生物化学原理课件（英文）：Chapter33 DNA replication

Chapter33 DNA ReplicationOutlineGeneral properties of DNA replicationMajor enzymes and proteins involved in DNA replicationDetailed mechanisms of DNA replication1.“-form” replication of genomic DNA in E. coli2.“ Rolling- circle” replication3.D-loop replication4.Replication of nuclear DNA in Eukaryotes5.Archaeal DNA replicationHigh fidelity of DNA ReplicationRegulation of DNA replicationHappy Birthday,Double Helix General Features of DNA Replication1.Many enzymes and proteins are required2.Template & dNTPs/Mg 2+ are required3.Semi-conservative4.DNA Unwinding is necessary5.A Primer with a free 3 -OH group is required6.Only in the 53direction7.Specific Origin of Replication - Ori C and ARS8.Bi-directional (With some exceptions)9.Semi-discontinuous10. 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 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 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). .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 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 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 the genetic information, in the form of the original sequence, intact. Three possible models for DNA replicationTesting Models for DNA replicationMatthew Meselson and Franklin Stahl (1958)Testing Models for DNA replicationDensity labeling experiment on E. coli DNAMeselson and Stahl Original DataSince DNA replication is semi-conservative, therefore the 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.Specific Origin of ReplicationEvidence of bidirectional replicationLabel at both replication forksEukaryotes have many origins of replicationPriming the Synthesis of DNADNA replicates Only in the 53directionddNTP can be used to prove the directionality of DNA replicationSequence properties of DNA replication origins DNA replication is semi-discontinuousContinuous synthesisDiscontinuous synthesisEnzymes and Proteins Involved in DNA Replication1.DNA dependent DNA polymerase (DNA pol)- catalyzes incorporation of nucleotides2.DNA Helicase- promotes strand separation, requires ATP and unwinds ds DNA at replication fork 3.Single-stranded DNA binding proteins（SSB）-keep strands apart, coat DNA and prevent re-association of strands and stimulate DNA polymerase 4.Primase- catalyzes formation of RNA primers5.DNA ligase -joins Okazaki fragments6.Topoisomerase- release stress of unwinding: relieves stress by breaking and sealing-otherwise DNA becomes too tightly coiled and stops the replicating fork 7.The Enzymes responsible for removing RNA primers 8.Uracil-DNA N-glycosylase: Removing the mis-incorporated dUMP during DNA replication 9.Telomerase-maintain telomeric DNA integrityDNA-dependent DNA polymerases Common Reaction Equation: Mg2+ DNA + Primer-OH + dNTP DNA/Primer-dNMP+PPi 5 3 Subsequent hydrolysis of PPi drives the reaction forward Bacterial DNA pol DNA pol I,II,III,IV and V Eukaryotic DNA pol DNA pol ,and A Mechanism for All PolymerasesThomas A. Steitz has suggested that biosynthesis of nucleic acids proceeds by an enzymatic mechanism that is universal among polymerases. His suggestion is based on structural studies indicating that DNA polymerases use a “two-metal-ion” mechanism to catalyze nucleotide addition during elongation of a growing polynucleotide chain. The incoming nucleotide has two Mg2+ ions coordinated to its phosphate groups, and these metal ions interact with two Asp residues that are highly conserved in DNA (and RNA) polymerases.One metal ion, designated A, interacts with the O atom of the free 3-OH group on the polynucleotide chain, lowering its affinity for its hydrogen. This interaction promotes nucleophilic attack of the 3-O on the phosphorus atom in the -phosphate of the incoming nucleotide. The second metal ion assists departure of the product pyrophosphate group from the incoming nucleotide. Together, the two metal ions stabilize the pentacovalent transition state on the -phosphorus atom.A “two-metal-ion” Mechanism for All PolymerasesE. coli DNA polymerases Identification Kornberg and DNA pol I (Kornberg enzyme) Structure and Function of DNA pol I A multi-functional enzyme DNA pol II and DNA pol III DNA pol IV and DNA pol V Conclusion 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 onceMg2+ (cofactor)ATP (energy source)free 3OH end (primer)In vitro assay for DNA synthesisUsed the assay to purify a DNA polymerizing enzyme DNA polIHow Amazing!DNA Pol I from E. coli is 928 aa monomer A single polypeptide with at least three different Enzymatic activities!1.a 3 to 5 exonuclease activity2. a 5 to 3 exonuclease activity3. a 5 to 3 DNA polymerizing activityThe 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 activities (on the large fragment, the so-called Klenow fragment”) Tom Steitz has determined the structure of the Klenow fragmentMore 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 again JProof reading activity is slow compared to polymerizing activity, but the stalling of DNAP I after insertion of an incorrect base allows the proofreading activity to catch up with the polymerizing activity and remove the incorrect base.Proof reading activity of the 3 to 5 exonucleaseJ5-exonuclease activity, working together with the polymerase, accomplishes nick translation Even More on Pol IIn 1969 John Cairns and Paula deLucia isolated a mutant bacterial strain with only 1% DNAP I activity (polA)1.mutant was super sensitive to UV radiation2.but otherwise the mutant was fine3.it could divideConclusion: DNAP I is NOT the principal replication enzyme in E. coliDNA Polymerase I is great, but.EDNAP I is too slow (600 dNTPs added/minute)EDNAP 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 mutantOther clues. functions in multiple processes that require only short lengths of DNA synthesishas 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 behindfor this it needs the nick-translation activityWhat does DNAP I do? A total of 5 different DNAPs have been reported in E. coli 1.DNAP I: does 90% of polymerizing activity 2.DNAP II: functions in DNA repair (proven in 1999)3.DNAP III: principal DNA replication enzyme 4.DNAP IV: functions in DNA repair (discovered in 1999)5.DNAP V: functions in DNA repair (discovered in 1999) The DNA Polymerase Family 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 overall. Genetic mutant(Ts)DNA Polymerase III ITS COMPLICATED!The subunits of E. coli DNA polymerase IIIThe 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. Operation of DNA Pol III holoenzymeComparison of E. coli DNA pol I, II, and IIIEukaryotic DNA polymeraseAction of Helicase (dnaB)Action of bacterial SSBAction of Topoisomerase I Action of Topoisomerase II Action of DNA LigaseThe “End-Replication Problem”LThe 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 cannot replicate all the way to the end of linear chromosome, since there is no DNA beyond the end for a priming event to fill in the gap between the last Okazaki fragment and the terminus. This leaves a 3 overhang. The “End-Replication Problem” and its solution1. Act as protective “caps” on the ends of chromosomes.2. They are composed of short, tandem repeats.3. In humans: 5-TTAGGG-3 repeated at the ends of each chromosome for a total length of 15 kilobases.4. Telomeres are non-coding DNA5. Therefore, if telomeres gradually get eroded by DNA replication, there is less harm to the organismTelomeresTelomerase = a protein componentwith 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, TelomeraseStructural model of TelomeraseJThe RNA component 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 repeat. Then the enzyme translocates again to the new end of the chromosome and repeats the process.How telomerase works:Action of telomeraseDetails of DNA ReplicationnThree steps 1) Initiation 2) Elongation 3) Termination and SeparationnDNA replication in E.coli- “form”nDNA replication in eukaryotesnD-loop replication 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 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)+ Tus (termination utilization substance) Separate Daughter DNAs: DNA Topo IV杨荣武杨荣武生物化生物化学原理学原理第二版第二版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 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 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 at each fork. Primase is associated with helicase.Primase lays down an on the leading strand. Primase lays down a primer on the laggingstrand. This a primosome Primers must 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 synthesizing leading & lagging strands.Tau subunit of Pol III binds to helicase.Coordination of replication of the leading and lagging strandsb b Clamp loader g Complex of Pol III holoenzyme( g 2 , d, d, c, psi) 1. Uses ATP to open dimer and position it at 3 -end of primer.2. “Loaded” clamp then binds Pol III core (and releases from ).3. Processive DNA synthesis. - loads b subunit dimer onto primerOrder of eventsRecycling phase1.Once Okazaki fragment completed, clamp releases from core.2. binds to .3. unloads b clamp from DNA.4. clamp recycles to next primer. Loading and recyling of clampTermination 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 substance (Tus) binds to ter region.*Tus prevents replication fork from passing by inhibiting helicase activity.Terminating DNA synthesis in prokaryotesFig. 21.27EEach fork stops at the Ter regions, which are 22 bp, 3 copies, and bind the Tus protein.Separation of two daughter DNAsEukaryotic DNA Replication Like E. coli, but more complex lEffect of Chromatin and Nucleosome on ReplicationlMultiple origins of replication lDNA replication occurs just at S phase of the cell cycle and is controlled by many proteins lOkazaki fragments are shorter than in ProkaryoteslReplication forks run a slower speed than in ProkaryoteslTwo rounds of replication cannot occur at the same timelTelomerase is requiredThe eukaryotic cell cycleLicensing: 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 the ORC and are essential for coating the DNA with MCM proteins. Only DNA coated with MCM proteins (there are 6 of them) can be replicated. Once replication begins in S phase, Cdt1 and Cdc6 leave the ORCs The MCM proteins leave in front of the advancing replication fork. MCM: Mini-Chromosome MaintenanceCdc: Cell Division CycleThe initiation of DNA replication in eukaryotic cellsProblematic issues with nucleosome replicationEukaryotic DNA PolymerasesA model for eukaryotic chromosome replication?Unwinding at origin of replication?DNA- prim initiates DNA synthesis?PCNA, RF-C, pold, bind?Polymerase switch on lagging strand DNA pol has a sliding clamp (PCNA) and a clamp-loader (RFC). It replicates the leading strandRPA = SSBDNA pol is not very processive, but has a built-in primase activity. It starts off replication at both strands, but the strands are probably extended by the more processive DNA pol.A model for eukaryotic DNA replicationActions of several eukaryotic replication proteinsRemoval of RNA primers in eukaryotesRolling circle Replication Examples: X174 phage Features 1) + strand nicked by gpA (gene protein A) 2) - strand acts as template 3) 3 end of + strand is extended for 1 genome length 4) full genome nicked off of + strand & circularizes as SS formModel of Rolling circle ReplicationD-loop ReplicationExamples: Mt DNA and Plastid DNAFeatures1. Two origins: one is for H strand and the 2. other for L strand.3. Both strands are synthesized continuously. 4. Form “D-loop” intermediate.5. Synthesis of the second strand is initiated after the fork synthesizing the first strand passes the origin of second strand synthesis. DiagramD-loop Replication of mtDNAReplication in Archaea1. The process of replication in archaebacteria has a number of features in common with replication in eukaryotic cells;2. many of the proteins taking part are more similar to those in eukaryotic cells than to those in bacteria. 3. Like bacteria, some archaea have a single replication origin, but the archaean Sulfolobus solfataricus has two origins of replication, similar to the multiple origins seen in eukaryotic genomes. 4. The replication origins of archaea do not contain the typical sequences recognized by bacterial initiator proteins; instead, they have sequences that are similar to those found in eukaryotic origins. 5. The initiator proteins of archaea also are more similar to those of eukaryotes than those of bacteria. 6. These similarities in replication between archaeal and eukaryotic cells reinforce the conclusion that the archaea are more closely related to eukaryotic cells than to the prokaryotic bacteria.High Fidelity of ReplicationCBalanced levels of dNTPs.CHigh selectivity of DNAPs based on Watson-Crick base pairing (to the template base)CProofreading of DNAPs by means of their 3 -5 exonuclease.CMismatch repair system.CRNA primers are removed by highly accurate Pol I enzyme.DNA polymerase error rates$Initial pairing error = 1/105 $After proofreading = 1/1071/108$mismatch repair = 1/10101/1011$Human genome = 3.2 x 109 bp$3 errors/replication!A Poem (English version)Replicative errors are no big deal,As long as editing is right.Even occurs one mistake,Repairing enzymes will correct it later.Rongwu Yang (Inspired by DNA replication)