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MMG /BIOC 352

MMG /BIOC 352. The Replisome: DNA Replication in E. coli and Eukaryotes. Spring 2006. Scott W. Morrical. Contact Information. Scott W. Morrical Given B407 656-8260 Scott.Morrical@uvm.edu. Lecture Outline:. Overview of DNA Replication Bacterial systems ( E. coli)

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MMG /BIOC 352

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  1. MMG /BIOC 352 The Replisome: DNA Replication in E. coli and Eukaryotes Spring 2006 Scott W. Morrical

  2. Contact Information Scott W. Morrical Given B407 656-8260 Scott.Morrical@uvm.edu

  3. Lecture Outline: Overview of DNA Replication Bacterial systems (E. coli) Eukaryotic systems (yeast/human) The E. coli Replisome Components & sub-assemblies Replisome structure/function Coordination of leading/lagging strand synthesis The Eukaryotic Replisome Polymerase switching Okazaki Maturation Initiation Mechanisms E. coli oriC paradigm Eukaryotic model Termination Mechanisms Tus-Ter Fidelity Mechanisms Proofreading Mismatch repair Processivity Mechanisms: Structure/Function of Sliding Clamps E. colib-clamp Eukaryotic PCNA Structure/Function of AAA+ Clamp Loaders E. coli g-complex Eukaryotic RFC Other AAA+ ATPase Machines

  4. Reference list for this topic: Ref 1: Johnson, A., and O’Donnell, M. (2005) Cellular DNA replicases: components and dynamics at the replication fork. Annu. Rev. Biochem. 74, 283-315. Ref 2: Davey, M.J., Jeruzalmi, D., Kuriyan, J., and O’Donnell, M. (2002) Motors and Switches: AAA+ machines within the replisome. Nat. Rev. Mol. Cell Biol. 3, 826-835. Ref 3: Kong, X.P., Onrust, R., O’Donnell. M. and Kuriyan, J. (1992) Three-dimensional structure of the beta subunit of E. coli DNA polymerase III holoenzyme: a sliding clamp. Cell 69, 425-437. Ref 4: Krishna. T.S., Kong, X.P., Gary, S., Burgers, P.M., and Kuriyan, J. (1994) Crystal structure of eukaryotic DNA polymerase processivity factor PCNA. Ref 5:Jeruzalmi, D., O’Donnell, M., and Kuriyan, J. (2001) Crystal structure of the processivity clamp loader gamma complex of E. coli DNA polymerase III. Cell 106, 429-421. Ref. 6:Bowman, G.D., O’Donnell, M., and Kuriyan, J. (2004) Structural analysis of a eukaryotic sliding DNA clamp-clamp loader complex.

  5. References (cont’d): Ref 7: Mendez, A., and Stillman, B. (2003) Perpetuating the double helix: molecular machines at eukaryotic DNA replication origins. Bioessays 25, 1158-1167. Ref 8: Neylon, C., Kralicek, A.V., Hill, T.M., and Dixon, N.E. (2005) Replication termination in Escherichia coli: structure and antihelicase activity of the Tus-Ter complex. Micr. Mol. Biol. Rev. 69, 501-526 Further Reading: Mammalian DNA mismatch repair. Buermeyer et al. (1999) Annu. Rev. Genet. 33, 533-564. Role of DNA mismatch repair defects in the pathogenesis of human cancer. Peltomaki (2003) J. Clinical Oncology 21, 1174-1179.

  6. DNA Chemistry 5’-end 3’-end Basepair A:T or G:C Backbone Phosphate 2’-deoxy- ribose 5’-end 3’-end

  7. DNA Replication Fork Chemical Inheritance-- DNA Replication • processive • 5’ to 3’ • semi-conservative • semi-discontinuous • high-fidelity

  8. E. Coli Chromosome 1 unique origin of bi-directional replication 10 polar termination sites

  9. Replication Progression of E. coli Chromosome oriC theta structure oriC oriC ter sequences

  10. Replication of Eukaryotic Chromosomes Many different origins on each chromosome firing simultaneously or in a programmed sequence.

  11. DNA Replication Fork • Major Protein Components: • DNA polymerase holoenzyme(s) • -- polymerase • -- proofreading exonuclease • -- sliding clamp • -- clamp loader complex • DNA helicase(s) • Primase • ssDNA binding protein • Other accessory factors needed for correct assembly, processive movement, and fidelity.

  12. Major Components of E. coli Replisome: PolIII-- DNA polymerase III holoenzyme (Pol III) DnaG primase DnaB helicase SSB-- ssDNA-binding protein Plus accessory proteins, loading factors

  13. Replisome Mol. Component Wt. [stoichiometry] Gene (kDa) Function Pol III holoenzyme 791.5 Dimeric, ATP-dependent, processive polymerase/clamp loader Pol III star 629.1 Dimeric polymerase/clamp loader Core 166.0 Monomeric polymerase/exonuclease a [2] dnaE 129.9 5’ --> 3’ DNA polymerase e [2] dnaQ 27.5 3’ --> 5’ exonuclease q [2] holE 8.6 Stimulates e exonuclease g/t complex 297.1 ATP-dependent clamp loader g/t [1/2] dnaX 47.5/71.1 ATPase, t organizes Pol III star and binds DnaB d [1] holA 38.7 Binds b clamp d’ [1] holB 36.9 Stator, stimulates g ATPase in ATP site 1 c [1] holC 16.6 Binds SSB y [1] holD 15.2 Connects c to clamp loader b [2 dimers] dnaN 40.6 Homodimeric processivity sliding clamp Primase [1] dnaG 65.6 Generates primers for Pol III holoenzyme DnaB helicase [6] dnaB 52.4 Unwinds duplex DNA 5’ --> 3’ ahead of the replication fork SSB [4] ssb 18.8 Melts 2o structure in ssDNA, binds clamp loader through c E. coli Replisome Stoichiometries

  14. E. coli b2 Sliding Clamp

  15. E. coli g Complex-- ATP-dependent clamp loading activity

  16. Clamp Loading Reaction

  17. Structural Organization of Pol III Holoenzyme

  18. DNA Flow in the E. coli Replisome

  19. Replisome Dynamics

  20. Replisome in Motion (zoom out)

  21. Replisome in Motion (zoom in)

  22. Functional Conservation of Replicase Sub-assemblies

  23. Model for Eukaryotic Replisome (Based on E. coli and T4 Phage Models)

  24. Polymerase Switching During Eukaryotic Lagging Strand Synthesis & Okazaki Maturation via RNaseH1 and Fen1/RTH1

  25. Okazaki Maturation Involving Helicase Strand Displacement & Flap Endonuclease Activity of Fen1/RTH1 E. coli: RNA primers removed by 5’ --> 3’ exo activity of DNA polymerase I (Pol I). Simultaneous fill-in with DNA (nick translation rxn) leaves nick that is sealed by ligase.

  26. Replication Initiation in Prokaryotes & Eukaryotes

  27. Direction-specific Termination of DNA Replication by E. coli Tus Protein Bound to a Ter Sequence

  28. Replication Fork Arrest by Correctly Oriented Tus-Ter Complex Final disentanglement of chromosomes by topoisomerases.

  29. Replication Fidelity Mechanisms: Spont. Error Frequency Pol 10-4 Pol + exo 10-7 Pol + exo + MMC 10-9 to 10-10

  30. Single base mismatches-- misincorporation by DNA polymerase, missed by proofreading exonuclease. Insertion-deletion loops (IDLs)-- caused by polymerase slippage on repetitive template, gives rise to Microsatallite Instability (MSI).

  31. E. coli Methyl-Directed Mismatch Repair System

  32. Eukaryotic Homologs of MutS and MutL

  33. Heterodimers of Eukaryotic MutS & MutL Homologs *Note: This is yeast nomenclature. Mlh1 paralogs have different names in yeast and humans. Mlh1-Mlh2 Msh2 Msh3 Mlh1-Mlh3 MutLb Msh2 Msh3 MutSb MutLa Rad1-Rad10 Mlh1-Pms1 Mlh1-Pms1 Mlh1-Mlh3 Msh2 Msh3 Msh2 Msh3 Msh2 Msh6 Msh4 Msh5 MutSa 2-4 b 1 b Non-homologous tail removal in recombination intermediates Insertion/deletion loop (IDL) removal Repair of base-base mismatches Promotion of meiotic crossovers

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