DNA polymerase IIIDNA polymerase III = DNA Pol IIIDNA Pol III: Low abundance but high processivityProcessivitySubunits of DNA Pol III and functionsSubassemblies have distinct functionsProcessivity factor beta2: Sliding clampGamma complex: Clamp loader/unloaderLoading the beta2 clampUnloading the beta2 clampModel for gamma complex loading beta clampAsymmetric dimer of DNA PolIII: simultaneous replication of both strands of DNAOne holoenzyme, 2 templatesSimultaneous replication of both strands of DNAEukaryotic replicative DNA polymerasesEukaryotic DNA polymerases in replicationSimilarities between bacterial and eukaryotic replication machineryEnzymes other than polymerases needed for replicationModel for replication fork movement in E. coliDNA helicasesAssay for helicase movement, #1Assay for helicase movement, #2Single-stranded binding protein (SSB)TopoisomerasesDNA ligasesMechanism of DNA ligasePrimasePrimers made by DnaGAssembly and migration of the primosomePrimosome has many proteinsAssay for assembly and migration of the primosomeSteps in priming and synthesisActivities of DnaB and PriA in replisomeRate of fork movementDNA polymerase IIIEnzyme used during replicationMultisubunit proteinHigh processivityDNA polymerase III = DNA Pol III•Discovered in extracts of polA- cells, i.e. lacking DNA Pol I•DNA Pol III is the replicative polymerase•Loss-of-function mutations in the genes encoding its subunits block DNA replication (dna mutants)•Highly processive•Multiple subunits•Also discovered DNA Pol II in polA- extracts (role in DNA repair)DNA Pol III: Low abundance but high processivityComparison Pol I Pol III core Pol III holomolecules per cell 400 40 10nts polymerized min-1(molecule enz)-1600 9000 42,000processivity [nts polymerized per initiation] 3-188 10 >1055' to 3' polymerase + + +3' to 5' exo, proofreading + + +5' to 3' exo + - -Processivity•Amount of polymerization catalyzed by an enzyme each time it binds to a template.•Measured in nucleotides polymerized per initiation•High processivity of DNA Pol III results from activities of non-polymerase subunitsFunctional Masscomponent Subunit (kDa) Gene ActivityCore polymerase  129.9 polC=dnaE 5' to 3' polymerase 27.5 dnaQ=mutD 3'-5' exonuclease 8.6 Stimulates  exonucleaseLinker protein  71.1 dnaX Dimerizes coresClamp loader  47.5 dnaX Binds ATP (aka  complex)  38.7 Binds to  (ATPase) ' 36.9 Binds to  and  16.6 Binds to SSB 15.2 Binds to  and Sliding clamp  40.6 dnaN Processivity factorSubunits of DNA Pol III and functionsSubassemblies have distinct functionsββαεθαεθδδ'ΨγχγττββLeading strand synthesisLagging strand synthesisPol III* subassembly lacks the beta sliding clamp.The γ complex loads and unloads the beta clamp, cyclically for lagging strand synthesis.Processivity factor beta2: Sliding clamp•The  subunit forms a homodimer.•The structure of this homodimer is a ring.•The ring encloses DNA, thereby clamping the DNA Pol III holoenzyme to the template.•An enzyme that is clamped on cannot come off easily, and thus will be highly processive.Gamma complex: Clamp loader/unloader•The  complex (2' ) loads the  dimer clamp onto a primer-template.–Bind the clamp ( dimer) onto the loader ( complex): need ATP–Exchange the clamp from the loader to the core: need ATP hydrolysis–Unload the clamp when polymerase reaches a previously synthesized Okazaki fragment: need ATP •The ATP-bound form of the  complex can bind the clamp•The ADP-bound form releases the clampLoading the beta2 clampATPPreviously synthesized Okazaki fragmentPrimerTemplatecoreβ2 clamp+load β clamp onto primer templatedissociate holder from clampATPexchange clamp onto Pol III coreATP hydrolysis by γ complexprocessive DNA synthesisADP + Piγ complexUnloading the beta2 clampprocessive DNA synthesisdissociate β clamp from Pol IIIexchange β clamp onto γ complexdissociate and recycle Pol III and clampbind ATPATPATPATP+Model for gamma complex loading beta clampJeruzalmi, O’Donnell and Kuriyan (2001) Cell 106: 429-441Asymmetric dimer of DNA PolIII: simultaneous replication of both strands of DNA•The 2 catalytic cores of DNA Pol III are joined by the tau subunits to make an asymmetric dimer.•Model: one holoenzyme synthesizes both strands at a replication fork.–One core synthesizes the leading strand–Other synthesizes the lagging strand.–If the template for lagging strand synthesis is looped around the enzyme, then both strands are synthesized in the direction of fork movement.One holoenzyme, 2 templates 5'3'5'helicasepolymerase for leading strand3'polymerase for lagging strandSSBcompleted Okazaki fragment #1primaseRNA primer =Simultaneous replication of both strands of DNA5'3'5'helicasepolymerase for leading strand3'polymerase for lagging strandSSBcompleted Okazaki fragment #1primaseRNA primer =5'3'5'3'Loop from the template for lagging strand ("trombone slide") gets longer as the replisome proceeds.completed Okazaki fragment #25'3'5'3'completed Okazaki fragment #2Large loop is released upon completion of the Okazaki fragment, and a smaller one is formed to allow elongation from the next primer.primer #3primer #4Eukaryotic replicative DNA polymerases•Nuclear DNA replication:: primase plus low processivity polymerase: both leading and lagging strand synthesis: may be used in lagging strand synthesisEukaryotic DNA polymerases in replication δ δ 5'3'5'helicaseDNA polymerase δ is required for both leading and lagging strand synthesis. 3'RFA = SSBcompleted Okazaki fragmentRNA primer =5'3'PCNA processivity factorSimilarities between bacterial and eukaryotic replication machineryFunction E. coli Pol III EukaryoticLeading and lagging asymmetric polymerase  strand synthesis dimerSliding clamp  subunit PCNAClamp loader -complex RFCPrimase DnaG polymerase Single strand binding SSB RFASwivel Gyrase (Topo II) Topo I or II (Maintain DNA topology)Enzymes other than polymerases needed for replicationHelicasesLigasesPrimosomeModel for replication fork movement in E. coli5'3'5'DnaB helicaseDNA polymerase III for leading strand3'DNA polymerase III for lagging strand; includes γ complexSSBcompleted Okazaki fragment DnaG primaseRNA primer =5'3'includes PriA, PriB, PriC, DnaT, DnaB helicase and DnaG primasePrimosome = 3'DNA PolIto exciseand replaceRNA primersDNA