapshot” of DNA replication,Pol III core dimer synthesizing leading & lagging strands.,Tau subunit of Pol III binds to

48、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.,- loads b subunit dimer onto primer,Order of events,Recycling phase,Once Oka

49、zaki 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 Replication,Termination occurs at ter region of E. coli chromosome. ter region rich in Gs and Ts, signals the end of replication. Terminator utilization sub

50、stance (Tus) binds to ter region.Tus prevents replication fork from passing by inhibiting helicase activity.,Terminating DNA synthesis in prokaryotes.,Fig. 21.27,Each fork stops at the Ter regions, which are 22 bp, 3 copies, and bind the Tus protein.,Eukaryotic DNA Replication,Like E. coli, but more