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