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 fork
Too slow and distributive
SSB and sliding clamp
Sliding clamp can’t get on
Clamp loader (/RFC)
+
Lagging strand contains RNA Pol I 5’-->3’ exo, RNAseH Lagging strand is nicked
DNA ligase
+
Helicase introduces + supercoils
Topoisomerase II
+
and products tangled
DNA polymerase holoenzyme
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Maturation of Okazaki fragments
Topoisomerases control chromosome topology
Catenanes/knots
Topos
Relaxed/disentangled
•Major therapeutic target - chemotherapeutics/antibacterials
•Type II topos transport one DNA through another
Starting and stopping summary
1. DNA replication is controlled at the initiation step.
2. DNA replication starts at specific sites in E. coli and yeast.
3. In E. coli, DnaA recognizes OriC and promotes loading of
the DnaB helicase by DnaC (helicase loader)
4. DnaA and DnaC reactions are coupled to ATP hydrolysis.
5. Bacterial chromosomes are circular, and termination
occurs opposite OriC.
6. In E. coli, the helicase inhibitor protein, tus, binds 7 ter DNA
sites to trap the replisome at the end.
7. Eukaryotic chromosomes are linear, and the chromosome
ends cannot be replicated by the replisome.
8. Telomerase extends the leading strand at the end.
9. Telomerase is a ribonucleoprotein (RNP) with RNA
(template) and reverse-transcriptase subunits.
Isolating DNA sequences that mediate initiation
Different origin sequences in different organisms
E. Coli (bacteria)
OriC
Yeast
ARS
(Autonomously Replicating Sequences)
Metazoans
????
Initiation in prokaryotes and eukaryotes
Bacteria
Eukaryotes
ORC + other proteins load
MCM hexameric helicases
MCM (helicase) + RPA (ssbp)
Primase + DNA pol 
PCNA:pol 
MCM (helicase) + RPA (ssbp)
PCNA:pol 
(clamp loader
Primase + DNA pol 
PCNA:pol 
DNA ligase
Initiation mechanism in bacteria -- 1
Crystal structure of DnaA:ATP revealed mechanism
of origin assembly
1.
2.
1. DnaA monomer (a) forms a polar filament (b).
2. DNA binding sites occur on the outside of the filament
(model).
Crystal structure of DnaA:ATP revealed mechanism
of origin assembly
1.
2.
1. The arrangement of DNA binding sites introduces positive supercoils by
wrapping DNA on the outside.
Compensating negative supercoils melt the replication bubble at the end.
2. Clamp deposition recruits Had, which promotes ATP hydrolysis and
progressive disassembly of the DnaA filament (hypothesis).
Initiation mechanism in bacteria -- 1
Initiation mechanism in bacteria -- 2
Initiation proteins in E. coli (bacteria)
10 ter sites opposite oriC coordinate the end game
Origin
Counterclockwise
fork
Tus protein binds
Ter sites and
inhibits the DnaB
helicase only
from one
direction!!!
Clockwise
fork trap
Clockwise
fork
Counterclockwise
fork trap
The ter/tus system is not essential in E. coli.
Unwinding ter from the “nonpermissive” direction
springs a “molecular mousetrap”
DNA
Half life (s)
Kd (nM)
130 (2 min)
1.6
terB
C6
C6
C6
<7 (FAST/permissive)
53
6900 (115 min, SLOW/ 0.4
nonpermissive)
Releasing C6 springs the trap
Mulcair et al. (2006) Cell 125, 1309-1319.
Unwinding ter from the “nonpermissive” direction
springs a “molecular mousetrap”
DNA
Half life (s)
Kd (nM)
130 (2 min)
1.6
terB
C6
<7 (FAST/permissive)
C6
53
DnaB
5’
3’
C6
6900 (115 min, SLOW/ 0.4
nonpermissive)
Releasing C6 springs the trap
Mulcair et al. (2006) Cell 125, 1309-1319.
Unwinding ter from the “nonpermissive” direction
springs a “molecular mousetrap”
Releasing C6 springs the trap
Mulcair et al. (2006) Cell 125, 1309-1319.
Unwinding ter from the nonpermissive direction
springs a “molecular mousetrap”
Releasing C6 springs the trap
Mulcair et al. (2006) Cell 125, 1309-1319.
Topoisomerase II unlinks the replicated
chromosomes
Topoisomerase II - Cuts DNA
and passes one duplex through
the other.
Class II topoisomerases include:
Topo IV and DNA gyrase
Summary: What problems do these proteins solve?
Tyr OH attacks
PO4 and forms a
covalent
intermediate
Structural
changes in the
protein open the
gap by 20 Å!
Summary: What problems do these proteins solve?
Function
E. coli
SV40 (simian virus 40)
Helicase
DnaB
T antigen
Primase
Primer removal
Primase (DnaG)
pol I’s 5’-3’exo
pol  primase
FEN 1 (also RNaseH)
Core
pol III (, ,  subunits)
pol , 
Clamp loader
 complex
RF-C
Sliding clamp

PCNA
ssDNA binding
SSB
RF-A
Remove +sc at fork (swivel)
gyrase
topo I or topo II
Decatenation
topo IV
topo II
Ligase
DNA ligase
DNA ligase I
Polymerase
… other model systems include bacteriophage T4 and yeast
The ends of (linear) eukaryotic chromosomes
cannot be replicated by the replisome.
Not enough
nucleotides for
primase to start
last lagging
strand fragment
Chromosome
ends shorten
every generation!
Telomere shortening signals trouble!
Telomere binding proteins (TBPs)
1. Telomere shortening
releases telomere binding
proteins (TBPs)
2. Further shortening affects
expression of telomereshortening sensitive genes
3. Further shortening leads to
DNA damage and mutations.
Telomerase replicates the ends (telomeres)
Telomere ssDNA
Telomerase extends
the leading strand!
Synthesis is in the
5’-->3’ direction.
Telomerase is a
ribonucleoprotein
(RNP). The enzyme
contains RNA and
proteins.
The RNA templates
DNA synthesis.
The proteins
include the
telomerase reverse
transcriptase TERT.
Telomerase cycles at the telomeres
TERT protein
Telomere ssDNA
TER RNA template
Telomerase extends a chromosome 3’ overhang
Conserved structures in TER and TERT
Core secondary
structures shared in
ciliate and
vertebrate
telomerase RNAs
(TERs). (Sequences
highly variable.)
148-209 nucleotides
1000s of nucleotides
1300 nucleotides
TERT protein
sequences
conserved
Starting and stopping summary
1. DNA replication is controlled at the initiation step.
2. DNA replication starts at specific sites in E. coli and yeast.
3. In E. coli, DnaA recognizes OriC and promotes loading of
the DnaB helicase by DnaC (helicase loader)
4. DnaA and DnaC reactions are coupled to ATP hydrolysis.
5. Bacterial chromosomes are circular, and termination
occurs opposite OriC.
6. In E. coli, the helicase inhibitor protein, Tus, binds 10 ter
DNA sites to trap the replisome at the end.
7. Eukaryotic chromosomes are linear, and the chromosome
ends cannot be replicated by the replisome.
8. Telomerase extends the leading strand at the end.
9. Telomerase is a ribonucleoprotein (RNP) with RNA
(template) and reverse-transcriptase subunits.