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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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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