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 ~1/10 9 .

Replication fork summary
1.
DNA polymerase can’t replicate a genome.
Problem Solution
No single stranded template Helicase
The ss template is unstable SSB (RPA (euks))
No primer
No 3’-->5’ polymerase
Too slow and distributive
Primase
Replication fork
ATP?
-
+
(+)
SSB and sliding clamp -
2. Replication fork is organized around an asymmetric, DNApolymerase 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 template
2. The ss template is unstable
3. No primer
4.
No 3’-->5’ polymerase
5. Too slow and distributive

Solution: the replication fork
1. No single-stranded template
2. The ss template is unstable
3. No primer
4.
No 3’-->5’ polymerase
5. Too slow and distributive
Schematic drawing of a replication fork

QuickTime™ and a
DV - PAL decompressor are needed to see this picture.

DNA replication factors were discovered using
“temperature sensitive” mutations
1. No single stranded template
2. The ss template is unstable
3. No primer
4.
No 3’-->5’ polymerase.
5. Too slow in vitro.
37 ºC
42 ºC
Mutations that inactivate the DNA replication machinery are lethal.
Temperature sensitive
(conditional) mutations allow isolation of mutations in essential genes.
42 ºC,
Mutant gene overexpressed

A hexameric replicative helicase unwinds DNA ahead of the replication fork
Helicase assay
1. No single stranded template
2. The ss template is unstable
3. No primer
4.
No 3’-->5’ polymerase.
5. Too slow in vitro.
ds DNA
Replicative DNA helicase is called DnaB in E. coli.
DnaB couples ATP binding and hydrolysis to DNA strand separation.
ss DNA

SSB (or RPA) cooperatively binds ss DNA template
1. No single stranded template
2. The ss template is unstable
3. No primer
4.
No 3’-->5’ polymerase.
5. Too slow in vitro.
SSB (single-strand binding protein (bacteria)) or RPA
(Replication Protein A
(eukaryotes)):
No ATP used.
Filament is substrate for DNA pol.
ss DNA + SSB ds DNA

SSB tetramer structure
1. No single stranded template
2. The ss template is unstable
3. No primer
4.
No 3’-->5’ polymerase.
5. Too slow in vitro.
SSB (bacteria) and RPA
(eukaryotes) form tetramers.
The C-terminus of SSB binds replication factors (primase, clamp loader (chi subunit))
C C
N
N
C C ds DNA
N
N
Conservation ss DNA + SSB
Positive potential

DNA synthesis is primed by a short RNA segment
1. No single stranded template
2. The ss template is unstable
3. No primer
4.
No 3’-->5’ polymerase.
5. Too slow in vitro.
Primase makes about 10-base RNA.
The product is a RNA/DNA hybrid.
RNA primer has a free 3’OH.
Primase: DNA-dependent RNA polymerase
Start preference for CTG on template
Uses ATP, which ends up across from T in the RNA/DNA hybrid.

Ribbon diagram
Model of
“primosome”:
DnaB helicase +
DnaG primase
DnaB helicase
Map of surface charge
DnaG primase

Primase passes the primed template to DNA polymerase
Leading strand: continuous
Lagging strand: discontinuous

DNA pol III “holoenzyme” is asymmetric
1. No single stranded template
2. The ss template is unstable
3. No primer
4.
No 3’-->5’ polymerase.
5. Too slow in vitro.
DNA pol III holoenzyme:
A molecular machine
Synthesizes
Leading
Strand
Synthesizes
Lagging
Strand
 binds SSB
 opens clamp (

)

Leading strand
Lagging strand

1. No single stranded template
2. The ss template is unstable
3. No primer
4.
No 3’-->5’ polymerase
5. Too slow and distributive

1. No single stranded template
2. The ss template is unstable
3. No primer
4.
No 3’-->5’ polymerase
5. Too slow and distributive

Sliding clamp wraps around DNA
C
N

“Palm”

“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 primer-template.
on the next

Replication fork summary
1.
DNA polymerase can’t replicate a genome.
Solution
No single stranded template Helicase
The ss template is unstable SSB (RPA (euks))
No primer
No 3’-->5’ polymerase
Too slow and distributive
Primase
Replication fork
SSB and sliding clamp
ATP?
+
-
(+)
-
2. Replication fork is organized around an asymmetric, DNApolymerase 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
No single stranded template Helicase
The ss template is unstable SSB (RPA (euks))
ATP?
+
-
No primer
No 3’-->5’ polymerase
Primase
Replication fork
(+)
Too slow and distributive
Sliding clamp can’t get on
SSB and sliding clamp -
Clamp loader (

/RFC)
+
Lagging strand contains RNA Pol I 5’-->3’ exo, RNAseH -
Lagging strand is nicked supercoils
DNA ligase
Helicase introduces positive Topoisomerase II
+
+
2. DNA replication is fast and processive

Sliding clamp wraps around DNA
C
N


/RFC clamp loader complex puts the clamp on DNA
6.
Sliding clamp can’t get on
7. Lagging strand contains RNA
8. Lagging strand is nicked
9. Helicase introduces + supercoils
 complex -- bacteria
RFC -- eukaryotes
(Replication Factor C)

RFC reaction
1. RFC + clamp + ATP opens clamp
2. 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 and mediates exchange of factors on the lagging strand
1. No single stranded template
2. The ss template is unstable
3. No primer
4.
No 3’-->5’ polymerase.
5. Too slow in vitro.
SSB (bacteria) and RPA
(eukaryotes) form tetramers.
The C-terminus of SSB binds replication factors (Primase, Clamp loader (chi subunit))
SSB:DNA binds primase
Primer:template:SSB
Binds 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 H remove RNA primers
6.
Sliding clamp can’t get on
7. Lagging strand contains RNA
8. Lagging strand is nicked
9. Helicase introduces + supercoils
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 H remove RNA primers
6.
Sliding clamp can’t get on
7. Lagging strand contains RNA
8. Lagging strand is nicked
9. Helicase introduces + supercoils
DNA polymerase I 5’-->3’ exo creates ss template.
Pol works on the PREVIOUS
Okazaki fragment!
OR RNaseH cleaves RNA:DNA --> ssDNA + rNMPs primer

1. Adenylylate the enzyme
2. Transfer AMP to the PO4 at the nick
3. Seal nick, releasing
AMP
Three steps in the DNA ligase reaction

6.
Sliding clamp can’t get on
7. Lagging strand contains RNA
8. Lagging strand is nicked
9. Helicase introduces + supercoils

“Topological” problems in DNA can be lethal
(+) supercoils
(-) supercoils
(+) supercoils
• Gene misexpression
• Chromosome breakage
• Cell death precatenanes catenanes

Topoisomerases control chromosome topology
Catenanes/knots
Topos
Relaxed/disentangled
• Major therapeutic target - chemotherapeutics/antibacterials
• Type II topos transport one DNA through another

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!

Type IIA topoisomerases comprise a homologous superfamily
ATPase DNA Binding/Cleavage
GyrB GyrA
Gyrase
(proks)
Topo II
(euks)

T-segment
G-segment
QuickTime™ and a
decompressor are needed to see this picture.
ADP
1
4 3
2
• “Two-gate” mechanism
•
Why is the reaction directional?
•
What are the distinct conformational states?

“Thumb” “Fingers”
“Palm”

Accessory factors summary
1.
DNA polymerase can’t replicate a genome.
Solution
No single stranded template Helicase
The ss template is unstable SSB (RPA (euks))
ATP?
+
-
No primer
No 3’-->5’ polymerase
Primase
Replication fork
(+)
Too slow and distributive
Sliding clamp can’t get on
SSB and sliding clamp -
Clamp loader (

/RFC)
+
Lagging strand contains RNA Pol I 5’-->3’ exo, RNAseH -
Lagging strand is nicked supercoils
DNA ligase
Helicase introduces positive Topoisomerase II
+
+
2. DNA replication is fast and processive