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4.5.Rep.Forks.Ppt [Read-Only] DNA polymerase summary 1. DNA replication is semi-conservative. 2. DNA polymerase enzymes are specialized for different functions. 3. DNA pol I has 3 activities: polymerase, 3’-->5’ exonuclease & 5’-->3’ exonuclease. 4. DNA polymerase structures are conserved. 5. But: Pol can’t start and only synthesizes DNA 5’-->3’! 6. Editing (proofreading) by 3’-->5’ exo reduces errors. 7. High fidelity is due to the race between addition and editing. 8. Mismatches disfavor addition by DNA pol I at 5 successive positions. The error rate is ~1/109. Replication fork summary 1. DNA polymerase can’t replicate a genome. Problem Solution ATP? No single stranded template Helicase + The ss template is unstable SSB (RPA (euks)) - No primer Primase (+) No 3’-->5’ polymerase Replication fork Too slow and distributive SSB and sliding clamp - 2. Replication fork is organized around an asymmetric, DNA- polymerase III dimer. 3. Both strands made 5’-->3’. 4. “Leading strand” is continuous; “lagging strand” is discontinuous. 1 DNA polymerase can’t replicate a genome! 1. No single stranded template 2. The ss template is unstable 3. No primer 4. No 3’-->5’ polymerase 5. Too slow and distributive Solution: the replication fork 1. No single-stranded template 2. The ss template is unstable 3. No primer 4. No 3’-->5’ polymerase 5. Too slow and distributive Schematic drawing of a replication fork 2 DNA polymerase holoenzyme DNA replication factors were discovered using “temperature sensitive” mutations 1. No single stranded template 2. The ss template is unstable 3. No primer 37 ºC 4. No 3’-->5’ polymerase. 5. Too slow in vitro. Mutations that inactivate 42 ºC the DNA replication machinery are lethal. 42 ºC, Temperature sensitive Mutant gene (conditional) mutations overexpressed allow isolation of mutations in essential genes. 3 A hexameric replicative helicase unwinds DNA ahead of the replication fork Helicase assay 1. No single stranded template 2. The ss template is unstable ds DNA 3. No primer 4. No 3’-->5’ polymerase. 5. Too slow in vitro. Replicative DNA helicase is called DnaB in E. coli. DnaB couples ATP binding and hydrolysis to DNA strand separation. ss DNA SSB (or RPA) cooperatively binds ss DNA template 1. No single stranded template 2. The ss template is unstable SSB (single-strand binding protein (bacteria)) or RPA (Replication 3. No primer Protein A (eukaryotes)): 4. No 3’-->5’ polymerase. No ATP used. 5. Too slow in vitro. Filament is substrate for DNA pol. ss DNA + SSB ds DNA 4 SSB tetramer structure 1. No single stranded template 2. The ss template is unstable SSB (bacteria) and RPA (eukaryotes) form tetramers. 3. No primer The C-terminus of SSB binds 4. No 3’-->5’ polymerase. replication factors (primase, clamp 5. Too slow in vitro. loader (chi subunit)) C C N N N N ss DNA + SSB C C ds DNA Conservation Positive potential DNA synthesis is primed by a short RNA segment 1. No single stranded template 2. The ss template is unstable 3. No primer Primase: DNA-dependent RNA 4. No 3’-->5’ polymerase. polymerase 5. Too slow in vitro. Primase makes about 10-base RNA. The product is a RNA/DNA hybrid. RNA primer has a free 3’OH. Start preference for CTG on template Uses ATP, which ends up across from T in the RNA/DNA hybrid. 5 DnaG primase defines a distinct polymerase family (DNA dependent RNA pol) Model of Ribbon “primosome”: diagram DnaB helicase + DnaG primase DnaB helicase Map of surface charge DnaG primase Primase passes the primed template to DNA polymerase Leading strand: continuous Lagging strand: discontinuous 6 DNA pol III “holoenzyme” is asymmetric 1. No single stranded template DNA pol III holoenzyme: 2. The ss template is unstable A molecular machine 3. No primer 4. No 3’-->5’ polymerase. 5. Too slow in vitro. Synthesizes Synthesizes Leading Lagging Strand Strand χ binds SSB δ opens clamp (β) Pol III dimer couples leading and lagging strand synthesis Leading Lagging strand strand 7 Replication fork 1. No single stranded template 2. The ss template is unstable 3. No primer 4. No 3’-->5’ polymerase 5. Too slow and distributive Replication fork 1. No single stranded template 2. The ss template is unstable 3. No primer 4. No 3’-->5’ polymerase 5. Too slow and distributive 8 Sliding clamp wraps around DNA N C Sliding clamps are structurally conserved “Palm” 9 Summary of the replication fork “Palm” Synthesis of Okazaki fragments by pol III holoenzyme When pol III reaches the primer of the previous Okazaki fragment, clamp loader removes β2 from the DNA template. As a result, the pol III on the lagging strand falls off the template. Clamp loader places β2 on the next primer-template. 10 Replication fork summary 1. 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 fork Too slow and distributive SSB and sliding clamp - 2. Replication fork is organized around an asymmetric, DNA- polymerase III dimer. 3. Both strands made 5’-->3’. 4. “Leading strand” is continuous; “lagging strand” is discontinuous. Replication fork summary 1. DNA polymerase can’t replicate a genome. Problem Solution ATP? No single stranded template Helicase + The ss template is unstable SSB (RPA (euks)) - No primer Primase (+) No 3’-->5’ polymerase Replication fork Too 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 positive Topoisomerase II + supercoils 2. DNA replication is fast and processive 11 Sliding clamp wraps around DNA N C γ/RFC clamp loader complex puts the clamp on DNA 6. Sliding clamp can’t get on γ complex -- bacteria 7. Lagging strand contains RNA RFC -- eukaryotes 8. Lagging strand is nicked (Replication Factor C) 9. Helicase introduces + supercoils 12 RFC reaction 1. RFC + clamp + ATP opens clamp 2. Ternary complex + DNA/RNA --> Closed clamp + RFC + ADP + Pi Schematic drawing of the RFC:PCNA complex on the primer:template RFC contains 5 similar subunits that spiral around RFC DNA. The RFC helix tracks the DNA or DNA/RNA helix PCNA DNA:RNA 13 RFC:PCNA crystal structure RFC PCNA DNA:RNA RFC:PCNA crystal structure SSB opens hairpins, maintains processivity and mediates exchange of factors on the lagging strand 1. No single stranded template SSB (bacteria) and RPA (eukaryotes) 2. The ss template is unstable form tetramers. The C-terminus of SSB binds 3. No primer replication factors (Primase, Clamp 4. No 3’-->5’ polymerase. loader (chi subunit)) 5. Too slow in vitro. SSB:DNA Primer:template:SSB Clamp loader exchanges binds primase Binds clamp loader with pol III on the clamp Primase - to - pol III switch 14 Synthesis of Okazaki fragments by pol III holoenzyme DNA polymerase 5’-->3’ exonuclease or RNase H remove RNA primers 6. Sliding clamp can’t get on 7. Lagging strand contains RNA 8. Lagging strand is nicked 9. Helicase introduces + supercoils DNA polymerase I 5’-->3’ exo creates ss template. Pol works on the PREVIOUS Okazaki fragment! OR RNaseH cleaves RNA:DNA --> ssDNA + rNMPs primer 15 DNA polymerase 5’-->3’ exonuclease or RNase H remove RNA primers 6. Sliding clamp can’t get on 7. Lagging strand contains RNA 8. Lagging strand is nicked 9. Helicase introduces + supercoils DNA polymerase I 5’-->3’ exo creates ss template. Pol works on the PREVIOUS Okazaki fragment! OR RNaseH cleaves RNA:DNA --> ssDNA + rNMPs primer DNA ligase seals the nicks 1. Adenylylate the enzyme 2. Transfer AMP to the PO4 at the nick 3. Seal nick, releasing AMP Three steps in the DNA ligase reaction 16 Maturation of Okazaki fragments All tied up in knots 6. Sliding clamp can’t get on 7. Lagging strand contains RNA 8. Lagging strand is nicked 9. Helicase introduces + supercoils 17 “Topological” problems in DNA can be lethal (+) supercoils (-) supercoils •Gene (+) supercoils misexpression •Chromosome breakage •Cell death precatenanes catenanes Topoisomerases control chromosome topology Catenanes/knots Topos Relaxed/disentangled •Major therapeutic target - chemotherapeutics/antibacterials •Type II topos transport one DNA through another 18 Topoisomerases cut one strand (I) or two (II) Topoisomerase I - Cuts ssDNA region (1A (proks)) or nicks DNA (1B (euks)) Topoisomerase II - Cuts DNA and passes one duplex through the other! Topoisomerase II is a dimer that makes two staggered cuts Tyr OH attacks PO4 and forms a covalent intermediate Structural changes in the protein open the gap by 20 Å! 19 Type IIA topoisomerases comprise a homologous superfamily ATPase DNA Binding/Cleavage GyrB GyrA Gyrase Topo II (proks) (euks) Type IIA topoisomerase mechanism T-segment G-segment 1 2 ADP 4 3 • “Two-gate” mechanism • Why is the reaction directional? • What are the distinct conformational states? 20 Summary of the replication fork “Fingers” “Thumb” “Palm” Accessory factors summary 1. 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 fork Too 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 positive Topoisomerase II + supercoils 2. DNA replication is fast and processive 21.
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