1Accessory factors summary1. DNA polymerase can’t replicate a genome.Solution ATP?No single stranded template Helicase +The ss template is unstable SSB (RPA (euks)) -No primer Primase (+)No 3’-->5’ polymerase Replication forkToo slow and distributive SSB and sliding clamp -Sliding clamp can’t get on Clamp loader (γ/RFC) +Lagging strand contains RNA Pol I 5’-->3’ exo, RNAseH -Lagging strand is nicked DNA ligase +Helicase introduces + supercoils Topoisomerase II +and products tangled2. DNA replication is fast and processiveDNA polymerase holoenzyme2Maturation of Okazaki fragmentsTopoisomerases control chromosome topologyCatenanes/knotsRelaxed/dis entangled•Major therapeutic target - chemotherapeutics/antibacterials•Type II topos transport one DNA through another Topos3Starting and stopping summary1. DNA replication is controlled at the initiation step.2. DNA replication starts at specific sites in E. coli and yeast.3. In E. coli, DnaA recognizes OriC and promotes loading of theDnaB helicase by DnaC (helicase loader)4. DnaA and DnaC reactions are coupled to ATP hydrolysis.5. Bacterial chromosomes are circular, and termination occursopposite OriC.6. In E. coli, the helicase inhibitor protein, tus, binds 7 ter DNAsites to trap the replisome at the end.7. Eukaryotic chromosomes are linear, and the chromosome endscannot be replicated by the replisome.8. Telomerase extends the leading strand at the end.9. Telomerase is a ribonucleoprotein (RNP) with RNA (template)and reverse-transcriptase subunits.Isolating DNA sequences that mediate initiation4Different origin sequences in different organismsE. Coli (bacteria)OriCYeastARS(Autonomously Replicating Sequences)Metazoans ????Initiation in prokaryotes and eukaryotesBacteriaEukaryotesORC + other proteins loadMCM hexameric helicasesMCM (helicase) + RPA (ssbp)Primase + DNA pol αPCNA:pol δ + RFCMCM (helicase) + RPA (ssbp)PCNA:pol δ + RFC (clamp loader)Primase + DNA pol αPCNA:pol δ + DNA ligase5Crystal structure of DnaA:ATP revealed mechanismof origin assembly1. DnaA monomer (a) forms a polar filament (b).2. DNA binding sites occur on the outside of the filament (model).1. 2.Crystal structure of DnaA:ATP revealed mechanismof origin assembly1. The arrangement of DNA binding sites introduces positive supercoils bywrapping DNA on the outside.Compensating negative supercoils melt the replication bubble at the end.2. Clamp deposition recruits Had, which promotes ATP hydrolysis andprogressive disassembly of the DnaA filament (hypothesis).1. 2.6Initiation mechanism in bacteria -- 1Initiation mechanism in bacteria -- 27Initiation proteins in E. coli (bacteria)10 ter sites opposite oriC coordinate the end gameThe ter/tus system is not essential in E. coli. Tus protein bindsTer sites andinhibits the DnaBhelicaseOriginCounterclockwiseforkClockwiseforkClockwisefork trapCounterclockwisefork trap8Unwinding ter from the “nonpermissive” directionsprings a “molecular mousetrap”Releasing C6 springs the trap DNA Half life (s) Kd (nM)130 (2 min) 1.653 (<1 min, FAST/ 53permissive)6900 (115 min, SLOW/ 0.4 nonpermissive)terBC6C6C6Mulcair et al. (2006) Cell 125, 1309-1319.Unwinding ter from the “nonpermissive” directionsprings a “molecular mousetrap”Releasing C6 springs the trap DNA Half life (s) Kd (nM)130 (2 min) 1.653 (<1 min, FAST/ 53permissive)6900 (115 min, SLOW/ 0.4 nonpermissive)terBC6C6C65’3’Mulcair et al. (2006) Cell 125, 1309-1319.9Unwinding ter from the “nonpermissive” directionsprings a “molecular mousetrap”Releasing C6 springs the trap Mulcair et al. (2006) Cell 125, 1309-1319.Unwinding ter from the nonpermissive directionsprings a “molecular mousetrap”Releasing C6 springs the trap Mulcair et al. (2006) Cell 125, 1309-1319.10Topoisomerase II unlinks the replicatedchromosomesTopoisomerase II - Cuts DNAand passes one duplex throughthe other.Class II topoisomerases include:Topo IV and DNA gyraseSummary: What problems do these proteins solve?Tyr OH attacksPO4 and forms acovalentintermediateStructuralchanges in theprotein open thegap by 20 Å!11DNA ligase IDNA ligaseLigasetopo IItopo IVDecatenationtopo I or topo IIgyraseRemove +sc at fork (swivel)RF-ASSBssDNA bindingPCNAβ Sliding clampRF-Cγ complex Clamp loaderpol δ, εpol III (α, ε, θ subunits) CorePolymerasepol α primaseFEN 1 (also RNaseH)Primase (DnaG)pol I’s 5’-3’exoPrimasePrimer removalT antigenDnaBHelicaseSV40 (simian virus 40)E. coliFunction… other model systems include bacteriophage T4 and yeastSummary: What problems do these proteins solve?The ends of (linear) eukaryotic chromosomescannot be replicated by the replisome.Not enoughnucleotides forprimase to startlast laggingstrand fragmentChromosome endsshorten everygeneration!12Telomere shortening signals trouble!1. Telomere shortening releasestelomere binding proteins (TBPs)2. Further shortening affectsexpression of telomere-shortening sensitive genes3. Further shortening leads toDNA damage and mutations.Telomere binding proteins (TBPs)Telomerase replicates the ends (telomeres)Telomere ssDNATelomerase extendsthe leading strand!Synthesis is in the5’-->3’ direction.Telomerase is aribonucleoprotein(RNP). The enzymecontains RNA andproteins.The RNA templatesDNA synthesis. Theproteins include thetelomerase reversetranscriptase TERT.13Telomerase cycles at the telomeresTelomere ssDNATERT proteinTER RNA templateTelomerase extends a chromosome 3’ overhang14Conserved structures in TER and TERTCore secondarystructures shared inciliate andvertebratetelomerase RNAs(TERs). (Sequenceshighly variable.)148-209 nucleotides1000s of nucleotidesTERT proteinsequences conserved1300 nucleotidesStarting and stopping summary1. DNA replication is controlled at the initiation step.2. DNA replication starts at specific sites in E. coli and yeast.3. In E. coli, DnaA recognizes OriC and promotes loading of theDnaB helicase by DnaC (helicase loader)4. DnaA and DnaC reactions are coupled to ATP hydrolysis.5. Bacterial chromosomes are circular, and termination occursopposite OriC.6. In E. coli, the helicase inhibitor protein, tus, binds 7 ter DNAsites to trap the replisome at the end.7. Eukaryotic chromosomes are linear, and the chromosome endscannot be replicated by the replisome.8. Telomerase extends the leading strand at the end.9. Telomerase is a ribonucleoprotein (RNP) with RNA (template)and reverse-transcriptase
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