A Replisome
5'
Primase
Primosome
3'
Helicase (DnaB)
Gyrase
DNA Polymerase I extends
one Okazaki fragment and
removes the RNA from
another.
DNA Ligase then joins
fragments together.
5'
3'
Sp i n n i n g
at 10,000
5'
rpm
DNA Polymerase III
acts here
ssDNA binding
protein (SSB)
5'
A p r ok ar y ot i c f or k i s tr av el l i n g at 50 to 100 k b / m i n u te.
3'
Eu k ar y oti c for k s t r av el at 0.5 - 5 k b / m i n u te.
Pol III*
Core
 complex
Pol III has a dimer of the “core subunits”,
which contain the polymerizing α subunits.
Fig. 21.17
 - Clamp – exists free and as subunit of Pol III holoenzyme
.
Donut-shaped
Dimer.
“Clamps” a
subunit onto
DNA, and
makes it
highly
processive.
Fig. 21.15 in Weaver
Fig. 21.16
The effect of  subunit
on the  clamp.
Can the  clamp can slide off the end of linear DNA?
Plasmid DNA with nick
 clamp
Assay
1. Load clamp onto circular plasmid DNA.
2. Treat DNA further.
3. Separate DNA-bound clamp from free clamp.
Based on Fig. 21.13
Fig. 21.11
Blue – control
Red – treated with the
indicated enzyme before
chromatography
First peak = protein-DNA
complex
Second peak = free 
protein
: many  dimers
can be loaded on the DNA,
and it will slide off linear
DNA
Based on Fig. 21.13 f,g
Yellow line- control red line- experimental
SSB can retain clamp, but linearize again after
loading SSB, clamp falls off.
Load holoenzyme onto DNA with SSB, if all 4
dNTPs added, clamp falls off (control- only 1
or 2 dNTPs retains clamp)
Clamp sliding off the ends of linear DNA can be stopped
by DNA binding proteins such as SSB and EBNA.
Clamp will slide off SSB-coated DNA if it is part of the
holoenzyme that is replicating DNA.
Pol III core
dimer
synthesizing
leading &
lagging
strands.
 (tau)
subunits
(2) of Pol III
bind to
helicase.
 Clamp loading
 complex of Pol III holoenzyme
( 2 , , ’, c,  )
- loads  subunit dimer onto DNA (at the primer) and
Pol core (and unloads it at the end of Okazaki
fragment)
Order of events:
1. Uses ATP to open  dimer and position it at 3’
end of primer.
2. “Loaded”  clamp then binds Pol III core (and
releases from ).
3. Processive DNA synthesis.
Recycling phase
1.
2.
3.
4.
Once Okazaki fragment completed, 
clamp releases from core.
 binds to  .
 unloads  clamp from DNA.
 clamp recycles to next primer.
Figure 21.25
Terminating DNA synthesis in
prokaryotes.
Each fork
stops at the
Ter regions,
which are 22
bp, 3 copies,
and bind the
Tus protein.
Fig. 21.26
Decatenation of Daughter DNAs
catenane
Decatenation is
performed by
Topoisomerase
IV in E. coli.
Topo IV is a
Type II
topoisomerase:
breaks and
rejoins 2
strands of a
duplex DNA.
Fig. 21.27
DNA replication in Eukaryotes
Eukaryotic DNA polymerases (5):
a - has primase activity
 - elongates primers, highly processive, can do
proofreading
 - DNA repair
 - DNA repair
 - replication of Mitochondrial (and/or Chloroplast
DNA in plants)
Eukaryotic DNA polymerases do NOT have 5'
to 3' exonuclease activity. A separate
enzyme, called FEN-1, is the 5' to 3'
exonuclease that removes the RNA primers.
Eukaryotes also have equivalents to the:
Sliding clamp – PCNA (a.k.a. proliferating cell
nuclear antigen)
SSB – RP-A
Problem for eukaryotes: Replicating the 5’ end
of the lagging strand (because chromosomes
are linear molecules)
A
B
C
D
A'
B'
C'
D'
A
B
C
D
A'
B'
C'
D'
3'
5'
Gap generated by removal of the RNA primer
3'
5'
Euk. chromosomes end with many copies of a special
“Telomeric” sequence.
A
A'
B
B'
C
C'
D
3'
5'
D'
(3 copies on this chromosome end)
Cells can lose some copies of the telomere w/out losing genes.
A
A'
B
B'
C
C'
D
D'
(Replication of this chromosome would
produce 1 that is shorter by 1 telomere)
3'
5'
Telomere Sequences
Organism
Tetrahymena, Paramecium, Oxytricha (all
are protozoa)
Saccharomyces (yeast)
telomere repeat
T2G4
Arabidopsis (plant)
T3AG3
Homo sapiens
T2AG3
(T G)1-3TG2-3
Telomeres form an unusual secondary structure.
Dashes are Ts
5’ 3’
Enzyme that adds new telomeric repeats
to 3’ ends of linear chromosomes.
Fig. 21.32
Proteins bind the 3’ SS overhang for protection.
More on the importance of Telomerase
• Apoptosis - Cells are very sensitive to chromosome ends
because they are highly recombinogenic. Telomeres
don’t trigger apoptosis.
• Aging - There are rapid aging diseases (e.g., Werner’s
Syndrome) where telomeres are shorter than normal.
• Cancer - Most somatic cells don’t have telomerase, but
tumor cells do. Over-expression of telomerase in a
normal cell, however, won’t turn it into a tumor cell.
• Plants - Transgenic Arabidopsis with the telomerase
gene turned off developed normally up to a point,
then became sick.
How is a Repl. origin selected?
Priming at the oriC (Bacterial)
Origin
Three 13-mers
GATCTnTTTATTT
GATCTnTTnTATT
GATCTCTTATTAG
9a-mer
TGTGGATAA
Three 9b-mers
TTATACACA
TTTGGATAA
TTATCCACA
GGATCCTGgnTATTAAAAAGAAGATCTnTTTATTTAGAGATCTGTTnTATT
sequence
GG . .
..
A
.
. C
G GC . .
..
T
.
. C
AG . .
..
.
. C
AG . .
..
.
. T
CGT A T
GA
T
A
C 13
9a
GTGATCTCTTATTAGGATCGGnnntnnnnTGTGGATAAgnngGATCCnnnn
sequence
..
CACTGCCC
CAAG
GGCT
..
CGCCAGGC
CCCG
TGTA
..
ACTCTCTA
GTCG
ACGA
..
GCTTGTCT
GTCA
GCGG
ATCGTGTTG
GTGATTATTCATA
Consensus
TTtAAGATCAAnnnnnTggnAAGGATCncTAnCTGTGAATGATCGGTGAT
sequence
. T .
CAACC GGA...
AT..A
A A .
TGCGT GGA...
AC..G
. T .
ACGCT AAG...
ACA.T
. T G
CCGTT AAG...
GC.TT
. A .
GAGAA GGCGTT
CT..C
9b
CCTGGnCCGTATAAGCTGGGATCAnAATGngGGnTTATACACAgCtCAAA
sequence
...A
G . AG..G
.
A.T
G..T
A C GGTAC
.
A.T
..TT
G . AA G
G
G.T
...T A
A . AA G
.
G.A
.A.C
TT . TG T
.
GGA
Consensus
9b
AAncgnACaaCGGTTaTTCTTTGGATAACTACCGGTTGATCCAAGCTTTt
sequence
.CTGA. AA.A
G .. .
. . ... .......CC
.GTGA. AA..
A .. .
. . ... ........C
.GCAT. TC..
A .. .
. . ... ........T
TTCAGG AA..
A A. .
. . ... ........T
.ACGC. TCG.
G .A G
G C TTA ACCAGAA.T
9b
nAnCAgAGTTATCCACAntnGAnnGcnn-GAT
sequence
TGA G..
GTA. TC.CAC-.
C.C G.T
ATG. TC.CAC-.
G.G GG.
GAA. AGCTGCG.
A.G T..
GAA. AA.TAT-.
A.G T..
TTCA CT.CCG-A
Escherichia
Salmonella
Enterobacter
Klebsiella
Erwinia
Consensus
Consensus
Consensus
Consensus
Eshcerichia
Salmonella
Enterobacter
Klebsiella
Erwinia
Sequence of Events at the Replication Origin
Primase (purple) with the first primers (arrows).
Order of events at OriC
1. Several copies of dnaA bind the four 9-mers; DNA
wraps around dnaA forming “Initial Complex”. This
requires ATP and a protein, Hu,that is already bound to
the DNA.
2. This triggers opening of the 13-mers (Open complex).
3. Two copies of dnaB (helicase) bind the 13-mers. This
requires dnaC (which does not remain with the
Prepriming Complex) and ATP.
4. Primase binds to dnaB (helicase) and the DNA.
5. dnaB:primase complex moves along the template 5’ >
3’ synthesizing RNA primers for Pol III to extend.