DNA Replication
Basic Mechanism and Enzymology
DNA Replication
– Semiconservative – each of two parental
strands serves as a template
– Half-discontinuous – DNA is made of short
pieces that are stitched together later
– Requires RNA primers
– Usually bidirectional
Hypotheses of Replication
– Semiconservative replication would
produce molecules with both old and new
DNA, but each molecule would be
composed of one old strand and one new
one
– Conservative replication would leave
intact the original DNA molecule and
generate a completely new molecule
– Dispersive replication would produce
two DNA molecules with sections of both
old and new DNA interspersed along
each strand
¾ The experimental evidences support the
semiconservative model for DNA replication
Hypotheses of Replication
– E.coli cells were grown in a
medium enriched with
heavy nitrogen isotope and
then transferred to the
normal (light) medium for
the various length of time.
– DNA was isolated and
subjected to the CsCl
gradient ultracentrifugation
Semiconservative Replication
– DNA replicates in a semiconservative
manner
– When parental strands separate each
strand serves as template for DNA
synthesis
DNA Replication Models
Semidiscontinuous Replication
• DNA polymerase synthesize DNA only in 5’→3’direction
– One strand (the leading strand) is replicated
continuously in the direction of the movement of the
replicating fork
– The other strand (the lagging strand) is replicated
discontinuously as 1-2 kb Okazaki fragments in the
opposite direction
– Both strands are replicated in 5’→3’-direction
• DNA replication is semidiscontinuous
Priming DNA Synthesis
• DNA polymerase cannot
initiate DNA synthesis
• DNase cannot completely
destroy Okazaki fragments
leaving short RNA peaces
• DNA synthesis is initiated
with RNA primers 10-12 nt
long
• DNA polymerase uses RNA
primer to start the synthesis
Direction of Replication
9 Replication starts at the defined sequence of
nucleotides called the origin of replication
9 Replicating fork represent a site of DNA replication
– Unidirectional replication: one replicating fork moves
away from another, which remains fixed in the origin of
replication
– Bidirectional replication: two replicating forks move in
opposite directions away from origin
9 Each DNA region served by one origin of replication is
called a replicon
Direction of Replication
Some linear DNA
viruses
Certain plasmids
Most common for
eukaryotes and
prokaryotes
Bidirectional Replication
• Also known as a θ model for DNA replication
• DNA replication begins with the creation of a
“bubble” – a small region where parental strands
have separated and progeny DNA has been
synthesized
• As the bubble expands, replicating DNA begins
to take on the θ shape
Bidirectional Replication
9 X and Y mark two replicating forks moving in opposite
directions from origin of replication
Demonstration of Bidirectional
Replication
Bidirectional Replication in
Eukaryotes
9 Eukaryotic
chromosomes have
multiple replicons
9 Multiple replicons
increase the speed of
DNA replication
Unidirectional Replication
– ColE1 is an example of a DNA molecule
that replicates unidirectionally
– ColE1 is a plasmid present in E. coli and
can replicate independently from cell’s
chromosome
Replication Origin
Replication origin consist of specific nucleotide sequences
recognized by initiator proteins.
– A-T rich (easier to separate)
–
100 bp (base pairs) in length
Prokaryotes
–Have one replication origin per chromosome (plasmid)
–Replication rate is on average of 500 nt/sec (up to 1000nt/sec)
Eukaryotes
–Have multiple replication sites on each chromosome
–Replication rate is on average of 50 nt/sec
–Replication origins are activated in clusters of 20 to 80 adjacent
origins forming replication units
The pattern of replication is controlled, temporally and spatially
Rolling Circle Replication
• Also known as a σ-model for
DNA replication
• Circular DNAs (phage φX174,
contains single-stranded DNA
genome) can replicate by a
rolling circle mechanism
– One strand of a dsDNA is
nicked and the 3’-end is
extended using the intact DNA
strand as a template; the 5’end is displaced later
Rolling Circle Replication
• In phage λ (contains double-stranded DNA genome),
displaced strand serves as the template for discontinuous,
lagging strand synthesis
• As the circle rolls right
– Leading strand elongates continuously
– Lagging strand elongates discontinuously
• Uses unrolled leading strand as a template
• Uses RNA primers for each Okazaki fragment
• The newly produced dsDNA reaches the size of many
genomes before one genome worth fragments are clipped off
E. coli DNA Polymerases
• There are 3 DNA polymerases found in E. coli:
– pol I:
• removes the RNA primer
• fills the gaps that naturally occur as primers are
removed
• has proofreading function
– pol II:
• is involved in UV-damaged DNA repair
• facilitate DNA synthesis directed by damaged templates
• has proofreading function
– pol III:
• is the most replication relevant polymerase
• has proofreading function.
DNA Polymerase I
• DNA polymerase I (pol I) is a versatile enzyme with
3 distinct activities
– DNA polymerase
– 3’→5’ exonuclease
– 5’→3’ exonuclease
• Mild proteolytic treatment of pol I results in 2
polypeptides
– Klenow fragment (polymerase and 3’→5’
exonuclease activities)
– Smaller fragment (5’→3’ exonuclease activity)
Klenow (Large) Fragment
3’→5’ exonuclease activity of pol I (Klenow
Fragment) is required for proofreading
– Wrong nucleotide added by pol I do not base pair
properly
– Pol I pauses, and using exonuclease activity
removes mispaired nucleotide
• Allows replication to continue
• Increases fidelity of replication
5’→3’ Exonuclease (Small Fragment)
• This activity allows pol I
to degrade a strand
ahead of advancing
polymerase
• Removes and replaces
a strand in one pass
• Basic functions are:
– RNA primers removal
– Nick repair
Polymerases II and III
• Pol I is mostly active in DNA repair
• Pol II activity is not required for DNA
replication, also involved in DNA repair
• Pol III is the only polymerase that is
required for DNA replication in bacteria
The Pol III Holoenzyme
• Pol III core is composed of 3 subunits:
– α-subunit (DNA polymerase)
– ε-subunit (3’→5’exonuclease)
– θ-subunit (the role is not clear)
• DNA-dependent ATPase activity is located in the
γ-complex containing 5 subunits (γ,δ, δ1,χ and ψ )
• β-subunit plus the other remaining 8 subunits
comprise the holoenzyme
The Pol III Holoenzyme
– The central role of the subunits not involved in a core
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.
The Pol III Holoenzyme
The processive activity of polymerase III is mediated by β
subunit
– 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.
– This ensures that the active sites of core polymerase remain
near the growing fork maximizing processivity of the enzyme
The Pol III Holoenzyme
Five subunits (γ,δ, δ1,χ and ψ) form so-called γ complex
that mediates two essential tasks:
– Loading of β subunit clamp onto the duplex DNA-primer
substrate in a reaction that requires hydrolysis of ATP
– unloading of β subunit clamp after strand of DNA has been
completed.
– Loading and unloading of the β subunit clamp requires
opening of the clamp ring
The final τ subunit dimerizes two core polymerases and
is essential to coordinate the synthesis of leading and
lagging strands.
The Pol III Holoenzyme
Subunit
α
ε
θ
γ
δ
δ’
χ
ψ
β
τ
Function
5’-3’ polymerization 3’5’ exonuclease
??
Loads enzyme on
template (Serves as
clamp loader)
Sliding clamp structure
(Processivity Factor)
Holds together the two core
polymerases at the
replication fork
Groupings
“Core” enzyme: Elongates
polynucleotide chain and
proofreads
γ complex
The Pol III Holoenzyme
Space-filling model based on Xray crystallographic studies of the
dimeric β subunit binding to DNA
duplex. Two β subunits (red and
yellow) form a donat-shaped
clamp that remains tightly bound
to DNA molecule but easily slides
along it.
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.
Fidelity of Replication
• DNA replication machinery has a built-in
proofreading system that requires priming
– Only correctly base-paired nucleotide can serve as a
primer for pol III holoenzyme
– If wrong nucleotide is incorporated accidentally replication
stalls until 3’→5’ exonuclease of pol III holoenzyme
removes it
• Primers are made of RNA and may contain errors.
Having primers made of RNA marks them for
degradation and replacement with DNA
Eukaryotic DNA Polymerases
Mammalian cells contain 5 different DNA polymerases:
Polymerase α - priming of replication of both strands (primase);
has low processivity
Polymerase δ - elongation of both strands;
has high processivity
Polymerase β - DNA repair;
not processive: adds only one nucleotide and
falls off the template
Polymerase ε - DNA repair
Polymerase γ - replication of mitochondrial DNA;
found only in mitochondria
Strand Separation
– DNA replication needs that the two DNA
strands at the fork become somehow
separated
– Strands separation requires enzyme, a
helicase that unwinds dsDNA at the replicating
fork (ex. E. coli dnaB gene)
– The helicase activity is ATP-dependent
Single-Strand DNA-Binding Proteins
• Prokaryotic ssDNA-binding proteins preferentially bind
to DNA in a single-stranded form
– SSBs trap unwound by helicase DNA in a singlestranded form, preventing it from annealing with its
partner
– SSBs stimulate DNA polymerase
• By coating ssDNA, SSBs protect it from degradation
• SSBs are essential for prokaryotic DNA replication, but
there is a limited number of eukaryotic SSBs known
(RF-A protein)
Topoisomerases
• When two strands of DNA separate during
replication they rotate around each other
• This introduces supercoils
to DNA
– The supercoils introduced
due to overwinding are
called positive
– The supercoils introduced
due to underwinding are
called negative
Topoisomerase Mechanism
• As helicase unwinds two parental strands it
introduces a compensating positive supercoiling
force
• Stress of this force must be overcome or DNA will
resist progression of replicating fork
• This stress releasing mechanism is called swivel
• DNA gyrase acts as swivel by pumping negative
supercoils into replicating DNA that neutralizes
positive supercoils
Supercoiling
– Supercoiling is a physical
rearrangement of the DNA
double helix that allows it to
conform more closely to the
ideal B-DNA structure under
circumstances in which it
otherwise might not.
– Supercoiling arises in any
helical or coiled structure in
which two ends are constrained
in some way.
Supercoiling
In a double helical molecule of DNA, the two polynucleotide
backbone chains have rotational freedom about one another as long
as the ends are not fixed
– If any part of a linear molecule of DNA is locally twisted, then
that twist can be redistributed throughout the molecule and it
can re-assume its most stable (Watson-Crick) conformation
If the ends of the molecule are fixed then any rotational freedom is
removed and as a result any local twisting or untwisting will cause a
stress on the structure
– The ends of a DNA double helix can be fixed in place if the
ends are joined to one another to form a circular molecule or if
the DNA is bound by protein.
Topoisomerases
•Type I Topoisomerases:
–Introduce temporary single stranded breaks in DNA
–Can relax only negative supercoils in bacteria and
negative and positive in eukaryotes
•Type II Topoisomerases:
–Introduce temporary break simultaneously in both
DNA chains
–Can relax positive supercoils
Topoisomerase I
– Enzyme binds to DNA molecule, cuts one strand, and generates a
phosphodiester bond between the released phosphate on the DNA and the
tyrosine residue in the enzyme
– The DNA strand that has not been cleaved is then passed through the
single-stranded break
– The cleaved strand is then released, forming the structure with the same
chemical bonds as in DNA, but with one less negative supercoil
Topoisomerase I
– Removal of supercoils by Topoisomerase I is energetically
favorable and reaction proceeds in absence of ATP
– Topoisomerase I can remove only one negative supercoil
during one reaction
DNA from SV 40 virions, under conditions
that will ensure max of supercoils
.
25 bands – number of possible
topoisomers
3 min 30 min
Topo I
Topoisomerase II
–Topoisomerase II enzyme have the ability to cut both
strands of a double-stranded DNA molecule, pass another
portion of the duplex through the cut, and reseal the cut in
process that utilizes ATP.
–Depending upon the substrate, these maneuvers will have
the effects of changing a positive supercoil into negative or of
increasing the number of negative supercoils by 2
Topoisomerase II
– Topo II enzymes from mammalian cells can not, like E. coli
DNA gyrase, increase the superhelical density at the
expense of ATP. Presumably no such activity is required in
eukaryotes, since binding of histones increases the potential
superhelicity.
– All type II topoisomerases catalyze catenation and
decatenation, that is linking and unlinking of two different
DNA duplexes.
–Type II topoisomerases are important for growing fork
movement and in resolving (untangling) finished
chromosomes (linear and circular) after DNA duplication
Topoisomerase II
(a) Introduction of negative supercoils. The initial folding introduces no stable
change, but a subsequent activity of gyrase produces a stable structure with
two negative supercoils. Eukaryotic Topo II enzymes cannot introduce
supercoils but can remove negative supercoils from DNA.
(b) Catenation and decatenation of two different DNA duplexes. Both
eukaryotic and prokaryotic enzymes can catalyse this reaction.
DNA Gyrase
– DNA gyrase is composed of two identical subunits
– Hydrolysis of ATP by gyrase’s inherent ATPase activity
powers conformational changes that are critical for enzyme
operation
– The enzyme functions to introduce negative supercoils at or
near the oriC site in the DNA template; DNA replication can
initiate only on a negatively supercoiled template
– Measurements of the degree of DNA supercoiling in E. coli
suggests that there is one negative supercoil for each 15-20
turns of the DNA helix
Model for DNA Gyrase Reaction
–The enzyme is a dimer of two
identical subunits
–Initially enzyme binds to one part
of a DNA strand, the G segment,
inducing a conformational change
in other enzyme domains
–Enzyme binds ATP and another
part of DNA strand, the T
segment.
Model for DNA Gyrase Reaction
–The G segment is cut by B’ domains
and the ends of the G DNA become
covalently linked to tyrosine residues in
these domains
–The T segment is passed through into
the lower jaws, where it is released
–The G segment is rejoined
–ATP is hydrolyzed restoring
conformational state required to accept
new T segment
–If needed, the segment-passing
process is repeated
Reading:
R. Weaver, Molecular Biology, 4th ed.
Chapter 20: pages 641-660