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

Contact Information Scott W. Morrical Given B

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. coli  -clamp Eukaryotic PCNA Structure/Function of AAA+ Clamp Loaders E. coli  -complex Eukaryotic RFC Other AAA+ ATPase Machines

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, 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, 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, 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, Ref. 6:Bowman, G.D., O’Donnell, M., and Kuriyan, J. (2004) Structural analysis of a eukaryotic sliding DNA clamp-clamp loader complex.

References (cont’d): Ref 7: Mendez, A., and Stillman, B. (2003) Perpetuating the double helix: molecular machines at eukaryotic DNA replication origins. Bioessays 25, 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, Further Reading: Mammalian DNA mismatch repair. Buermeyer et al. (1999) Annu. Rev. Genet. 33, Role of DNA mismatch repair defects in the pathogenesis of human cancer. Peltomaki (2003) J. Clinical Oncology 21,

DNA Chemistry A:T or G:C Basepair 3’-end5’-end Backbone Phosphate 2’-deoxy- ribose 5’-end 3’-end

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

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

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

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

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.

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

ReplisomeMol. ComponentWt. [stoichiometry]Gene(kDa)Function Pol III holoenzyme Dimeric, ATP-dependent, processive polymerase/clamp loader Pol III star629.1Dimeric polymerase/clamp loader Core166.0Monomeric polymerase/exonuclease  [2]dnaE129.95’ --> 3’ DNA polymerase  [2]dnaQ27.53’ --> 5’ exonuclease  [2]holE8.6Stimulates  exonuclease  /  complex297.1ATP-dependent clamp loader  /  [1/2]dnaX47.5/71.1ATPase,  organizes Pol III star and binds DnaB  [1]holA38.7Binds  clamp  ’ [1]holB36.9Stator, stimulates  ATPase in ATP site 1  [1]holC16.6Binds SSB  [1]holD15.2Connects  to clamp loader  [2 dimers]dnaN40.6Homodimeric processivity sliding clamp Primase [1]dnaG65.6Generates primers for Pol III holoenzyme DnaB helicase [6]dnaB52.4Unwinds duplex DNA 5’ --> 3’ ahead of the replication fork SSB [4]ssb18.8Melts 2 o structure in ssDNA, binds clamp loader through  E. coli Replisome Stoichiometries

E. coli  2 Sliding Clamp

E. coli  Complex-- ATP-dependent clamp loading activity

Clamp Loading Reaction

Structural Organization of Pol III Holoenzyme

DNA Flow in the E. coli Replisome

Replisome Dynamics

Replisome in Motion (zoom out)

Replisome in Motion (zoom in)

Functional Conservation of Replicase Sub-assemblies

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

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

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.

Replication Initiation in Prokaryotes & Eukaryotes

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

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

Replication Fidelity Mechanisms: Spont. Error Frequency Pol10 -4 Pol + exo10 -7 Pol + exo + MMC10 -9 to

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).

E. coli Methyl-Directed Mismatch Repair System

Eukaryotic Homologs of MutS and MutL

Mlh1-Pms1 Heterodimers of Eukaryotic MutS & MutL Homologs Msh2Msh3 Mlh1-Mlh2 Msh2Msh3 Mlh1-Mlh3 Msh2Msh3 Mlh1-Pms1 Msh2Msh6 Rad1-Rad10 Msh2Msh3Msh4Msh5 Mlh1-Mlh3 Non-homologous tail removal in recombination intermediates Insertion/deletion loop (IDL) removal Repair of base-base mismatches Promotion of meiotic crossovers MutS  MutS  MutL  MutL  *Note: This is yeast nomenclature. Mlh1 paralogs have different names in yeast and humans. 1 b 2-4 b