DNA Replication IBiochemistry 302Overview of Information MetabolismWatson Crick prediction:Each stand of parent DNA serves as a template for synthesis of a new complementary daughter strandPlausible models of gene replicationProof of semiconservative DNA replicationDNA Replication: the early years…What was learn and not learnedBiochemical & genetic approaches led to notion of bidirectional replication from a fixed originTermination and re-initiation occur on opposite sides of E. coli chromosomeBasic features of DNA replicationEnzymes and other proteins involved in DNA replication(some multi-subunit complexes)Requirement for 5?? to 3? synthesis imposes a topological problemAnatomy of a replication fork: leading and lagging strand synthesisDirectionality of DNA synthesis: a very simple model of a replication forkChromosomes from different cell types exhibit different replication parametersBasic features of E. coli DNA replication continued….DNA Pol I: the first polymerase(discovered by A. Kornberg, 1950s Nobel prize 1959)Closed right hand structure common among Klenow family of DNA Pols (active site lies in palm domain)Molecular basis of primer-template recognition and proofreadingDNA polymerase active site opens and closesTwo metal ion mechanism of catalysis by DNA polymerases (T. Steitz model)Lesson learned from structural analysesSummary of DNA Replication IDNA Replication IBiochemistry 302Bob KelmJanuary 26, 2004Overview of Information Metabolism• Replication (copying)• Transcription (readout)• Translation (decoding)• Template-dependent (DNA or RNA)– Initiation (point of regulation, generally)– Chain elongation– TerminationFig. 4-23Watson Crick prediction:Each stand of parent DNA serves as a template for synthesis of a new complementary daughterstrandFig. 4.12Plausible models of gene replicationFig. 4.13with respect to parental duplex DNAProof of semiconservative DNA replicationMeselson-Stahl, 1958 Differential isotopic labeling of E. coli DNA coupled with density gradient centrifugation to equilibriumheavylightFig. 4.14hybrid¾ L¼ HDNA Replication: the early years…What was learn and not learned• Watson and Crick model predicted semi-conservative DNA replication.• Meselson-Stahl experiment biochemically confirmed this notion.– Did not answer whether replication was sequential (with DNA unwinding) and ordered (in terms of direction).– Could not answer whether DNA replication was initiated at only one or more fixed points (or origins) on a chromosome.Biochemical & genetic approaches led to notion of bidirectional replication from a fixed origin• First studied by whole chromosome radiolabeling experiments. John Cairns visualized nascent E. coliDNA synthesis.• Subsequent work utilized denaturation mapping to visualize bidirectional replication of λ phage DNA.• Other genetic experiments relied on the copy number of inherited marker genes to establish bidirectionality.Figure shows multiple sequential initiation with solid line radiolabeled.Fig. 24.14Termination and re-initiation occur on opposite sides of E. coli chromosomeradioautogramFig. 24.12Higher specific activity [3H]thymidine added at end of replication cycleBasic features of DNA replication• Semi-conservative• Ordered and sequential– Starts at a fixed point. – Synthesis of new daughter strands is in 5′→3′direction and follows parental DNA unwinding.• Utilizes “activated” substrates (dNTPs)• Discontinuous– Daughter strands grow in opposite directions to maintain anti-parallel polarity w/ template strand.• Extremely accurateEnzymes and other proteins involved in DNA replication(some multi-subunit complexes)• DNA Polymerases• ssDNA-binding proteins• Helicases• Primase• Topisomerases• DNA LigaseBasic chemistry of DNA synthesis• Incoming dNTP is positioned by base-pairing with template nucleotide.• DNA Pol catalyzes reaction between the terminal 3′ OH on the primer strand and the 5′ α PO3of dNTP to form 3′− 5′ PDE bond.• Release and hydrolysis of pyrophosphate drives the reaction energetically.• Consequently, chain growth can only occur in one direction.Fig. 24.2Requirement for 5 ′ to 3′ synthesis imposes a topological problem• Two strands of DNA duplex are anti-parallel but both parental strands are replicated in the same “fork.”• If fork movement is to occur in a concerted manner, there must be a mechanism for replicating each strand in 3′→ 5′ direction.• Solution: Two DNA polymerase molecules (Pol III, E. coli) one catalyzing forward (leading strand) synthesis and the other backward (lagging strand) synthesis.Anatomy of a replication fork: leading and lagging strand synthesisOverall growth of lagging strand pieces (Okazaki fragments) is 3′→5′.3′3′5′5′UnwindingPrimingExtendingEditingStitchingFig. 24.3continuousdiscontinuousFigure from Kornberg and Baker “DNA Replication”Directionality of DNA synthesis: a very simple model of a replication forkDNA polymerase III, replicates as leading and lagging strand dimerLeading strandLagging strandRNA primerprimasomeFork movementLagging strand synthesis requires repeated RNA priming due to physical constraint imposed by PolIII dimerization at the fork.Fig. 24.1Chromosomes from different cell types exhibit different replication parameters • Bacterial chromosomes: single origin (E. coli ~4.6 x 106bp)• Eukaryotes: multiple origins on a single chromosome (103-104 total /cell) • Replication in bacteria is 10-fold faster than in eukaryotes: 850 nt/sec/fork vs 60-90 nt/sec/fork• Doubling time of bacteria ∼20-30 minutes vs 24 hours in a “typical” eukaryotic cell (HeLa)• E. coli can completely replicate its genome in 40 min, 8 hours for a HeLa cell (S phase of interphase)• Bacteria re-initiates more frequently than the doubling time – Daughter cell receives a chromosome well into its next round of replication to adapt to rapid growth.– “Replicon firing” earlier in the cell cycle implicates initiation as the key control point in DNA replication.Basic features of E. coli DNA replication continued….• Replication is an enzymatic reaction.– Substrates: dNTPs (also need primer-template)– Enzymes: Polymerases (Pol III: rapid synthesis and Pol I: editing) along with topoisomerases, helicases, primase, and DNA ligase • Three main phases:– Initiation: formation of specific protein:DNA complex at specific origins (ori): highly regulated–