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DNA polymerase summary

DNA polymerase summary. DNA replication is semi-conservative. DNA polymerase enzymes are specialized for different functions. DNA pol I has 3 activities: polymerase, 3’-->5’ exonuclease & 5’-->3’ exonuclease. DNA polymerase structures are conserved.

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DNA polymerase summary

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  1. DNA polymerase summary DNA replication is semi-conservative. DNA polymerase enzymes are specialized for different functions. DNA pol I has 3 activities: polymerase, 3’-->5’ exonuclease & 5’-->3’ exonuclease. DNA polymerase structures are conserved. But: Pol can’t start and only synthesizes DNA 5’-->3’! Editing (proofreading) by 3’-->5’ exo reduces errors. High fidelity is due to the race between addition and editing. Mismatches disfavor addition by DNA pol I at 5 successive positions. The error rate is ~1/109.

  2. Replication fork summary • DNA polymerase can’t replicate a genome. • ProblemSolution 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.

  3. DNA polymerase can’t replicate a genome! No single stranded template The ss template is unstable No primer No 3’-->5’ polymerase Too slow and distributive

  4. Solution: the replication fork No single-stranded template The ss template is unstable No primer No 3’-->5’ polymerase Too slow and distributive Schematic drawing of a replication fork

  5. DNA polymerase holoenzyme

  6. DNA replication factors were discovered using “temperature sensitive” mutations No single stranded template The ss template is unstable No primer No 3’-->5’ polymerase. Too slow in vitro. 37 ºC Mutations that inactivate the DNA replication machinery are lethal. Temperature sensitive (conditional) mutations allow isolation of mutations in essential genes. 42 ºC 42 ºC, Mutant gene overexpressed

  7. A hexameric replicative helicase unwinds DNA ahead of the replication fork Helicase assay No single stranded template The ss template is unstable No primer No 3’-->5’ polymerase. Too slow in vitro. ds DNA Replicative DNA helicase is called DnaB in E. coli. DnaB couples ATP binding and hydrolysis to DNA strand separation. ss DNA

  8. SSB (or RPA) cooperatively binds ss DNA template No single stranded template The ss template is unstable No primer No 3’-->5’ polymerase. Too slow in vitro. SSB (single-strand binding protein (bacteria)) or RPA (Replication Protein A (eukaryotes)): No ATP used. Filament is substrate for DNA pol. ss DNA + SSB ds DNA

  9. SSB tetramer structure No single stranded template The ss template is unstable No primer No 3’-->5’ polymerase. Too slow in vitro. SSB (bacteria) and RPA (eukaryotes) form tetramers. The C-terminus of SSB binds replication factors (primase, clamp loader (chi subunit)) C C N N N N ss DNA + SSB C C Conservation Positive potential ds DNA

  10. DNA synthesis is primed by a short RNA segment No single stranded template The ss template is unstable No primer No 3’-->5’ polymerase. Too slow in vitro. Primase: DNA-dependent RNA polymerase 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.

  11. DnaG primase defines a distinct polymerase family (DNA dependent RNA pol) Model of “primosome”: DnaB helicase +DnaG primase Ribbon diagram DnaB helicase Map of surface charge DnaG primase

  12. Primase passes the primed template to DNA polymerase Leading strand: continuous Lagging strand: discontinuous

  13. DNA pol III “holoenzyme” is asymmetric DNA pol III holoenzyme: A molecular machine No single stranded template The ss template is unstable No primer No 3’-->5’ polymerase. Too slow in vitro. Synthesizes Leading Strand Synthesizes Lagging Strand binds SSB  opens clamp ()

  14. Pol III dimer couples leading and lagging strand synthesis Leading strand Lagging strand

  15. Replication fork No single stranded template The ss template is unstable No primer No 3’-->5’ polymerase Too slow and distributive

  16. Replication fork No single stranded template The ss template is unstable No primer No 3’-->5’ polymerase Too slow and distributive

  17. Sliding clamp wraps around DNA N C

  18. Sliding clamps are structurally conserved “Palm”

  19. Summary of the replication fork “Palm”

  20. Synthesis of Okazaki fragments by pol III holoenzyme When pol III reaches the primer of the previous Okazaki fragment, clamp loader removes 2 from the DNA template. As a result, the pol III on the lagging strand falls off the template. Clamp loader places 2 on the next primer-template.

  21. Replication fork summary • 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.

  22. Replication fork summary • DNA polymerase can’t replicate a genome. • ProblemSolution 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 (/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

  23. Sliding clamp wraps around DNA N C

  24. /RFC clamp loader complex puts the clamp on DNA  complex -- bacteria RFC -- eukaryotes Sliding clamp can’t get on Lagging strand contains RNA Lagging strand is nicked Helicase introduces + supercoils (Replication Factor C)

  25. RFC reaction RFC + clamp + ATP opens clamp Ternary complex + DNA/RNA --> Closed clamp + RFC + ADP + Pi

  26. Schematic drawing of the RFC:PCNA complex on the primer:template RFC contains 5 similar subunits that spiral around DNA. The RFC helix tracks the DNA or DNA/RNA helix RFC PCNA DNA:RNA

  27. RFC:PCNA crystal structure RFC PCNA DNA:RNA RFC:PCNA crystal structure

  28. SSB opens hairpins, maintains processivity andmediates exchange of factors on the lagging strand No single stranded template The ss template is unstable No primer No 3’-->5’ polymerase. Too slow in vitro. SSB (bacteria) and RPA (eukaryotes) form tetramers. The C-terminus of SSB binds replication factors (Primase, Clamp loader (chi subunit)) SSB:DNA binds primase Primer:template:SSB Binds clamp loader Clamp loader exchanges with pol III on the clamp Primase - to - pol III switch

  29. Synthesis of Okazaki fragments by pol III holoenzyme

  30. DNA polymerase 5’-->3’ exonuclease or RNase Hremove RNA primers Sliding clamp can’t get on Lagging strand contains RNA Lagging strand is nicked 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

  31. DNA polymerase 5’-->3’ exonuclease or RNase Hremove RNA primers Sliding clamp can’t get on Lagging strand contains RNA Lagging strand is nicked 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

  32. DNA ligase seals the nicks Adenylylate the enzyme 2. Transfer AMP to the PO4 at the nick 3. Seal nick, releasing AMP Three steps in the DNA ligase reaction

  33. Maturation of Okazaki fragments

  34. All tied up in knots Sliding clamp can’t get on Lagging strand contains RNA Lagging strand is nicked Helicase introduces + supercoils

  35. Gene misexpression • Chromosome breakage • Cell death “Topological” problems in DNA can be lethal (+) supercoils (-) supercoils (+) supercoils precatenanes catenanes

  36. Relaxed/disentangled Topoisomerases control chromosome topology Catenanes/knots Topos • Major therapeutic target - chemotherapeutics/antibacterials • Type II topos transport one DNA through another

  37. 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!

  38. 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!

  39. Type IIA topoisomerases comprise a homologous superfamily ATPase DNA Binding/Cleavage GyrB GyrA Gyrase (proks) Topo II (euks)

  40. ADP Type IIA topoisomerase mechanism T-segment G-segment 1 2 4 3 • “Two-gate” mechanism • Why is the reaction directional? • What are the distinct conformational states?

  41. Summary of the replication fork “Fingers” “Thumb” “Palm”

  42. Accessory factors summary • 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 (/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

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