DNA Replication IIBiochemistry 302Conceptual model for proofreading based on kinetic considerationsFollowing in Dad’s footsteps…Summary of E. coli DNA PolymerasesSubunit composition of DNA Pol III holoenzyme (10 different polypeptides)Model for converting DNA Pol III from a distributive to a processive enzyme: clamp loadingA slightly more detailed view of the replication fork (J. Biol. Chem. 273:24550, 1998)Overview of E. coli replication fork proteins and their functional rolesMechanism of action of type I and II topoisomerasesImportant biochemical features of replication fork proteinsImportant functional features of replication fork proteins continued:Important functional features of replication fork proteins continued:Summary of key fork-proximal events in E. coli DNA replicationStructural requirements for initiation of DNA replication in E. coliModel for initiation of DNA replication in E. coli (only one origin, oriC)Termination of DNA replication in E. coli occurs at specific sitesCharacteristics of initiation of DNA replication in eukaryotesKinetic features/enzymology of eukaryotic DNA replicationProperties of eukaryotic DNA polymerases (different enzymes for leading/lagging strand synthesis?)Problematic issues with nuclear genomes and linear chromosomes: nucleosome replication and 5? gapsMechanisms for ensuring fidelity of DNA replicationSummary of DNA Replication IIDNA Replication IIBiochemistry 302Bob KelmJanuary 28, 2004Conceptual model for proofreading based on kinetic considerationsFig. 24.44stalling → transient melting → exonuclease site occupancyFollowing in Dad’s footsteps…• Original A. Kornberg E. coli DNA Pol I is a lousy replicative enzyme.– 400 molecules/cell but < 10 replication forks/cell– Vmax∼20 nt/sec– Processivity of ∼3-200 nt/encounter• So there must be more processive DNA Pol enzymes or accessory factors….J. Cairns,1969 isolated E. coli ts mutant w/o Pol I polymerase and 3′ exonuclease activity but w/ 5′ exo activity.– Live, divide, and replicate DNA normally– Sensitive to UV light and alkylating agents• R. Kornberg eventually isolated Pol II and Pol III.Summary of E. coli DNA PolymerasesTable 24.2 DNA Polymerases of E. coliSubunit composition of DNA Pol III holoenzyme (10 different polypeptides)• Subunits α, ε, θ = core polymerase; α = PolC; ε = 3′exonuclease; θ ?• Dimeric τ dimerizes the holoenzyme holding the lead and lag strand Polymerases together at the rep fork.• The β subunitor sliding clamp tethers the enzyme to DNA to ↑processivity from 10 to 105.• The γ or clamp loadercomplex: γ (2), δ, δ′, χ, and ψloads β onto DNA.• The χ subunit mediates switch from RNA primers to DNA.αβτ(tau)εθ (theta)γδ δχ(chi)ψ(psi)‘Fig. 24.19Model for converting DNA Pol III from a distributive to a processive enzyme: clamp loadingγδδ′χψβHingorani and O’Donnell (1998) J. Biol. Chem. 273:24550Fig. 24.21“Primed” DNA strand• 1: ATP binding to γ dimer promotes conformational change (opening) of clamp loader complex. • 2: Opening permits clamp loader complex binding to DNA and δ - βinteraction.• 3: γ forces the subunits of β apart.• 4: β encircles DNA and ATP hydrolysis drives ring closure and dissociation of clamp loader complex.A slightly more detailed view of the replication fork (J. Biol. Chem. 273:24550, 1998)• Clamp loading need only occur once per round of DNA replication on leading strand.• Clamp loading must occur multiple times on lagging strand because Pol III needs to rebind at the initiation of synthesis of each Okazaki fragment.DiscontinuousContinuousFigure from Ken MariansOverview of E. coli replication fork proteins and their functional roles• Topoisomerase I: relieve superhelical stress• Helicases: helical unwinding in fixed direction DnaB 5′→3′; Rep 3′→5′• Primase (DnaG): RNA primer synthesis – 5 mer in T4, 11 mer in E. coli. DnaB and DnaG interact.• SSB: maintain template strands in ssDNA configuration • DNA Pol III holoenzyme: lead/lag strand synthesis• DNA Pol I: removal of RNA primers, gap filling• DNA ligase: joins nicks in dsDNA following Pol I proofreadingMechanism of action of type I and II topoisomerasesrelaxationCatenation/decatenationknotting/unknottingType I (change L by 1) Double-strand break(topo II and IV)DNA gyraseFig. 24.33Fig. 24.30Type II (change L by 2)Single-strand break (topo I and III)Important biochemical features of replication fork proteins• Helicases (dsDNA unwinding)– Helicases usually bind to ssDNA near duplex region and move in a fixed direction either 5′→3′ or 3′→′5. – Movement requires ATP hydrolysis (some use dTTP).– Different mechanisms account for movement of homo-oligomeric dimers (E. coli) vs hexameric or ring helicases (T7). • Ring helicases wrap around one strand displacing the other strand while rotating along the DNA. • Homodimeric helicases “roll” along the DNA mediated by sequential ATP binding and hydrolysis.Fig. 24.27Rep helicase actionImportant functional features of replication fork proteins continued:• Primase (DnaG):– Creates new RNA primers for each round of discontinuous synthesis & first round of leading strand synthesis). – Requires the action of the DnaB helicase. In T4 and E. coli, primers are 5 and 11 nt in length, respectively.• SSB – Binds ssDNA in a highly cooperative but non-specific manner as a tetramer.– Required for DNA replication, repair, and recombination. – Does not denature dsDNA per se but binds to ssDNA after dsDNA is partially unwound.Fig. 24.6Important functional features of replication fork proteins continued:• Pol III and Pol I– III:replicative DNA synthesis– I:RNA primer removal and gap filling• DNA ligase– Seals dsDNA “nicks” between Okazaki fragments.– Nucleotides must be adjacent and properly base-paired.– Activates the 5′terminal phosphate on the DNA substrate by adenylation.Fig. 24.24Summary of key fork-proximal events in E. coli DNA replicationHead of the fork: Topoisomerases relieve stress; helicases melt the duplex, SSB coats ssDNALeading strand: Addition of new dNTPs to 3′ end of primer - synthesis in same direction as fork movementLagging strand: Primase synthesizes RNA primers, Pol III extends to previous Okazaki fragment, dissociates, then reassembles at the 3′ end of a new RNA primerGap filling: Removal of RNA primers and filling in of gaps by Pol I, Nick