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

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

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)

QuickTime™ and aAnimation decompressor

are needed to see this picture.

QuickTime™ and aAnimation decompressor

are needed to see this picture.

Replisome in Motion (zoom in)

Functional Conservation of Replicase Sub-assemblies

Eukaryotic Replisome Components

S. cerevisiae (kDa) H. sapiens (kDa) Function and remarks [S. pombe name]

RFCa RFC (277.7)a RFC (314.9)a Pentameric clamp loadera

RFC1 (94.9) p140 (128.2) Binds ATP; phosphorylated RFC2 (39.7) p37 (39.2) Binds ATP RFC3 (38.2) p36 (40.6) Binds ATP RFC4 (36.1) p40 (39.7) Binds ATP RFC5 (39.9) p38 (38.5) Binds ATP or ADP

PCNAa PCNA (28.9) a PCNA (28.7) 87 kDa a Homotrimeric sliding clamp a

Pol a Pol (220.2) a Pol (238.7) a Replicative DNA polymerase a

Pol3 (124.6) p125 (123.6) DNA polymerase, 3'-5' exo, binds PCNA; subunit A [S.p. Pol3]

Pol31 (55.3) p50 (51.3) Structural subunit; subunit B [S.p. Cdc1] Pol32 (40.3) p66 (51.4) Binds PCNA; subunit C [S.p. Cdc27];

binds Pol large subunit — p12 (12.4) Structural, stimulates processivity;

subunit D [S.p. Cdm1]

Pol a Pol (378.7) a Pol (350.3) a Replicative DNA polymerase a

Pol2 (255.7) p261 (261.5) DNA polymerase, 3'-5' exo [S.p. Pol2/cdc20] Dpb2 (78.3) p59 (59.5) Binds polymerase subunit [S.p. Dpb2] Dpb3 (22.7) p17 (17.0) Binds Dpb4 Dpb4 (22.0) p12 (12.3) Present in ISW2/yCHRAC chromatin

remodeling complex [S.p. Dpb4]

Pol a Pol (355.6) a Pol (340.6) a DNA polymerase/primase a

Pol1 (166.8) p180 (165.9) DNA polymerase Pol12 (78.8) p68 (66.0) Structural subunit Pri2 (62.3) p55 (58.8) Interacts tightly with p48 Pri1 (47.7) p48 (49.9) RNA primase catalytic subunit

aInformation concerns a protein complex.

Eukaryotic Replisome Components (cont’d)

S. cerevisiae (kDa) H. sapiens (kDa) Function and remarks [S. pombe name]

MCM a MCM (605.6) a MCM (535) a Putative 3'-5' replicative helicase a

Mcm2 (98.8) Mcm2 (91.5) Phosphorylated by Dbf4-dependent kinase Mcm3 (107.5) Mcm3 (91.0) Ubiquitinated, acetylated Mcm4 (105.0) Mcm4 (96.6) Helicase with MCM6,7; phosphorylated by CDK;

aka Cdc54 Mcm5 (86.4) Mcm5 (82.3) Aka Cdc46; Bob1 is a mutant form Mcm6 (113.0) Mcm6 (92.3) Helicase with MCM4,7 Mcm7 (94.9) Mcm7 (81.3) Helicase with MCM4,6; ubiquitinated

RPA a RPA (114) a RPA (100.5) a Single-stranded DNA-binding protein a

RPA70 (70.3) RPA70 (70.3) Binds DNA, stimulates Pol RPA30 (29.9) RPA30 (29) Binds RPA70 and 14, phosphorylated RPA14 (13.8) RPA14 (13.5) Binds RPA30

a Information concerns a protein complex.

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.

DNA Replication:

Initiation, Termination, &Fidelity Mechanisms

Scott Morrical

Initiation of E. coli DNA Replication at oriC-- roles of DnaA Initiator Protein and DnaC Helicase Loader

DnaA-- initiator protein

oriC-- replicator sequence

Factors Required for Activation of Eukaryotic Origins

Shameless Speculation About Helicase LoadingMechanisms at Eukaryotic Origins

Replication Termination:Direction-specific Termination of DNA Replication

by E. coli Tus Protein Bound to a Ter Sequence

E. Coli Chromosome1 unique origin of bi-directional replication

10 polar termination sites

Replication Fork Arrest by Correctly Oriented Tus-TerComplex

Alternative Models for the Direction-Specificity of Fork Arrest by Tus-Ter

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.

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Insertion-deletion loops (IDLs)-- caused by polymerase slippage onrepetitive template, gives rise to Microsatallite Instability (MSI).

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.QuickTime™ and a

TIFF (Uncompressed) decompressorare needed to see this picture.

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

Sliding Processivity Clampsof DNA Polymerases:

X-ray Structure of-subunit of E. coli DNA Polymerase III

BACKGROUND:

1. Biochemical studies established that beta is essential for processive DNA synthesis by Pol III.

2. Beta exists as a stable dimer in solution.

3. Beta dimer has no intrinsic affinity for DNA, yet in the presence of gamma complex + ATP, beta dimer forms and extremely stable complex with circular, but not with linear, primed DNA molecules.

4. Proposal by O’Donnell & coworkers: 2 is topologically linked, not thermodynamically bound to DNA, and forms a sliding clamp that tethers Pol III.

3’

3’5’

5’

2 Pol III core

Kong, Onrust, O’Donnell, & Kuriyan (1992) “Three-dimensional structure of the betasubunit of E. coli DNA polymerase III holoenzyme: a sliding DNA clamp”. Cell 69, 425-37.

2.5Å resolution

Non-crystallographic 2-foldrotational axis of symmetryperpendicular to face of ring and passing through center of hole.

• Highly symmetrical; almost hexagonal symmetry.

• Protomers interact head-to-tail in 2 interfaces on opposite sides of the ring.

Space-filling Model of Beta Dimer with B-form DNAModeled In

O.D. ≈ 80 Å, I.D. ≈ 35 Å-- easily accommodates B- or A-form (RNA/DNA hybrid) duplex (~25 Å O.D.) without steric repulsions.

Thickness ≈ 34 Å, equal to ~1 full helical repeat of B-form dsDNA.

Connectivity of Beta Subunit

Green (N-terminus)

light blue

purple

red

yellow (C-terminus)

• Dimer interfaces are between domains colored green/blue and red/yellow.

• Domains are numbered 1,2,3 and 1’, 2’, 3’ in the two monomers.

Unexpected Features of the 2 Structure:

1. Internal symmetry.

-- each monomer consists of 3 structural domains of identical chain topology and very similar 3-D structure.

-- this was surprising because there are no internal regions of a.a. sequence homology.

2. Each domain is roughly 2-fold symmetric in architecture, with an outer layer of 2 -sheets providing a scaffold that supports 2 -helices.

Unexpected Features of the 2 Structure (cont’d):

3. Replication of this motif around a circle (2 subunits x 3 domains/subunit) results in a rigid molecule with 12 -helices lining the inner surface of the ring, and with 6 “seamless” interlocking -sheets forming the outer surface.

4. Symmetry of domains gives rise to highly symmetrical and roughly hexagonal star shape of the dimer.

60o rotation about dimer axis superimposes domains.

-- different a.a. sequences, but 80% structurally analogous at C’s.

-- hence the approximately hexagonal symmetry.

Simple Principle Underlying 2 Architecture:

• The 2 outer strands of -sheets in one domain form hydrogen-bonding interactions with corresponding strands in 2 adjacent domains, continued around the circle.

• No distinctions apparent between such -sheet extensions across internal domain boundaries as opposed to intermolecular contacts (i.e. the 2 dimer interfaces also form continuous antiparallel -sheets).

• These interactions lead to a completely closed circle with 6 “seamless” -sheets on the outer surface.

Spatial Arrangement of -helices Within 2 DimerWith Respect To B-form DNA

(assuming duplex is perpendicular to plane of ring)

1. Each of the 12 -helices has similar tilt w.r.t. the axis of the ring, due to their symmetrical arrangement.

Spatial Arrangement of -helices Within 2 DimerWith Respect To B-form DNA (Cont’d)(assuming duplex is perpendicular to plane of ring)

2. The axis of each -helix is almost exactly perpendicular to the local direction of the sugar-phosphate backbone.

--> the helices span the major & minor grooves.

--> effectively prevents entry of the protein into either groove, which should facilitate rapid motion along the duplex

Spatial Arrangement of -helices Within 2 DimerWith Respect To B-form DNA (Cont’d)(assuming duplex is perpendicular to plane of ring)

3. If one a-helix spans the major groove, the one directly across the ring spans the minor groove.

--> damps out variation in interaction energy with sugar-phosphate backbone as the protein moves across the grooves of DNA.

Electrostatic Potential of 2 Dimer

2 contains 38 Asp, 58 Glu, 24 Lys, 50 Arg, and 14 His residues

--> net charge of -15 to -22

• Highly asymmetric electrostatic field:

-- outer edge and both faces strongly negatively charged, but asymmetric from face-to-face.

-- asymmetry may correctly orient 2 w.r.t. primer terminus, complex, and Pol III core.

Electrostatic Potential of 2 Dimer (Cont’d)

2 contains 38 Asp, 58 Glu, 24 Lys, 50 Arg, and 14 His residues

--> net charge of -15 to -22

• Inner surface of hole highly positively-charged:

-- favorable interaction with DNA backbone --> “float on electrostatic cloud”.

-- ??? stabilize dimer around DNA, since electrostatic interactions in dimer interface might not withstand repulsive interactions with DNA ???

Contributions to Stability of Dimer Interface*** Highly Electrostatic ***

1. Continuation, across molecular boundary, of hydrogen-bonded -sheet structure.

--> ≥ 4 strong hydrogen bonds at each of 2 interfaces. (Indistinguishable from continuation of -sheet across interdomain boundaries.)

2. Small hydrophobic core (in white):

Phe-106|Ile-278

Ile-272|Leu-273

packagainst

Contributions to Stability of Dimer Interface (Cont’d)*** Highly Electrostatic ***

3. Electrostatics: potential intermolecular ion pairs:

Lys-74 … Arg-96 … Arg-103 … Arg-105

Glu-298 … Glu-300 … Glu-301 … Glu-303 … Glu-304

4. Charge complementarity at each interface: All positively-charged residues from one monomer, all negatively charged from the other.

5. Relatively little surface area is buried upon dimerization, ~8%.

Conservation of Structure inSliding Processivity Clamps

of DNA Polymerases

dimer trimer

Chain Topology of PCNA Monomer

Domain 2

Domain 1

T4 Gp45 Yeast PCNA

Superposition of Domain 1 FromT4 Gp45 (red) and Yeast PCNA (yellow)

Electrostatic Surface Potentials

Gp45

PCNA

All Ring Holes Accommodate B-form dsDNAor RNA/DNA Hybrid Without Steric Hindrance

ArchaealClamp

EukaryoticClamp

BacterialClamp

P. furiosusPCNA

S. cerevisiaePCNA

E. coliBeta

Main Chain Hydrogen Bonds Between Strandsat PCNA Subunit Interface

P. furiosus yeast human