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DNA polymerase summary

1. DNA replication is semi-conservative.2. DNA polymerase enzymes are specialized for different

functions.3. DNA pol I has 3 activities: polymerase, 3’-->5’ exonuclease &

5’-->3’ exonuclease.4. DNA polymerase structures are conserved.5. But: Pol can’t start and only synthesizes DNA 5’-->3’!6. Editing (proofreading) by 3’-->5’ exo reduces errors.7. High fidelity is due to the race between addition and editing.8. Mismatches disfavor addition by DNA pol I at 5 successive

positions. The error rate is ~1/109.

Replication fork summary

1. DNA polymerase can’t replicate a genome.Problem Solution ATP?

No single stranded template Helicase +The ss template is unstable SSB (RPA (euks)) -No primer Primase (+)No 3’-->5’ polymerase Replication forkToo slow and distributive SSB and sliding clamp -

2. Replication fork is organized around an asymmetric, DNA-polymerase III dimer.

3. Both strands made 5’-->3’.4. “Leading strand” is continuous; “lagging strand” is

discontinuous.

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DNA polymerase can’t replicate a genome!

1. No single stranded template2. The ss template is unstable3. No primer4. No 3’-->5’ polymerase5. Too slow and distributive

Solution: the replication fork

1. No single-stranded template2. The ss template is unstable3. No primer4. No 3’-->5’ polymerase5. Too slow and distributive

Schematic drawing of a replication fork

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DNA polymerase holoenzyme

DNA replication factors were discovered using“temperature sensitive” mutations

1. No single stranded template2. The ss template is unstable3. No primer4. No 3’-->5’ polymerase.5. Too slow in vitro.

Mutations that inactivatethe DNA replicationmachinery are lethal.

Temperature sensitive(conditional) mutationsallow isolation of mutationsin essential genes.

37 ºC

42 ºC

42 ºC,Mutant geneoverexpressed

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A hexameric replicative helicase unwinds DNA aheadof the replication fork

1. No single stranded template2. The ss template is unstable3. No primer4. No 3’-->5’ polymerase.5. Too slow in vitro.

Replicative DNA helicase iscalled DnaB in E. coli.

DnaB couples ATP bindingand hydrolysis to DNAstrand separation.

Helicase assay

ds DNA

ss DNA

SSB (or RPA) cooperatively binds ss DNA template

1. No single stranded template2. The ss template is unstable3. No primer4. No 3’-->5’ polymerase.5. Too slow in vitro.

SSB (single-strand binding protein(bacteria)) or RPA (ReplicationProtein A (eukaryotes)):No ATP used.Filament is substrate for DNA pol.

ds DNA

ss DNA + SSB

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SSB tetramer structure

1. No single stranded template2. The ss template is unstable3. No primer4. No 3’-->5’ polymerase.5. Too slow in vitro.

SSB (bacteria) and RPA (eukaryotes)form tetramers.

The C-terminus of SSB bindsreplication factors (primase, clamploader (chi subunit))

ds DNA

ss DNA + SSB

N

C

N

C

N

C

N

C

Conservation Positive potential

1. No single stranded template2. The ss template is unstable3. No primer4. No 3’-->5’ polymerase.5. Too slow in vitro.

DNA synthesis is primed by a short RNA segment

Primase makes about 10-base RNA. The product is a RNA/DNA hybrid.RNA primer has a free 3’OH.

Uses ATP, which ends up acrossfrom T in the RNA/DNA hybrid.

Primase: DNA-dependent RNApolymerase

Start preference for CTG on template

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DnaG primase defines a distinct polymerasefamily (DNA dependent RNA pol)

Map ofsurfacecharge

Ribbondiagram

Model of“primosome”:DnaB helicase +DnaG primase

DnaB helicase

DnaG primase

Primase passes the primed template to DNA polymerase

Lagging strand:discontinuous

Leading strand:continuous

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1. No single stranded template2. The ss template is unstable3. No primer4. No 3’-->5’ polymerase.5. Too slow in vitro.

DNA pol III “holoenzyme” is asymmetric

DNA pol III holoenzyme:A molecular machine

χ binds SSB δ opens clamp (β)

SynthesizesLaggingStrand

SynthesizesLeadingStrand

Pol III dimer couples leading and laggingstrand synthesis

Leadingstrand

Laggingstrand

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Replication fork

1. No single stranded template2. The ss template is unstable3. No primer4. No 3’-->5’ polymerase5. Too slow and distributive

Replication fork

1. No single stranded template2. The ss template is unstable3. No primer4. No 3’-->5’ polymerase5. Too slow and distributive

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Sliding clamp wraps around DNA

N

C

Sliding clamps are structurally conserved

“Palm”

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Summary of the replication fork

“Palm”

Synthesis of Okazaki fragments by pol III holoenzyme

When pol III reaches the primer of theprevious Okazaki fragment, clamploader removes β2 from the DNAtemplate. As a result, the pol III on thelagging strand falls off the template.

Clamp loader places β2 on the nextprimer-template.

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Replication fork summary

1. DNA polymerase can’t replicate a genome.Solution ATP?

No single stranded template Helicase +The ss template is unstable SSB (RPA (euks)) -No primer Primase (+)No 3’-->5’ polymerase Replication forkToo slow and distributive SSB and sliding clamp -

2. Replication fork is organized around an asymmetric, DNA-polymerase III dimer.

3. Both strands made 5’-->3’.4. “Leading strand” is continuous; “lagging strand” is

discontinuous.

Replication fork summary

1. DNA polymerase can’t replicate a genome.Problem Solution ATP?

No single stranded template Helicase +The ss template is unstable SSB (RPA (euks)) -No primer Primase (+)No 3’-->5’ polymerase Replication forkToo slow and distributive SSB and sliding clamp -Sliding clamp can’t get on Clamp loader (γ/RFC) +Lagging strand contains RNA Pol I 5’-->3’ exo, RNAseH -Lagging strand is nicked DNA ligase +Helicase introduces positive Topoisomerase II +

supercoils

2. DNA replication is fast and processive

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Sliding clamp wraps around DNA

N

C

γ/RFC clamp loader complex puts the clamp on DNA

6. Sliding clamp can’t get on7. Lagging strand contains RNA8. Lagging strand is nicked9. Helicase introduces + supercoils

γ complex -- bacteriaRFC -- eukaryotes(Replication Factor C)

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RFC reaction

1. RFC + clamp + ATP opens clamp2. Ternary complex + DNA/RNA --> Closed clamp + RFC + ADP + Pi

Schematic drawing of the RFC:PCNA complex onthe primer:template

RFC contains 5 similarsubunits that spiral aroundDNA.The RFC helix tracks theDNA or DNA/RNA helix

RFC

PCNA

DNA:RNA

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RFC:PCNA crystal structure

RFC:PCNA crystal structure

RFC

PCNA

DNA:RNA

SSB opens hairpins, maintains processivity andmediates exchange of factors on the lagging strand1. No single stranded template2. The ss template is unstable3. No primer4. No 3’-->5’ polymerase.5. Too slow in vitro.

SSB (bacteria) and RPA (eukaryotes)form tetramers.The C-terminus of SSB bindsreplication factors (Primase, Clamploader (chi subunit))

SSB:DNAbinds primase

Primer:template:SSBBinds clamp loader

Clamp loader exchanges with pol III on the clamp

Primase - to - pol III switch

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Synthesis of Okazaki fragments by pol III holoenzyme

DNA polymerase 5’-->3’ exonuclease or RNase Hremove RNA primers

DNA polymerase I 5’-->3’ exocreates ss template.Pol works on the PREVIOUSOkazaki fragment!

6. Sliding clamp can’t get on7. Lagging strand contains RNA8. Lagging strand is nicked9. Helicase introduces + supercoils

OR RNaseH cleaves RNA:DNA --> ssDNA + rNMPs primer

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DNA polymerase 5’-->3’ exonuclease or RNase Hremove RNA primers

DNA polymerase I 5’-->3’ exocreates ss template.Pol works on the PREVIOUSOkazaki fragment!

6. Sliding clamp can’t get on7. Lagging strand contains RNA8. Lagging strand is nicked9. Helicase introduces + supercoils

OR RNaseH cleaves RNA:DNA --> ssDNA + rNMPs primer

DNA ligase seals the nicks

1. Adenylylate theenzyme

2. Transfer AMP tothe PO4 at thenick

3. Seal nick, releasingAMP

Three steps in the DNA ligase reaction

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Maturation of Okazaki fragments

All tied up in knots

6. Sliding clamp can’t get on7. Lagging strand contains RNA8. Lagging strand is nicked9. Helicase introduces + supercoils

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“Topological” problems in DNA can be lethal

•Genemisexpression

•Chromosomebreakage

•Cell death

(+) supercoils

(-) supercoils

(+) supercoils

precatenanes

catenanes

Topoisomerases control chromosome topologyCatenanes/knots

Relaxed/disentangled

•Major therapeutic target - chemotherapeutics/antibacterials

•Type II topos transport one DNA through another

Topos

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Topoisomerases cut one strand (I) or two (II)

Topoisomerase I - Cuts ssDNA region (1A (proks)) or nicks DNA (1B (euks))

Topoisomerase II - Cuts DNA and passes one duplex through the other!

Topoisomerase II is a dimer that makes twostaggered cuts

Tyr OH attacksPO4 and forms acovalentintermediate

Structuralchanges in theprotein open thegap by 20 Å!

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ATPase DNA Binding/Cleavage

GyrAGyrB

Type IIA topoisomerases comprise ahomologous superfamily

Gyrase(proks)

Topo II(euks)

Type IIA topoisomerase mechanism

• “Two-gate” mechanism• Why is the reaction directional?

• What are the distinctconformational states?

ADP

G-segment

T-segment

1 2

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Summary of the replication fork

“Palm”

“Fingers” “Thumb”

Accessory factors summary

1. DNA polymerase can’t replicate a genome.Solution ATP?

No single stranded template Helicase +The ss template is unstable SSB (RPA (euks)) -No primer Primase (+)No 3’-->5’ polymerase Replication forkToo slow and distributive SSB and sliding clamp -Sliding clamp can’t get on Clamp loader (γ/RFC) +Lagging strand contains RNA Pol I 5’-->3’ exo, RNAseH -Lagging strand is nicked DNA ligase +Helicase introduces positive Topoisomerase II +

supercoils

2. DNA replication is fast and processive