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Berkeley MCELLBI 110 - Accessory factors summary

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PowerPoint PresentationAccessory factors summarySlide 3Maturation of Okazaki fragmentsSlide 5Starting and stopping summaryIsolating DNA sequences that mediate initiationDifferent origin sequences in different organismsInitiation in prokaryotes and eukaryotesCrystal structure of DnaA:ATP revealed mechanism of origin assemblySlide 11Initiation mechanism in bacteria -- 1Initiation mechanism in bacteria -- 2Initiation proteins in E. coli (bacteria)10 ter sites opposite oriC coordinate the end gameUnwinding ter from the “nonpermissive” direction springs a “molecular mousetrap”Slide 17Slide 18Unwinding ter from the nonpermissive direction springs a “molecular mousetrap”Topoisomerase II unlinks the replicated chromosomesSummary: What problems do these proteins solve?Slide 22The ends of (linear) eukaryotic chromosomes cannot be replicated by the replisome.Telomere shortening signals trouble!Telomerase replicates the ends (telomeres)Telomerase cycles at the telomeresSlide 27Conserved structures in TER and TERTSlide 29Accessory 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 (/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 holoenzymeQuickTime™ and aDV - PAL decompressorare needed to see this picture.Maturation of Okazaki fragmentsTopoisomerases control chromosome topologyCatenanes/knotsRelaxed/disentangled•Major therapeutic target - chemotherapeutics/antibacterials•Type II topos transport one DNA through another ToposStarting 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 the DnaB helicase by DnaC (helicase loader)4. DnaA and DnaC reactions are coupled to ATP hydrolysis.5. Bacterial chromosomes are circular, and termination occurs opposite OriC.6. In E. coli, the helicase inhibitor protein, tus, binds 7 ter DNA sites to trap the replisome at the end.7. Eukaryotic chromosomes are linear, and the chromosome ends cannot 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 initiationDifferent origin sequences in different organisms E. Coli (bacteria)OriCYeastARS(Autonomously Replicating Sequences)Metazoans ????Initiation in prokaryotes and eukaryotesBacteriaEukaryotesORC + other proteins loadMCM hexameric helicasesMCM (helicase) + RPA (ssbp)Primase + DNA pol PCNA:pol MCM (helicase) + RPA (ssbp)PCNA:pol  (clamp loader)Primase + DNA pol PCNA:pol DNA ligaseCrystal structure of DnaA:ATP revealed mechanism of 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 mechanism of origin assembly1. The arrangement of DNA binding sites introduces positive supercoils by wrapping DNA on the outside. Compensating negative supercoils melt the replication bubble at the end.2. Clamp deposition recruits Had, which promotes ATP hydrolysis and progressive disassembly of the DnaA filament (hypothesis). 1. 2.Initiation mechanism in bacteria -- 1Initiation mechanism in bacteria -- 2Initiation 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 binds Ter sites and inhibits the DnaB helicaseOriginCounterclockwiseforkClockwiseforkClockwisefork trapCounterclockwisefork trapUnwinding ter from the “nonpermissive” direction springs 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” direction springs 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.Unwinding ter from the “nonpermissive” direction springs a “molecular mousetrap”Releasing C6 springs the trap Mulcair et al. (2006) Cell 125, 1309-1319.Unwinding ter from the nonpermissive direction springs a “molecular mousetrap”Releasing C6 springs the trap Mulcair et al. (2006) Cell 125, 1309-1319.Topoisomerase II unlinks the replicated chromosomesTopoisomerase II - Cuts DNA and passes one duplex through the other.Class II topoisomerases include:Topo IV and DNA gyraseSummary: What problems do these proteins solve?Tyr OH attacks PO4 and forms a covalent intermediateStructural changes in the protein open the gap by 20 Å!Function E. coli SV40 (simian virus 40)Helicase DnaB T antigenPrimasePrimer removalPrimase (DnaG)pol I’s 5’-3’exopol  primaseFEN 1 (also RNaseH)Polymerase Core pol III (, ,  subunits)pol ,  Clamp loader complexRF-C Sliding clampPCNAssDNA binding SSB RF-ARemove +sc at fork (swivel) gyrase topo I or topo IIDecatenation topo IV topo IILigase DNA ligase DNA ligase I… other model systems include bacteriophage T4 and yeastSummary: What problems do these proteins solve?The ends of (linear) eukaryotic chromosomes cannot be replicated by the replisome.Not enough nucleotides for primase to start last lagging strand fragmentChromosome ends shorten every generation!Telomere shortening signals trouble!1. Telomere shortening releases telomere binding proteins (TBPs)2. Further shortening affects expression of telomere-shortening sensitive genes3. Further shortening leads to DNA damage and mutations.Telomere binding proteins (TBPs)Telomerase replicates the ends (telomeres)Telomere ssDNATelomerase extends the leading strand!Synthesis is in the 5’-->3’ direction.Telomerase is a ribonucleoprotein (RNP). The enzyme contains RNA and proteins.The RNA templates DNA synthesis.


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Berkeley MCELLBI 110 - Accessory factors summary

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