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MMG /BIOC 352
Spring 2006
The Replisome: DNA Replication in E. coli
and Eukaryotes
Scott W. Morrical
Contact Information
Scott W. MorricalGiven B407656-8260
Scott.Morrical@uvm.edu
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, 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: asliding 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 theprocessivity 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.
References (cont’d):
Ref 7: Mendez, A., and Stillman, B. (2003) Perpetuating the double helix: molecularmachines 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.
DNA Chemistry
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A:T or G:CBasepair
3’-end5’-end
Backbone
Phosphate2’-deoxy-
ribose
5’-end3’-end
Chemical Inheritance-- DNA Replication
DNA Replication Fork • processive
• 5’ to 3’
• semi-conservative
• semi-discontinuous
• high-fidelity
E. Coli Chromosome1 unique origin of bi-directional replication
10 polar termination sites
Replication Progression of E. coli Chromosome
oriC
ter sequences
oriC
oriC
thetastructure
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
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 [2] dnaE 129.9 5’ --> 3’ DNA polymerase [2] dnaQ 27.5 3’ --> 5’ exonuclease [2] holE 8.6 Stimulates exonuclease / complex 297.1 ATP-dependent clamp loader / [1/2] dnaX 47.5/71.1 ATPase, organizes Pol III star and binds DnaB [1] holA 38.7 Binds clamp ’ [1] holB 36.9 Stator, stimulates ATPase in ATP site 1 [1] holC 16.6 Binds SSB [1] holD 15.2 Connects to clamp loader [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
E. coli Replisome Stoichiometries
E. coli 2 Sliding Clamp
E. coli Complex-- ATP-dependent clamp loading activity
Clamp Loading Reaction
Structural Organization ofPol III Holoenzyme
DNA Flow in the E. coli Replisome
Replisome Dynamics
Replisome in Motion (zoom out)
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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 Replicationby E. coli Tus Protein Bound to a Ter Sequence
Replication Fork Arrest by Correctly Oriented Tus-TerComplex
Final disentanglement of chromosomes by topoisomerases.
Replication Fidelity Mechanisms:Spont. Error Frequency
Pol 10-4
Pol + exo 10-7
Pol + exo + MMC 10-9 to 10-10
Single base mismatches-- misincorporation by DNA polymerase,missed by proofreading exonuclease.
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Insertion-deletion loops (IDLs)-- caused by polymerase slippage onrepetitive template, gives rise to Microsatallite Instability (MSI).
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E. coliMethyl-DirectedMismatch RepairSystem
Eukaryotic Homologs of MutS and MutL
Mlh1-Pms1
Heterodimers of Eukaryotic MutS & MutL Homologs
Msh2 Msh3
Mlh1-Mlh2
Msh2 Msh3
Mlh1-Mlh3
Msh2 Msh3
Mlh1-Pms1
Msh2 Msh6
Rad1-Rad10
Msh2 Msh3 Msh4 Msh5
Mlh1-Mlh3
Non-homologoustail removal inrecombinationintermediates
Insertion/deletionloop (IDL)
removal
Repair ofbase-base mismatches
Promotion ofmeiotic crossovers
MutS
MutS
MutL
MutL
*Note: This is yeast nomenclature.Mlh1 paralogs have different namesin yeast and humans.
1 b2-4 b