mmg /bioc 352 spring 2006 the replisome: dna replication in e. coli and eukaryotes scott w. morrical
TRANSCRIPT
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MMG /BIOC 352
Spring 2006
The Replisome: DNA Replication in E. coli
and Eukaryotes
Scott W. Morrical
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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
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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.
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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.
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DNA Chemistry
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A:T or G:CBasepair
3’-end5’-end
Backbone
Phosphate2’-deoxy-
ribose
5’-end3’-end
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Chemical Inheritance-- DNA Replication
DNA Replication Fork • processive
• 5’ to 3’
• semi-conservative
• semi-discontinuous
• high-fidelity
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E. Coli Chromosome1 unique origin of bi-directional replication
10 polar termination sites
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Replication Progression of E. coli Chromosome
oriC
ter sequences
oriC
oriC
thetastructure
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Replication of Eukaryotic Chromosomes
Many different origins on each chromosome firing simultaneously or in a programmed sequence.
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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.
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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
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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
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E. coli 2 Sliding Clamp
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E. coli Complex-- ATP-dependent clamp loading activity
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Clamp Loading Reaction
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Structural Organization ofPol III Holoenzyme
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DNA Flow in the E. coli Replisome
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Replisome Dynamics
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Replisome in Motion (zoom out)
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Replisome in Motion (zoom in)
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Functional Conservation of Replicase Sub-assemblies
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Model for Eukaryotic Replisome(Based on E. coli and T4 Phage Models)
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Polymerase Switching During Eukaryotic Lagging Strand Synthesis& Okazaki Maturation via RNaseH1 and Fen1/RTH1
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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.
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Replication Initiation in Prokaryotes & Eukaryotes
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Direction-specific Termination of DNA Replicationby E. coli Tus Protein Bound to a Ter Sequence
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Replication Fork Arrest by Correctly Oriented Tus-TerComplex
Final disentanglement of chromosomes by topoisomerases.
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Replication Fidelity Mechanisms:Spont. Error Frequency
Pol 10-4
Pol + exo 10-7
Pol + exo + MMC 10-9 to 10-10
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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
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Eukaryotic Homologs of MutS and MutL
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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