2. 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 ~1/10 9.

3. Replication fork summary 1.DNA polymerase can’t replicate a genome. ProblemSolutionATP? No single stranded templateHelicase + The ss template is unstableSSB (RPA (euks)) - No primerPrimase (+) No 3’-->5’ polymeraseReplication fork Too slow and distributiveSSB 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.

4. 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

5. 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

6. DNA polymerase holoenzyme

7. DNA replication factors were discovered using “temperature sensitive” mutations 1.No single stranded template 2.The ss template is unstable 3.No primer 4.No 3’-->5’ polymerase. 5.Too slow in vitro. Mutations that inactivate the DNA replication machinery are lethal. Temperature sensitive (conditional) mutations allow isolation of mutations in essential genes. 37 ºC 42 ºC 42 ºC, Mutant gene overexpressed

8. A hexameric replicative helicase unwinds DNA ahead of 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 in vitro. Replicative DNA helicase is called DnaB in E. coli. DnaB couples ATP binding and hydrolysis to DNA strand separation. Helicase assay ds DNA ss DNA

9. SSB (or RPA) cooperatively binds ss DNA template 1.No single stranded template 2.The ss template is unstable 3.No primer 4.No 3’-->5’ polymerase. 5.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. ds DNA ss DNA + SSB

10. SSB tetramer structure 1.No single stranded template 2.The ss template is unstable 3.No primer 4.No 3’-->5’ polymerase. 5.Too slow in vitro. SSB (bacteria) and RPA (eukaryotes) form tetramers. The C-terminus of SSB binds replication factors (primase, clamp loader (chi subunit)) ds DNA ss DNA + SSB N C N C N C N C ConservationPositive potential

11. 1.No single stranded template 2.The ss template is unstable 3.No primer 4.No 3’-->5’ polymerase. 5.Too slow in vitro. DNA synthesis is primed by a short RNA segment Primase makes about 10-base RNA. The product is a RNA/DNA hybrid. RNA primer has a free 3’OH. Uses ATP, which ends up across from T in the RNA/DNA hybrid. Primase: DNA-dependent RNA polymerase Start preference for CTG on template

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

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

14. 1.No single stranded template 2.The ss template is unstable 3.No primer 4.No 3’-->5’ polymerase. 5.Too slow in vitro. DNA pol III “holoenzyme” is asymmetric DNA pol III holoenzyme: A molecular machine  binds SSB  opens clamp (  ) Synthesizes Lagging Strand Synthesizes Leading Strand

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

16. 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

17. 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

18. Sliding clamp wraps around DNA N C

19. Sliding clamps are structurally conserved “Palm”

20. Summary of the replication fork “Palm”

21. 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.

22. Replication fork summary 1.DNA polymerase can’t replicate a genome. SolutionATP? No single stranded templateHelicase + The ss template is unstableSSB (RPA (euks)) - No primerPrimase (+) No 3’-->5’ polymeraseReplication fork Too slow and distributiveSSB 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.

23. Replication fork summary 1.DNA polymerase can’t replicate a genome. ProblemSolutionATP? No single stranded templateHelicase + The ss template is unstableSSB (RPA (euks)) - No primerPrimase (+) No 3’-->5’ polymeraseReplication fork Too slow and distributiveSSB and sliding clamp - Sliding clamp can’t get onClamp loader (  /RFC) + Lagging strand contains RNAPol I 5’-->3’ exo, RNAseH - Lagging strand is nickedDNA ligase + Helicase introduces positive Topoisomerase II + supercoils 2.DNA replication is fast and processive

24. Sliding clamp wraps around DNA N C

25.  /RFC clamp loader complex puts the clamp on DNA 6.Sliding clamp can’t get on 7.Lagging strand contains RNA 8.Lagging strand is nicked 9.Helicase introduces + supercoils  complex -- bacteria RFC -- eukaryotes (Replication Factor C)

26. RFC reaction 1.RFC + clamp + ATP opens clamp 2.Ternary complex + DNA/RNA --> Closed clamp + RFC + ADP + Pi

27. 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

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

29. SSB opens hairpins, maintains processivity and mediates exchange of factors on the lagging strand 1.No single stranded template 2.The ss template is unstable 3.No primer 4.No 3’-->5’ polymerase. 5.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

30. Synthesis of Okazaki fragments by pol III holoenzyme

31. DNA polymerase 5’-->3’ exonuclease or RNase H remove RNA primers DNA polymerase I 5’-->3’ exo creates ss template. Pol works on the PREVIOUS Okazaki fragment! 6.Sliding clamp can’t get on 7.Lagging strand contains RNA 8.Lagging strand is nicked 9.Helicase introduces + supercoils OR RNaseH cleaves RNA:DNA --> ssDNA + rNMPs primer

32. DNA polymerase 5’-->3’ exonuclease or RNase H remove RNA primers DNA polymerase I 5’-->3’ exo creates ss template. Pol works on the PREVIOUS Okazaki fragment! 6.Sliding clamp can’t get on 7.Lagging strand contains RNA 8.Lagging strand is nicked 9.Helicase introduces + supercoils OR RNaseH cleaves RNA:DNA --> ssDNA + rNMPs primer

33. 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

34. Maturation of Okazaki fragments

35. 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

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

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

38. 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!

39. 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!

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

41. Type IIA topoisomerase mechanism “Two-gate” mechanism Why is the reaction directional? What are the distinct conformational states? ADP G-segment T-segment 12 34

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

43. Accessory factors summary 1.DNA polymerase can’t replicate a genome. SolutionATP? No single stranded templateHelicase + The ss template is unstableSSB (RPA (euks)) - No primerPrimase (+) No 3’-->5’ polymeraseReplication fork Too slow and distributiveSSB and sliding clamp - Sliding clamp can’t get onClamp loader (  /RFC) + Lagging strand contains RNAPol I 5’-->3’ exo, RNAseH - Lagging strand is nickedDNA ligase + Helicase introduces positive Topoisomerase II + supercoils 2.DNA replication is fast and processive