mmg /bioc 352
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MMG /BIOC 352. The Replisome: DNA Replication in E. coli and Eukaryotes. Spring 2006. Scott W. Morrical. Contact Information. Scott W. Morrical Given B407 656-8260 [email protected]. Lecture Outline:. Overview of DNA Replication Bacterial systems ( E. coli) - PowerPoint PPT PresentationTRANSCRIPT
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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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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.
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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.
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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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DNA Replication:
Initiation, Termination, &Fidelity Mechanisms
Scott Morrical
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Initiation of E. coli DNA Replication at oriC-- roles of DnaA Initiator Protein and DnaC Helicase Loader
DnaA-- initiator protein
oriC-- replicator sequence
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Factors Required for Activation of Eukaryotic Origins
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Shameless Speculation About Helicase LoadingMechanisms at Eukaryotic Origins
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Replication Termination:Direction-specific Termination of DNA Replication
by E. coli Tus Protein Bound to a Ter Sequence
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E. Coli Chromosome1 unique origin of bi-directional replication
10 polar termination sites
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Replication Fork Arrest by Correctly Oriented Tus-TerComplex
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Alternative Models for the Direction-Specificity of Fork Arrest by Tus-Ter
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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
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Sliding Processivity Clampsof DNA Polymerases:
X-ray Structure of-subunit of E. coli DNA Polymerase III
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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
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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.
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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.
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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.
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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.
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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.
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60o rotation about dimer axis superimposes domains.
-- different a.a. sequences, but 80% structurally analogous at C’s.
-- hence the approximately hexagonal symmetry.
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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.
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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.
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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
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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.
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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.
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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 ???
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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
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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%.
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Conservation of Structure inSliding Processivity Clamps
of DNA Polymerases
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dimer trimer
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Chain Topology of PCNA Monomer
Domain 2
Domain 1
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T4 Gp45 Yeast PCNA
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Superposition of Domain 1 FromT4 Gp45 (red) and Yeast PCNA (yellow)
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Electrostatic Surface Potentials
Gp45
PCNA
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All Ring Holes Accommodate B-form dsDNAor RNA/DNA Hybrid Without Steric Hindrance
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ArchaealClamp
EukaryoticClamp
BacterialClamp
P. furiosusPCNA
S. cerevisiaePCNA
E. coliBeta
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Main Chain Hydrogen Bonds Between Strandsat PCNA Subunit Interface
P. furiosus yeast human