DNA replication - 2
The DNA replication machinery
DNA polymerases are unable to melt duplex DNA (I.e. break
certain hydrogen bonds) in order to separate strands that are to be
copied
All DNA polymerases so far discovered can only elongate a preexisting DNA or RNA strand, the primer; they can not initiate
chains.
The two strands in the DNA duplex are opposite (5’→3 and 3’
→5’) in chemical polarity, but all DNA polymerases catalyze
nucleotide addition at the 3’hydroxyl end of a growing chain – only
5’→3 direction.
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DnaA protein initiates replication in E.coli
Genetic studies suggested that initiation of replication at oriC in
E.coli is dependent upon protein coded by dnaA gene. DnaA
protein binds with oriC.
Although DnaA can bind to duplex E.coli origin DNA in the
relaxed-circle form, it can initiate replication only when the DNA is
negatively supercoiled.
The reason – negative supercoiles are tightly wound and are easier
to melt locally (thus providing a single-stranded template region)
than DNA molecules w/o supercoiles.
Supercoiling is controlled by enzymes called topoisomerases.
Binding of DnaA to oriC 9-mers facilitates melting of duplex DNA,
which occurs at oriC 13-mers. This process requires ATP and
yields so called open complex.
DnaA protein initiates replication in E.coli
DnaA binds oriC.
Study of t sensitive mutants of DnaA– cells grew at 30 C, but not at
39-42 C.
Genetic studies of recombinant E. coli pointed that DnaA binds oriC,
forming initial complex, and melts DNA at 9-mers and 13-mers.
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Further melting of the two strands in E.coli chromosome to generate
unpaired template strands is mediated by the protein product of the
dnaB locus - a helicase that is essential for DNA replication.
One molecule of DnaB,
subunits, clamps
DnaB, a hexamer of identical subunits
around each of the two single strands in the open complex formed
between the DnaA and oriC.
This binding requires ATP and the protein product of the dnaC
locus.
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The function of DnaC is to deliver DnaB to the template. One
DnaB hexamer clamps around each single strand of DNA at oriC,
forming the prepriming complex. DnaB is a helicase
helicase, and the two
molecules then proceed to unwind the DNA in opposite directions
away from the origin.
DnaB is a helicase that melts duplex DNA
Helicases constitute a class of enzymes
that can move along a DNA duplex
utilizing the energy of ATP hydrolysis to
separate the strands.
SSB protein - binds ssDNA
Helicases exhibit directionality with
respect to unwinding reaction.
DnaB moves along the single strand of
DNA to which it binds in the direction of
it’s free 3’ end – it unwinds DNA 5’→3’
direction.
DnaB, like many other proteins that act on
DNA, is processive. Because it forms the
clamp around ssDNA DnaB does not fall
off until it reaches the end of the strand or
is “unloaded” by other protein.
Other kinds of helicases unwind in
opposite direction, moving along the
strand to which they are bound toward the
free 5’ end.
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E. coli primase catalyzes formation of RNA primers for for DNA synthesis
E. coli primase catalyzes formation of RNA primers for for DNA synthesis
Primase
Primase
Catalyzes the formation of an RNA strand, complementary and
antiparallel to a single DNA strand:
oRNA strand grows 5'--> 3'
ocomplementary to the DNA, read 3'-->5'
Process:
•Primer --> a short length of RNA-DNA duplex (about 10
nucleotides in length)
DNA polymerase attaches to the duplex
DNA polymerase forms a new DNA strand, starting at the 3'-end
of the RNA strand.
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E. coli primase catalyzes formation of RNA primers for DNA synthesis
The primers used during DNA replication in eukaryotes and
prokaryotes are short RNA molecules whose synthesis is catalyzed
by the RNA polymerase primase.
Primase is usually recruited to a segment of single-stranded DNA
by first binding to DnaB hexamer already attached at that site. The
term primosome is now generally used to denote a complex between
primase and helicase, sometimes with other proteins.
In initiation of E. coli DNA replication, a primosome is formed
by binding of primases to DnaB in prepriming complex.
After bound primases synthesize short primer RNAs
complementary to both strands of duplex DNA , they dissociate
from the single stranded template.
E. coli primase catalyzes formation of RNA primers for for DNA synthesis
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DNA replication is
continuous on the
leading strand (1
primer); and
discontinuous on the
lagging strand – many
primers.
When newly formed
fragment approaches
the 5’ end of the other
one DNA polymerase
I takes over. It has
exonuclease activity –
removes RNA primer
and fills the gap by
adding
deoxynucleotides.
Steps in the discontinuous synthesis of the lagging strand
strand: this process
requires multiple primers, two DNA polymerases, a ligase that joins the 3’
hydroxyl end of one Okazaki fragment with the 5’ phosphate of the adjacent
fragment.
Replication, Okazaki fragments
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Ligation reaction:
During this reaction ligase transiently attaches covalently to the 5’
phosphate on one stand, thus activating the phosphate group.
E. coli DNA ligase uses NAD+ as a cofactor, generating NMN and
AMP. Bacteriophage T4 ligase, commonly used in DNA cloning,
uses ATP, generating PPi and AMP.
Polymerases
DNA polymerases are important enzymes involved in DNA
replication.
Three polymerases have been purified from E.coli.
In addition to important role in filling the gaps between
Okazaki fragments, DNA polymerase I is the most important
enzyme for gap filling during DNA repair.
DNA polymerase II functions in the inducible SOS response;
this polymerase fills the gap and appears to facilitate DNA
synthesis directed by damaged templates.
DNA polymerase III catalyzes chain elongation at the
growing fork of E. coli.
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DNA polymerase I
1957 – Arthur Kornberg isolated an enzyme (DNA polymerase I)
from E. coli that was able to direct DNA synthesis in vitro.
Major requirements for in vitro DNA synthesis were:
1. All four deoxyribonucleoside triphosphates (dATP, dCTP,
dGTP, dTTP = dNTP).
2. Template DNA
The chemical reaction catalysed by DNA polymerase I
(dNMP)x
(dNMP)x
+ P-P
dNTP +
Deoxyribose
nucleoside
triphosphate
(A,T,C,G)
(dNMP)n
DNA template (dNMP)x
and its partial
complement (dNMP)n
(dNMP)n+1
Mg+
DNA
polymerase I
Inorganic
pyrophosphate
Complement to template
strand is extended by one
nucleotide (n+1)
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DNA Polymerases II and III
1969 – Peter DeLucia and John Cairns discovered a mutant strain
of E. coli that was deficient in polymerase I activity.
Observation: the mutant strain duplicated its DNA and reproduced
itself but cells are highly deficient in DNA repair (UVsensitive).
Conclusions:
1. At least one more enzyme is able to replicate E. coli DNA.
2. DNA polymerase I may serve a secondary (at least for
replication) function which is associated with DNA fidelity.
Two other unique DNA polymerases have been isolated
Role of polymerases in vivo
Polymerase I :
-removes the RNA primer;
-fills the gaps that naturally occur as primers are removed;
-has proofreading function.
Polymerase II:
-is involved in UV-damaged DNA repair;
-has proofreading function.
Polymerase III:
-is the most replication relevant polymerase;
-has proofreading function.
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Properties of Three Bacterial DNA Polymerases
Initiation of chain synthesis
5’-3’ polymerization
3’-5’ exonuclease activity
5’-3’ exonuclease activity
Molecules of polymerase/cell
Synthesis from
Intact DNA
Primed single strands
Primed single strands plus SSB
Protein
In vitro chain elongation rate
Mutation lethal?
I
+
+
+
400
II
+
+
?
III
+
+
15
+
-
-
+
600
+
?
-
+
30000
+
DNA Polymerase III Holoenzyme
The DNA polymerase III holoenzyme is a very large (>600
kDa), highly complexed protein composed of 10 different
polypeptides. The so called core polymerase is composed of
3 subunits.
The α subunit contains active site for nucleoride addition,
and the ε subunit is a 3’-5’ exonuclease that removes
incorrectly added (mispaired) nucleotides at the end of
growing chain. The function of θ is still unknown.

The central role of the remaining subunits is to convert the
Polymerase III from distributive enzyme which falls the
template after forming short stretches of 10-50 nucleotides
to processive enzyme which can form stretches of up to 5 x
105 nucleotides before being released from the template.
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DNA Polymerase III Holoenzyme
The key to the processive activity of polymerase III is β subunit that forms a donut-shaped dimer around the DNA duplex and
then associates with and holds the catalytic core polymerase near
the 3’ terminus of growing strand.
Once associated with DNA , the β subunit functions like a “clamp”
which can slide freely along the DNA as the associated core
polymerase moves. In this way active sites of core polymerase
remain near the growing fork and the processivity of the enzyme
is maximized.
DNA Polymerase III Holoenzyme
Out of the six remaining subunits 5 (γγ,δδ, δ1,χ and ψ) form socalled γ complex that mediates two essential tasks:
1) Loading of β subunit clamp onto the duplex DNA-primer
substrate in a reaction that requires hydrolysis of ATP;
2) unloading of β subunit clamp after a strand of DNA has been
completed. Loading and unloading of the β subunit clamp
require opening of the clamp ring, but exactly how the γ
complex does it is still unknown.
The final τ subunit acts to dimerize two core polymerases and is
essential to coordinate the synthesis of leading and lagging
strands.
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Subunits of DNA Polymerase III Holoenzyme
Subunit
Function
Groupings
α
5’-3’ polymerization “Core” enzyme:
Elongates polynucleotide
ε
3’-5’ exonuclease
chain and proofreads
θ
??
γ
Loads enzyme on
δ
template (Serves
δ’
γ complex
as
clamp
loader)
χ
ψ
β
τ
Sliding clamp structure
(Processivity Factor)
Holds together the two core
polymerases at the replication fork
Subunits of DNA Polymerase III Holoenzyme
Space-filling model based on X-ray
crystallographic studies of the
dimeric β subunit binding to DNA
duplex. Two β subunits (red and
yellow) form a donat-shapes clamp.
That remains tightly bound to a
closed circular DNA molecule bur
readily slides off.
Schematic diagram of proposed
association of the core polymerase
with the β subunit clamp at the
primer-template terminus. This
interaction keeps the core from
falling off the template and
positions is near the point of
nucleotide addition.
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Leading and lagging strands are synthesized concurrently
Leading and
lagging strands are
linked together by
a τ subunit dimer.
Two molecules of
core polymerase
are bound at each
growing fork: one
at leading strand,
the other one at
lagging strand.
1) A single DnaB helicase moves along the lagging strand towards its 3’ end and
melts the duplex DNA at fork. 2) One core polymerase (core1) quickly adds
nucletides at 3’ end of the leading strand as its single-stranded template is
uncovered by the helicase action of DnaB. This leading strand polymerase,
together with its β subunit clamp remains bound to DNA, synthesizing leading
strand continuously.
Leading and lagging strands are synthesized concurrently
3) Second core
polymerase (core2)
synthesise the
lagging strand
discontinuously as
an Okazaki
fragment. The two
core polymerases
are linked by a
dimeric τ protein.
4) As each segment of the ss template for the lagging strand is uncovered, it
becomes coated with the SSB protein and forms a loop. Once synthesis of an
Okazaki fragment is completed, the lagging strand polymerase dissociates form
DNA but core remains bound to the τ dimer. The released polymerase
subsequently rebinds with the assistance of the another β clamp in the region of
the other Okazaki fragment.
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Leading and lagging strands are synthesized concurrently
Two molecules of core polymerase are bound at each growing
fork: one at leading strand, the other one at lagging strand.
The core polymerase synthesizing the leading strand moves,
together with its β subunit clamp, along its template in the direction
of movement of the fork, elongating the leading strand.
 It follows closely the movement of DnaB protein that melts the
duplex DNA of the fork.
 Since the core polymerase remains attached to the duplex DNA
the leading strand is synthesized continuously.
Leading and lagging strands are synthesized concurrently
The other core-polymerase molecule, which elongates the lagging
strand, moves with its its β subunit clamp in the direction opposite to
the fork movement.
 As elongation of the lagging strand proceeds, the size of the DNA
loop between the fork and this core polymerase increases.
Eventually core polymerase synthesizing the lagging strand will
complete an Okazaki fragment , then it dissociates from the DNA
template but the τ-subunit dimer remains to link it to the fork
proteins.
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Leading and lagging strands are synthesized concurrently
 Simultaneously, primase binds to the site adjacent to the DnaB
helicase on the single-stranded segment of the lagging strand
template and initiates synthesis of another RNA primer.
 The resulting DNA primer complex attracts another β subunit
clamp to this segment of lagging strand template, followed by rebinding of the core polymerase, which is still attached to the complex.
This polymerase then proceeds to elongate the RNA primer into
another Okazaki fragment.
As each Okazaki fragment nears completion, the RNA primer is
remover by the 5’→3’ exonuclease activity of DNA polymerase I.
 This enzyme also fills the gaps between the lagging strand
fragments, which are ligated together by DNA ligase.
Leading and lagging strands are synthesized concurrently
Although the two core polymerase molecules are linked by τsubunit dimer, they are oriented in opposite directions.
 Thus, the 3’ growing ends of both leading and lagging strands are
close together but offset from each other. For this reason the point
of the template from which the lagging strand is being copied is
displaced from the point in the template at which leading strand
copying is occurring.
Nonetheless, the two core polymerases can add
deoxyribonucleotides to the growing strands at the same time and
rate, so that leading and lagging strand synthesis occur
s concurrently.
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Leading and lagging strands are synthesized concurrently
 One τ-subunit also contacts the DnaB helicase at the fork. This
interaction strongly increases normally slow unwinding activity of
the helicase.
 Thus, there is a physical and functional link between the two
major replication machines at the fork – the two core polymerases
and the primosome complex of DnaB and primase.
Synthesis of leading and
lagging strands
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Cycling of polIII complex
Summary of
lagging strand
replication
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References:
R. Weaver, Molecular Biology, 2002
Lodish et al., Molecular cell biology, 2000.
T.Brown, Genomes, 1999.
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