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:

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.

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

DNA polymerase holoenzyme

QuickTime™ and aDV - PAL decompressor

are needed to see this picture.

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 inactivate the DNA replication machinery are lethal.

Temperature sensitive (conditional) mutations allow isolation of mutations in essential genes.

37 ºC

42 ºC

42 ºC,Mutant geneoverexpressed

A hexameric replicative helicase unwinds DNA ahead of the replication fork


Replicative DNA helicase is called DnaB in E. coli.

DnaB couples ATP binding and hydrolysis to DNA strand separation.

Helicase assay

ds DNA

ss DNA

SSB (or RPA) cooperatively binds ss DNA template


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

ds DNA

ss DNA + SSB

SSB tetramer structure


SSB (bacteria) and RPA (eukaryotes)

form tetramers.The C-terminus of SSB binds replication factors (primase, clamp loader (chi subunit))

ds DNA

ss DNA + SSB

N

C

N

C

N

C

N

C

Conservation Positive potential


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 across from T in the RNA/DNA hybrid.

Primase: DNA-dependent RNA polymerase

Start preference for CTG on template

DnaG primase defines a distinct polymerase family (DNA dependent RNA pol)

Map ofsurfac

e charg

e

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


DNA pol III “holoenzyme” is asymmetric

DNA pol III holoenzyme:A molecular machine

binds SSB opens clamp ()

SynthesizesLaggingStrand

SynthesizesLeadingStrand

Pol III dimer couples leading and lagging strand synthesis

Leadingstrand

Laggingstrand

Replication fork


Replication fork


Sliding clamp wraps around DNA

N

C

Sliding clamps are structurally conserved

“Palm”

Summary of the replication fork

“Palm”

Synthesis of Okazaki fragments by pol III holoenzyme

When pol III reaches the primer of the previous Okazaki fragment, clamp loader removes 2 from the DNA template. As a result, the pol III on the lagging strand falls off the template.

Clamp loader places 2 on the next primer-template.


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



-No primer Primase


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.


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



-No primer Primase


clamp - Sliding clamp can’t get on Clamp loader

(/RFC) +Lagging strand contains RNAPol I 5’-->3’ exo,

RNAseH -Lagging strand is nicked DNA ligase

+Helicase introduces positive Topoisomerase II

+supercoils

2. DNA replication is fast and processive

Sliding clamp wraps around DNA

N

C

/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

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

RFC reaction

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

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

RFC contains 5 similar subunits that spiral around DNA.The RFC helix tracks the DNA or DNA/RNA helix

RFC

PCNA

DNA:RNA

RFC:PCNA crystal structure

RFC:PCNA crystal structure

RFC

PCNA

DNA:RNA

SSB opens hairpins, maintains processivity andmediates exchange of factors on the lagging strand


SSB (bacteria) and RPA (eukaryotes) form tetramers.The C-terminus of SSB binds replication factors (Primase, Clamp loader (chi subunit))

SSB:DNAbinds primase

Primer:template:SSBBinds clamp loader

Clamp loader exchanges with pol III on the clamp

Primase - to - pol III switch

Synthesis of Okazaki fragments by pol III holoenzyme

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

DNA polymerase I 5’-->3’ exo creates ss template.Pol works on the PREVIOUS Okazaki fragment!


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

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

DNA polymerase I 5’-->3’ exo creates ss template.Pol works on the PREVIOUS Okazaki fragment!


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

DNA ligase seals the nicks

1. Adenylylate theenzyme

2. Transfer AMP tothe PO4 at the nick

3. Seal nick, releasingAMP

Three steps in the DNA ligase reaction

Maturation of Okazaki fragments

All tied up in knots


“Topological” problems in DNA can be lethal

•Gene misexpression

•Chromosome breakage

•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

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 two staggered cuts

Tyr OH attacks PO4 and forms a covalent intermediate

Structural changes in the protein open the gap by 20 Å!

ATPase DNA Binding/Cleavage

GyrAGyrB

Type IIA topoisomerases comprise a homologous superfamily

Gyrase(proks)

Topo II(euks)

Type IIA topoisomerase mechanism

• “Two-gate” mechanism

• Why is the reaction directional?

• What are the distinct conformational states?

ADP

G-segment

T-segment

QuickTime™ and a decompressor

are needed to see this picture.

1 2

34

Summary of the replication fork

“Palm”

“Fingers” “Thumb”

Accessory factors summary

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



-No primer Primase


clamp - Sliding clamp can’t get on Clamp loader

(/RFC) +Lagging strand contains RNAPol I 5’-->3’ exo,

RNAseH -Lagging strand is nicked DNA ligase

+Helicase introduces positive Topoisomerase II

+supercoils

2. DNA replication is fast and processive

Documents

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: