The Replication of DNA

Disclaimer: All the content material (Photographs and the text) used in this presentation are from different sources, I acknowledge them and they are used only
for teaching purpose to have better insight and understanding of the topic.
Watson and Crick 1953 article in Nature
 Double helix structure of DNA

“It has not escaped our notice that the specific pairing we have postulated
immediately suggests a possible copying mechanism for the genetic
material.” Watson & Crick
Directionality of DNA
• You need to nucleotide
PO4
number the
carbons!
• it matters!
N base

5 CH2
This will be O
IMPORTANT!!
4 ribose 1

3 2
OH
The DNA backbone 5
PO4
• Putting the DNA backbone
together base
5 CH2
• refer to the 3 and 5 ends of
O
the DNA
4 1
• the last trailing carbon C
3 2
O
–O P O

Sounds trivial, but…

O base
this will be 5 CH
2
IMPORTANT!! O
4 1

3 2
OH
3
 Anti-parallel strands

• Nucleotides in DNA backbone

are bonded from phosphate to
sugar between 3 & 5 carbons 5 3
• DNA molecule has “direction”
• complementary strand runs in
opposite direction

3 5
Bonding in DNA
hydrogen
bonds
5 3

covalent
phosphodiester
bonds

3
5

….strong or weak bonds?

How do the bonds fit the mechanism for copying DNA?
Base pairing in DNA
• Purines
• adenine (A)
• guanine (G)
• Pyrimidines
• thymine (T)
• cytosine (C)
• Pairing
• A:T
• 2 bonds
• C:G
• 3 bonds
Copying DNA

• Replication of DNA
• base pairing allows each
strand to serve as a
template for a new strand
• new strand is 1/2 parent
template &
1/2 new DNA
 Mechanism of DNA Replication
❖ Conservative model: According to this hypothesis, both strands of parental
DNA remain together following DNA replication. In this model, the original
arrangement of parental strands is completely conserved, while the two
newly made daughter strands also remain together following replication.

❖ Semiconservative model: In this mechanism, the double stranded DNA is

half conserved following the replication process. In other words, the newly
made double-stranded DNA contains one parental strand and one daughter
strand.

❖ Dispersive model: It proposes that segments of parental DNA and newly

made DNA are interspersed in both strands following the replication
process. Or In which each strand of each daughter molecule is composed
partially of the original polynucleotide and partially of newly synthesized
polynucleotide.
Alternative models of DNA replication
 DNA Replication
❖Matthew Meselson & Franklin Stahl, 1958 investigated
the process of DNA replication considered 3 possible
mechanisms:
– conservative model
– semiconservative model
– dispersive model
 Meselson and Stahl’s Experiment
❖ To determine which of the three models of replication applied to E. coli cells,
Matthew Meselson and Franklin Stahl needed a way to distinguish old and new
DNA.
❖ They did so by using two isotopes of nitrogen, 14N (the common form) and 15N
(a rare, heavy form).
❖ Meselson and Stahl grew a culture of E. coli in a medium that contained 15N as
the sole nitrogen source; after many generations, all the E. coli cells had 15N
incorporated into all of the purine and pyrimidine bases of their DNA.
❖ Meselson and Stahl took a sample of these bacteria, switched the rest of the
bacteria to a medium that contained only 14N, and then took additional samples
of bacteria over the next few cellular generations.
❖ In each sample, the bacterial DNA that was synthesized before the change in
medium contained 15N and was relatively heavy, whereas any DNA synthesized
after the switch contained 14N and was relatively light.
❖ Meselson and Stahl distinguished between the heavy 15N laden DNA and the
light 14N-containing DNA with the use of equilibrium density gradient
centrifugation.
1958: Matthew Meselson & Frank Stahl’s Experiment
Equilibrium density gradient centrifugation
❖ In this technique, a centrifuge tube is filled with a heavy salt solution of
cesium chloride (CsCl) and a substance of unknown density—in this case,
DNA fragments.
❖ The tube is then spun in a centrifuge at high speeds. After several days of
spinning, a gradient of density develops within the tube, with high density at
the bottom and low density at the top.
❖ The density of the DNA fragments matches that of the salt: light molecules
rise and heavy molecules sink.
❖ Meselson and Stahl found that DNA from bacteria grown only on medium
containing 15N produced a single band at the position expected of DNA
containing only 15N.
❖ DNA from bacteria transferred to the medium with 14N and allowed one round
of replication also produced a single band but at a position intermediate
between that expected of DNA containing only 15N and that expected of DNA
containing only 14N.
❖ This result is inconsistent with the conservative replication model, which
predicts one heavy band (the original DNA molecules) and one light band (the
new DNA molecules).
❖ A single band of intermediate density is predicted by both the
semiconservative and the dispersive models.
❖ To distinguish between these two models, Meselson and Stahl grew the
bacteria in medium containing 14N for a second generation.
❖ After a second round of replication in medium with 14N, two bands of equal
intensity appeared, one in the intermediate position and the other at the
position expected of DNA containing only 14N.
❖ All samples taken after additional rounds of replication produced the same two
bands, and the band representing light DNA became progressively stronger.
❖ Meselson and Stahl’s results were exactly as expected for semiconservative
replication and are incompatible with those predicated for both conservative
and dispersive replication.
1958: Matthew Meselson & Frank Stahl’s Experiment
Semiconservative model of DNA replication
❖ The molecule exhibiting intermediate density is
also consistent with dispersive replication.
However Meselson and Stahl ruled out this mode
of replication on the basis of two observation.
❖ After 1st generation of replication in an 14N
containing medium, they isolated the hybrid
molecule and heat denatured it. When the densities
of the single strands of the hybrid were
determined, they exhibited either an 15N profile or
an 14N profile but not an intermediate density. This
observation is consistent with the
semiconservative mode, but inconsistent with
dispersive mode.
❖ Further if replication were dispersive, all
generation after t=0 would demonstrate DNA of an
intermediate density. In each generation after the
first, the ratio of 15N/14N would decrease and the
hybrid band would become lighter and lighter,
eventually approaching the 14N band. This was not
observed.
Replicon is any piece of DNA which replicates as a
single unit. It contains an origin and sometimes a
terminus

Origin is the DNA sequence where a replicon initiates

its replication.

Terminus is the DNA sequence where a replicon

usually stops its replication
Prokaryotic genome: a single circular
DNA = a single replicon

Eukaryotic genome: multiple linear

chromosomes & multiple replicons on
each chromosome
1955: Arthur Kornberg

Worked with E. coli.

Discovered the mechanisms of DNA synthesis.

Four components are required:

1. dNTPs: dATP, dTTP, dGTP, dCTP

(deoxyribonucleoside 5’-triphosphates)
(sugar-base + 3 phosphates)
2. DNA template
3. DNA polymerase (Kornberg enzyme)
4. Mg 2+ (optimizes DNA polymerase activity)

1959: Arthur Kornberg (Stanford University)

& Severo Ochoa (NYU)
 DNA Template

• Each strand of the parent DNA is used as a template to make

the new daughter strand
• DNA replication makes 2 new complete double helices each
with 1 old and 1 new strand
 THE CHEMISTRY OF DNA SYNTHESIS
DNA Synthesis Requires Deoxynucleoside Triphosphates and a Primer:Template Junction
✓ For the synthesis of DNA to proceed, two key substrates must be present.
First, new synthesis requires the four deoxynucleoside triphosphates—dGTP,
dCTP, dATP, and dTTP.
✓ Nucleoside triphosphates have three phosphoryl groups that are attached via
the 5’-hydroxyl of the 2’-deoxyribose.
✓ The phosphoryl group proximal to the deoxyribose is called the α-phosphate,
whereas the middle and distal groups are called the ꞵ--phosphate and the γ -
phosphate, respectively.
✓ The second essential substrate for DNA synthesis is a particular arrangement
of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) called as
primer : template junction.
✓ As suggested by its name, the primer: template junction has two key
components. The template provides the ssDNA that directs the addition of
each complementary deoxynucleotide.
❖ The primer is complementary to, but shorter than, the template.
❖ The primer must have an exposed 3’-OH adjacent to the single-strand region of the
template. It is this 3’-OH that will be extended by nucleotide addition.
❖ Formally, only the primer portion of the primer:template junction is a substrate for
DNA synthesis because only the primer is chemically modified during DNA
synthesis.
❖ The template provides only the information necessary to select which nucleotides
are added. Nevertheless, both a primer and a template are essential for all DNA
synthesis.

Substrates required for DNA synthesis. (a) The general structure of the 2-deoxynucleoside triphosphates. The positions of the
α-phosphate, ꞵ-phosphate, and γ -phosphate are labeled. (b) The structure of a generalized primer:template junction. The
shorter primer strand is completely annealed to the longer DNA strand and must have a free 3’-OH adjacent to an ssDNA
region of the template. The longer DNA strand includes a region annealed to the primer and an adjacent ssDNA region that
acts as the template for new DNA synthesis. New DNA synthesis extends the 3’ end of the primer.
 DNA Is Synthesized by Extending the 3’ End of the Primer
❖ The chemistry of DNA synthesis requires that the new chain grows by
extending the 3’ end of the primer.
❖ Indeed, this is a feature of the synthesis of both RNA and DNA. The
phosphodiester bond is formed in which the hydroxyl group at the 3’ end of the
primer strand attacks the α-phosphoryl group of the incoming nucleoside
triphosphate.
❖ The leaving group for the reaction is pyrophosphate, which is composed of the
ꞵ-phosphate and γ-phosphate of the nucleotide substrate. Pyrophophatase
rapidly hydrolysed pyrophosphate into two phosphate molecules.
❖ The template strand directs which of the four nucleoside triphosphates is
added. The nucleoside triphosphate that base-pairs with the template strand is
highly favoured for addition to the primer strand.
❖ Recall that the two strands of the double helix have an antiparallel orientation.
This arrangement means that the template strand for DNA synthesis has the
opposite orientation of the growing DNA strand.
Diagram of the mechanism of DNA synthesis. DNA synthesis is initiated when the 3’-OH of the primer
mediates the nucleophilic attack of the a-phosphate of the incoming dNTP. This results in the
extension of the 3’ end of the primer by one nucleotide and releases one molecule of pyrophosphate.
Pyrophosphatase rapidly hydrolyzes released pyrophosphate into two phosphate molecules.
 Kornberg’s Enzyme DNA Polymerase
❖ Soon after Kornberg’s initial discovery, several other forms of DNA polymerase
were detected.
❖ In E. coli, the enzyme discovered by Kornberg turned out not to be responsible
for DNA replication in intact cells.
❖ This fact first became apparent when it was discovered that mutant strains of
bacteria lacking the Kornberg enzyme can still replicate their DNA and
reproduce normally.
❖ Eventually several other bacterial enzymes that synthesize DNA were
identified.
❖ These additional enzymes are named using Roman numerals (for example,
DNA polymerases II, III, IV, and V) to distinguish them from the original
Kornberg enzyme, now called DNA polymerase I.
❖ When the rates at which the various DNA polymerases synthesize DNA in a test
tube were first compared, only DNA polymerase III was found to work fast
enough to account for the rate of DNA replication in intact cells, which
averages about 50,000 base pairs/minute in bacteria.
❖ Such observations suggested that DNA polymerase III is the main enzyme
responsible for DNA replication in bacterial cells, but the evidence would be
more convincing if it could be shown that cells lacking DNA polymerase III are
unable to replicate their DNA.
❖ One powerful approach to do so involves the use of temperature-sensitive
mutants, which are bacteria that produce proteins that function properly at
normal temperatures but become seriously impaired when the temperature is
altered slightly.
❖ For example, mutant bacteria have been isolated in which DNA polymerase III
behaves normally at 37°C but loses its function when the temperature is raised
to 42°C.
❖ Such bacteria grow normally at 37°C but lose the ability to replicate their DNA
when the temperature is elevated to 42°C, indicating that DNA polymerase III
plays an essential role in the process of normal DNA replication.
❖ DNA polymerases show an impressive ability to distinguish between
ribonucleoside and deoxyribonucleoside triphosphates (rNTPs and dNTPs).
❖ Although rNTPs are present at approximately 10-fold higher concentration in the
cell, they are incorporated at a rate that is more than 1000-fold lower than dNTPs.
❖ This discrimination is mediated by the steric exclusion of rNTPs from the DNA
polymerase active site.
❖ In DNA polymerase, the nucleotide-binding pocket cannot accommodate a 2’-OH
on the in-coming nucleotide.
❖ This space is occupied by two amino acids that make van der Waals contacts with
the sugar ring.
❖ Changing these amino acids to other amino acids with smaller side chains (e.g.,
by changing a glutamate to an alanine) results in a DNA polymerase with
significantly reduced discrimination between dNTPs and rNTPs.
❖ Nucleotides that meet some but not all of the requirements for use by DNA
polymerase can inhibit DNA synthesis by terminating elongation.
❖ Such nucleotides represent an important class of drugs used to treat cancer and
viral infections.
Correctly paired bases are required for
DNA-polymerase catalyzed nucleotide
addition.
(a)Schematic diagram of the attack of a
primer 3’-OH end on a correctly base-
paired dNTP.
(b)Schematic diagram of the
consequence of incorrect base
pairing on catalysis by DNA
polymerase. In the example shown,
the incorrect A:A base pair displaces
the a-phosphate of the incoming
nucleotide. This incorrect alignment
reduces the rate of catalysis
dramatically, resulting in the DNA
polymerase preferentially adding
correctly base paired dNTPs.
 DNA Topoisomerases
❖ E. coli chromosome contains a circular molecule of DNA. With the E. coli DNA
spinning at 3000 revolutions per minute to allow the unwinding of the parental
strands during replication, what provides the swivel or axis of rotation that
prevents the DNA from becoming tangled (positively supercoiled) ahead of the
replication fork? The required axes of rotation during the replication of circular
DNA molecules are provided by enzymes called DNA topoisomerases.
❖ The topoisomerases catalyze transient breaks in DNA molecules but use
covalent linkages to themselves to hold on to the cleaved molecules. The
topoisomerases are of two types:
(1) DNA topoisomerase I enzymes produce temporary single-strand breaks or
nicks in DNA, and
(2) DNA topoisomerase II enzymes produce transient double strand breaks in
DNA.
❖ Topoisomerase I activities remove supercoils from DNA one at a time, whereas
topoisomerase II enzymes remove and introduce supercoils two at a time.
❖ DNA topoisomerase I:
✓ Topoisomerase I activities remove
supercoils from DNA one at a time.
✓ The transient single-strand break produced
by the activity of topoisomerase I provides
an axis of rotation that allows the segments
of DNA on opposite sides of the break to
spin independently, with the phosphodiester
bond in the intact strand serving as a swivel.
✓ Topoisomerase I enzymes are energy-
efficient.
✓ They conserve the energy of the cleaved
phosphodiester linkages by storing it in
covalent linkages between themselves and
the phosphate groups at the cleavage sites;
they then reuse this energy to reseal the
breaks.
❖ DNA topoisomerase II:
✓ Topoisomerase II enzymes induce transient
double-strand breaks and add negative
supercoils or remove positive supercoils two
at a time by an energy (ATP)-requiring
mechanism.
✓ They carry out this process by cutting both
strands of DNA, holding on to the ends at the
cleavage site via covalent bonds, passing
the intact double helix through the cut, and
resealing the break.
✓ In addition to relaxing supercoiled DNA and
introducing negative supercoils into DNA,
topoisomerase II enzymes can separate
interlocking circular molecules of DNA.
✓ The best-characterized type II topoisomerase
is an enzyme named DNA gyrase in E. coli.
✓ DNA gyrase is a tetramer with two α subunits encoded by the gyrA gene (originally
nalA, for nalidixic acid) and two ꞵ subunits specified by the gyrB gene (formerly cou,
for coumermycin).
✓ Nalidixic acid and coumermycin are antibiotics that block DNA replication in E. coli by
inhibiting the activity of DNA gyrase.
✓ Nalidixic acid and coumermycin inhibit DNA synthesis by binding to the α and ꞵ
subunits, respectively, of DNA gyrase. Thus, DNA gyrase activity is required for DNA
replication to occur in E. coli.
✓ Recall that chromosomal DNA is negatively supercoiled in E. coli.
✓ The negative supercoils in bacterial chromosomes are introduced by DNA gyrase, with
energy supplied by ATP. This activity of DNA gyrase provides another solution to the
unwinding problem.
✓ Instead of creating positive supercoils ahead of the replication fork by unwinding the
complementary strands of relaxed DNA, replication may produce relaxed DNA ahead
of the fork by unwinding negatively supercoiled DNA.
✓ Because super helical tension is reduced during unwinding—that is, strand separation
is energetically favoured—the negative supercoiling behind the fork may drive the
unwinding process.
 DNA Replication
DNA replication includes:
• initiation – replication begins at an origin of
replication
• elongation – new strands of DNA are
synthesized by DNA polymerase
• termination – replication is terminated
differently in prokaryotes and eukaryotes

48
Prokaryotic DNA
Replication
Bidirectional replication of a circular bacterial replicon

• All prokaryotic chromosomes and

many bacteriophage and viral DNA
molecules are circular and comprise
single replicons.
• There is a single termination site
roughly 180o opposite the unique
origin.
❖ There are, however, several different ways in which semiconservative
replication can take place, differing principally in the nature of the template
DNA—that is, whether it is linear or circular.
❖ Individual units of replication are called replicons, each of which contains a
replication origin. Replication starts at the origin and continues until the entire
replicon has been replicated.
❖ Bacterial chromosomes have a single replication origin, whereas eukaryotic
chromosomes contain many.
❖ Theta replication: A common type of replication that takes place in circular
DNA, such as that found in E. coli and other bacteria, is called theta replication
because it generates a structure that resembles the Greek letter theta (θ).
❖ In theta replication, double-stranded DNA begins to unwind at the replication
origin, producing singles stranded nucleotide strands that then serve as
templates on which new DNA can be synthesized.
❖ The unwinding of the double helix generates a loop, termed a replication
bubble.
❖ Unwinding may be at one or both ends of the bubble, making it progressively
larger.
❖ DNA replication on both of the template strands is simultaneous with
unwinding. The point of unwinding, where the two single nucleotide strands
separate from the double-stranded DNA helix, is called a replication fork.
❖ If there are two replication forks, one at each end of the replication bubble, the
forks proceed outward in both directions in a process called bidirectional
replication, simultaneously unwinding and replicating the DNA until they
eventually meet.
❖ If a single replication fork is present, it proceeds around the entire circle to
produce two complete circular DNA molecules, each consisting of one old and
one new nucleotide strand.
❖ John Cairns provided the first visible evidence of theta replication in 1963 by
growing bacteria in the presence of radioactive nucleotides. After replication,
each DNA molecule consisted of one “hot” (radioactive) strand and one “cold”
(nonradioactive) strand.
 John Cairns Experiment to show Theta Replication
❖ In 1963, John Cairns tested this prediction by allowing replicating DNA in
bacterial cells to incorporate tritiated thymidine ([3H] thymidine) - the thymine
nucleotide labelled with a radioactive hydrogen isotope called tritium.
❖ Theoretically, each newly synthesized daughter molecule should then contain
one radioactive (“hot”) strand (with 3H) and another nonradioactive (“cold”)
strand (with 2H).
❖ After varying intervals and varying numbers of replication cycles in a “hot”
medium, Cairns carefully lysed the bacteria and allowed the cell contents to
settle onto a piece of filter paper, which was put on a microscope slide.
❖ Finally, Cairns covered the filter with photographic emulsion and exposed it in
the dark for 2 months.
❖ This procedure, called autoradiography, allowed Cairns to develop a picture of
the location of 3H in the cell material. As 3H decays, it emits a beta particle (an
energetic electron).
❖ The photographic emulsion detects a chemical reaction that takes place
wherever a beta particle strikes the emulsion.
❖ The emulsion can then be developed like a photographic print so that the
emission track of the beta particle appears as a black spot or grain.
❖ After one replication cycle in [3H] thymidine, a ring of dots appeared in the
autoradiograph.
❖ Cairns interpreted this ring as a newly formed radioactive strand in a circular
daughter DNA molecule. It is thus apparent that the bacterial chromosome is
circular.
❖ In the second replication cycle, the forks predicted by the model were indeed
seen. Furthermore, the density of grains in the three segments was such that
the interpretation shown in Figure could be made: the thick curve of dots
cutting through the interior of the circle of DNA would be the newly
synthesized daughter strand, this time consisting of two radioactive strands.
❖ Cairns saw all sizes of these moon-shaped, autoradiographic patterns,
corresponding to the progressive movement of the replication forks, around
the ring. Structures of the sort shown in Figure b are called theta (θ) structures.
A replicating bacterial chromosome has
two replication forks.
(a) Left: Autoradiograph of a bacterial
chromosome after one replication in tritiated
thymidine. According to the semiconservative
model of replication, one of the two strands
should be radioactive. Right: Interpretation of
the autoradiograph. The orange helix
represents the tritiated strand.

(b)Left: Autoradiograph of a bacterial

chromosome in the second round of
replication in tritiated thymidine. In this theta
(θ) structure, the newly replicated double helix
that crosses the circle could consist of two
radioactive strands (if the parental strand were
the radioactive one).
Right: The double thickness of the radioactive
tracing on the autoradiogram appears to
confirm the interpretation shown here.
The process of bacterial
chromosome replication.
(a) An overview of the process
of bacterial chromosome
replication.
(b) A replicating E. coli
chromosome visualized by
autoradiography and
transmission electron
microscopy (TEM). This
chromosome was
radiolabeled by growing
bacterial cells in media
containing radiolabeled
thymidine. The diagram at
the right shows the
locations of the two
replication forks. The
chromosome is about one-
third replicated. New
strands are shown in blue.
Bi-directional replication in E. coli
Significant Findings of Cairns Experiment

❖ Bacteria DNA is Circular

❖ It has single origin
❖ Replication is bidirectional and
❖ It undergoes semiconservative mode of
Replication
Replication of circular DNA in
E. coli :

1. Two replication forks result in a

theta-like () structure.

2. As strands separate, positive

supercoils form elsewhere in the
molecule.

3. Topoisomerases relieve tensions

in the supercoils, allowing the
DNA to continue to separate.
 Prokaryotic
Replication

Disclaimer: All the content material (Photographs and the text) used in this presentation are from different sources, I acknowledge them and they are used only
for teaching purpose to have better insight and understanding of the topic.
❖ DNA polymerase III, the “replicase” in E. coli, is a multimeric enzyme (an
enzyme with many subunits) with a molecular mass of about 900,000 daltons
in its complete or holoenzyme form.
❖ The holoenzyme contains at least seven different subunits. The minimal core
that has catalytic activity in vitro contains three subunits these are alpha (α),
epsilon (ε) and theta (θ).
❖ α alpha (the product of dnaE gene),is the catalytic subunit.
❖ ε (epsilon) (the product of dnaQ), it is responsible for proofreading 3’ to 5’
activity of the polymerase.
❖ The function of θ (theta) (the product of holE) subunit remains to be
established.
❖ The auxiliary subunits, β (beta), γ (gamma) and δ (delta), encoded by dnaN,
dnaZ and dnaX respectively are required for the functioning of polymerase on
natural chromosome.
❖ Addition of the τ (tau) subunit (the dnaX product) results in polymerase
dimerization, perhaps forming a structure that can coordinate leading and
lagging strand synthesis at the replication fork and thus helps in increased
activity of polymerase.
❖ The catalytic core synthesizes rather short DNA strands because of its
tendency to fall off the DNA template.
❖ In order to synthesize the long DNA molecules, present in chromosomes, this
frequent dissociation of the polymerase from the template must be eliminated.
❖ The β (beta) subunit (the dnaN gene product) of DNA polymerase III forms a
dimeric clamp that keeps the polymerase from falling off the template DNA.
❖ The β-dimer forms a ring that encircles the
replicating DNA molecule and allows DNA
polymerase III to slide along the DNA while
remaining tethered to it.
❖ The DNA polymerase III holoenzyme, which
is responsible for the synthesis of both
nascent DNA strands at a replication fork,
contains at least 20 polypeptides.
❖ The structural complexity of the DNA
polymerase III holoenzyme is illustrated in
Figure.
four
 Prokaryotic DNA Replication

The enzymes for DNA replication are contained within the

replisome.
The replisome consists of
• the primosome - composed of primase and helicase
• 2 DNA polymerase III molecules
The replication fork moves in 1 direction, synthesizing both
strands simultaneously.

80
 Initiation
❖ The replication of the E. coli chromosome
begins at oriC, the unique sequence at which
replication is initiated, with the formation of a
localized region of strand separation called the
replication bubble.
❖ This replication bubble is formed by the
interaction of prepriming proteins with oriC
(Figure).
❖ The first step in prepriming appears to be the
binding of four molecules of the dnaA gene
product—DnaA protein—to the four 9-base-pair
(bp) repeats in oriC.
❖ Next, DnaA proteins bind cooperatively to form
a core of 20 to 40 polypeptides with oriC DNA
wound on the surface of the protein complex.
❖ Strand separation begins within the three
tandem 13-bp repeats in oriC and spreads until
the replication bubble is created.
❖ A complex of DnaB protein (the hexameric DNA helicase) and DnaC protein
(six molecules) joins the initiation complex and contributes to the formation
of two bidirectional replication forks.
❖ The DnaT protein also is present in the prepriming protein complex, but its
function is unknown.
❖ Other proteins associated with the initiation complex at oriC are DnaJ
protein, DnaK protein, PriA protein, PriB protein, PriC protein, DNA-binding
protein HU, DNA gyrase, and single-strand DNA binding (SSB) protein.
❖ In some cases, however, their functional involvement in the prepriming
process has not been established; in other cases, they are known to be
involved, but their roles are unknown.
❖ Thus an initiator protein (known as DnaA in E. coli) binds to oriC and causes
a short section of DNA to unwind.
❖ This unwinding allows helicase and other single-strand-binding proteins to
attach to the polynucleotide strand.
UNWINDING DNA WITH HELICASES, DNA-
BINDING PROTEINS, AND
TOPOISOMERASES
❖ Unwinding:
✓ Because DNA synthesis requires a
single-stranded template and because
double-stranded DNA must be
unwound before DNA synthesis can
take place, the cell relies on several
proteins and enzymes to accomplish
the unwinding.
❖ DNA helicase:
✓ The major replicative DNA helicase in
E. coli is the product of the dnaB gene.
DNA helicases unwind DNA molecules
using energy derived from ATP.
✓ Initiator proteins and DNA helicase binds to the DNA at the replication fork and
untwist the DNA using energy derived from ATP (adenosine triphosphate).
(Hydrolysis of ATP causes a shape change in DNA helicase)
✓ A DNA helicase breaks the hydrogen bonds that exist between the bases of the two
nucleotide strands of a DNA molecule.
✓ Helicase cannot initiate the unwinding of double-stranded DNA; the initiator protein
first separates DNA strands at the origin, providing a short stretch of single-
stranded DNA to which a helicase binds.
✓ Helicase binds to the lagging-strand template at each replication fork and moves in
the 5→3′ direction along this strand, thus also moving the replication fork.
❖ Single-strand-binding proteins:
✓ After DNA has been unwound by helicase, single-strand-binding proteins (SSBs)
attach tightly to the exposed single-stranded DNA.
✓ These proteins protect the single stranded nucleotide chains and prevent the
formation of secondary structures such as hairpins that interfere with replication.
✓ Unlike many DNA-binding proteins, SSBs are indifferent to base sequence: they
will bind to any single-stranded DNA.
✓ Single- strand-binding proteins form tetramers (groups of four); each tetramer
covers from 35 to 65 nucleotides.
The formation of
functional template DNA
requires
(a)DNA helicase, which
unwinds the parental
double helix, and
(b) single-strand DNA-
binding (SSB) protein,
which keeps the
unwound DNA strands
in an extended form.
In the absence of SSB
protein, DNA single
strands can form
hairpin structures by
intrastrand base-pairing
(b, top), and the hairpin
structures will retard or
arrest DNA synthesis.
 Origin of replication (e.g., the prokaryote example):

✓ Begins with double-helix denaturing into single-strands thus exposing the

bases.
✓ Exposes a replication bubble from which replication proceeds in both
directions.

~245 bp in E. coli
DNA gyrase:
✓ Another protein essential for the unwinding process is the enzyme DNA
gyrase, a topoisomerase. Topoisomerases control the supercoiling of DNA.
✓ In replication, DNA gyrase reduces the torsional strain (torque) that builds up
ahead of the replication fork as a result of unwinding.
✓ It reduces torque by making a double-stranded break in one segment of the
DNA helix, passing another segment of the helix through the break, and then
resealing the broken ends of the DNA.
✓ This action removes a twist in the DNA and reduces the supercoiling.
✓ A group of antibiotics called 4-quinolones kill bacteria by binding to DNA
gyrase and inhibiting its action.
✓ The inhibition of DNA gyrase results in the cessation of DNA synthesis and
bacterial growth.
✓ Many bacteria have acquired resistance to quinolones through mutations in
the gene for DNA gyrase.
DNA helicase unwinds DNA by binding to the lagging strand template at each replication
fork and moving in the 5′→3′ direction.
 Elongation
❖ During the elongation phase of replication, single-stranded DNA is used as a
template for the synthesis of DNA. This process requires a series of enzymes.
❖ Synthesis of primers:
✓ All DNA polymerases require a nucleotide with a 3′-OH group to which a new
nucleotide can be added.
✓ Because of this requirement, DNA polymerases cannot initiate DNA synthesis
on a bare template; rather, they require a primer - an existing 3′-OH group to get
started. How, then, does DNA synthesis begin?
✓ An enzyme called primase synthesizes short stretches of nucleotides, or
primers, to get DNA replication started.
✓ New DNA chain is initiated by a short RNA primer synthesized by DNA primase.
The E. coli DNA primase is the product of the dnaG gene. In prokaryotes, these
RNA primers are 10 to 60 nucleotides long.
✓ Primase synthesizes a short stretch of RNA nucleotides (about 10–12
nucleotides long), which provides a 3-OH group to which DNA polymerase can
attach DNA nucleotides.
✓ (Because primase is an RNA polymerase, it does not require a 3-OH group to
which nucleotides can be added.)
✓ All DNA molecules initially have short RNA primers embedded within them;
these primers are later removed and replaced by DNA nucleotides.
✓ On the leading strand, where DNA synthesis is continuous, a primer is required
only at the 5’ end of the newly synthesized strand.
✓ On the lagging strand, where replication is discontinuous, a new primer must
be generated at the beginning of each Okazaki fragment.
✓ Primase forms a complex with helicase at the
replication fork and moves along the
template of the lagging strand.
✓ The single primer on the leading strand is
probably synthesized by the primase –
helicase complex on the template of the
lagging strand of the other replication fork, at
the opposite end of the replication bubble.
Supercoiled DNA relaxed by gyrase & unwound by helicase + proteins:

5’ SSB Proteins
Okazaki Fragments
1 ATP

Polymerase III 2
Helicase
Lagging strand 3 +
Initiator Proteins

3’
primase base pairs

Polymerase III 5’

RNA primer replaced by polymerase I

& gap is sealed by ligase

Leading strand
RNA Primer

3’
❖ DNA synthesis by DNA polymerases:
❖ After DNA is unwound and a primer has been added, DNA polymerases
elongate the new polynucleotide strand by catalysing DNA polymerization.
❖ The best-studied polymerases are those of E. coli, which has at least five
different DNA polymerases.
❖ Two of them, DNA polymerase I and DNA polymerase III, carry out DNA
synthesis in replication; the other three have specialized functions in DNA
repair.
❖DNA polymerase III :
✓ It is a large multiprotein complex that acts as the main workhorse of replication.
DNA polymerase III synthesizes nucleotide strands by adding new nucleotides to
the 3’ end of a growing DNA molecule.
✓ This enzyme has two enzymatic activities.
▪ Its 5’→3’polymerase activity allows it to add new nucleotides in the 5’→3’
direction.
▪ Its 3′→5′ exonuclease activity allows it to remove nucleotides in the 3′→5′
direction, enabling it to correct errors.
✓ If a nucleotide having an incorrect base is inserted into the growing DNA
molecule, DNA polymerase III uses its 3′→5’ exonuclease activity to back up and
remove the incorrect nucleotide. It then resumes its 5′→3′ polymerase activity.
✓ These two functions together allow DNA polymerase III to efficiently and
accurately synthesize new DNA molecules.
✓ DNA polymerase III has high processivity, which means that it is capable of
adding many nucleotides to the growing DNA strand without releasing the
template: it normally holds on to the template and continues synthesizing DNA
until the template has been completely replicated.
✓ In case of DNA polymerase, the degree of processivity is defined as the
average no. of nucleotide added each time the enzyme binds a primer :
template junction. They are capable of adding as many as 1000 nucleotides per
second to the primer strand.
✓ The high processivity of DNA polymerase III is ensured by one of the
polypeptides that constitutes the enzyme.
✓ This polypeptide, is termed as β subunit, serves as a clamp for the polymerase
enzyme: it encircles the DNA and keeps the DNA polymerase attached to the
template strand during replication.
✓ DNA polymerase III adds DNA nucleotides to the primer, synthesizing the DNA
of both the leading and the lagging strands.

❖ DNA Polymerase I:

✓ The first E. coli polymerase to be discovered, DNA polymerase I, also has 5′→3′
polymerase and 3′→5’ exonuclease activities, permitting the enzyme to
synthesize DNA and to correct errors.
✓ Unlike DNA polymerase III, however, DNA polymerase I also possesses 5′→3′
exonuclease activity, which is used to remove the primers laid down by
primase and to replace them with DNA nucleotides by synthesizing in a 5′→3′
direction.
✓ DNA polymerase I has lower processivity than DNA polymerase III. The removal
and replacement of primers appear to constitute the main function of DNA
polymerase I.
✓ After DNA polymerase III has initiated synthesis at the primer and moved
downstream, DNA polymerase I removes the RNA nucleotides of the primer,
replacing them with DNA nucleotides. DNA polymerases II, IV, and V function in
DNA repair.
❖ Despite their differences, all of E. coli’s DNA polymerases
1. synthesize any sequence specified by the template strand;
2. synthesize in the 5′→3′ direction by adding nucleotides to a 3′-OH group;
3. use dNTPs to synthesize new DNA;
4. require a primer to initiate synthesis;
5. catalyze the formation of a phosphodiester bond by joining the 5′-phosphate
group of the incoming nucleotide to the 3’-OH group of the preceding
nucleotide on the growing strand, cleaving off two phosphates in the
process;
6. produce newly synthesized strands that are complementary and antiparallel
to the template strands; and
7. are associated with a number of other proteins.
 Polymerase & Proteins Coordinated

• One polymerase complex apparently synthesizes leading/lagging strands simultaneously.

❖ The initiation of Okazaki fragments on the lagging
strand is carried out by the primosome, a protein
complex containing DNA primase and DNA
helicase.
❖ The primosome moves along a DNA molecule,
powered by the energy of ATP.
❖ As it proceeds, DNA helicase unwinds the parental
double helix, and DNA primase synthesizes the
RNA primers needed to initiate successive Okazaki
fragments.
❖ The RNA primers are covalently extended with the
addition of deoxyribonucleotides by DNA
polymerase III.
❖ DNA topoisomerases provide transient breaks in
the DNA that serve as swivels for DNA unwinding
and keep the DNA untangled.
❖ Single-strand DNA binding protein coats the
unwound pre replicative DNA and keeps it in an
extended state for DNA polymerase III.
❖ The RNA primers are replaced with DNA by DNA
polymerase I, and the single-strand nicks left by
polymerase I are sealed by DNA ligase.
 Mistakes during Replication

• Base pairing rules must be maintained

• Mistake = genome mutation, may have consequence on daughter
cells
• Only correct pairings fit in the polymerase active site
• If wrong nucleotide is included
• Polymerase uses its proofreading ability to cleave the
phosphodiester bond of improper nucleotide
• Activity 3’ → 5’
• And then adds correct nucleotide and proceeds down the chain
again in the 5’ → 3’ direction
Proofreading
❖ DNA ligase:
✓ After DNA polymerase III attaches a DNA nucleotide to the 3’-OH group on the
last nucleotide of the RNA primer, each new DNA nucleotide then provides the
3’-OH group needed for the next DNA nucleotide to be added.
✓ This process continues as long as template is available.
✓ DNA polymerase I follows DNA polymerase III and, using its 5’→3′
exonuclease activity, removes the RNA primer.
✓ It then uses its 5′→3′ polymerase activity to replace the RNA nucleotides with
DNA nucleotides.
✓ DNA polymerase I attaches the first nucleotide to the OH group at the 3′ end
of the preceding Okazaki fragment and then continues, in the 5′→3′ direction
along the nucleotide strand, removing and replacing, one at a time, the RNA
nucleotides of the primer.
✓ After polymerase I has replaced the last nucleotide of the RNA primer with a
DNA nucleotide, a nick remains in the sugar–phosphate backbone of the new
DNA strand.
✓ The 3’-OH group of the last nucleotide to have been added by DNA
polymerase I is not attached to the 5′-phosphate group of the first nucleotide
added by DNA polymerase III.
✓ This nick is sealed by the enzyme DNA ligase, which catalyzes the formation
of a phosphodiester bond without adding another nucleotide to the strand.
DNA ligase seals the gaps between
Okazaki fragments with a
phosphodiester bond
Elongation at the replication fork:
✓ Now that the major enzymatic components of elongation—DNA polymerases,
helicase, primase, and ligase—have been introduced, let’s consider how these
components interact at the replication fork.
✓ Because the synthesis of both strands takes place simultaneously, two units
of DNA polymerase III must be present at the replication fork, one for each
strand.
✓ In one model of the replication process, the two units of DNA polymerase III
are connected; the lagging-strand template loops around so that it is in
position for 5′→3′ replication. In this way, the DNA polymerase III complex is
able to carry out 5′→3′ replication simultaneously on both templates, even
though they run in opposite directions.
✓ After about 1000 bp of new DNA has been synthesized, DNA polymerase III
releases the lagging strand template, and a new loop forms. Primase
synthesizes a new primer on the lagging strand and DNA polymerase III then
synthesizes a new Okazaki fragment.
❖ As a replication fork moves along a parental double helix, two
DNA strands (the leading strand and the lagging strand) are
replicated in the highly coordinated series of reactions described
above.

❖ The complete replication apparatus moving along the DNA

molecule at a replication fork is called the replisome.

❖ The replisome contains the DNA polymerase III holoenzyme; one

catalytic core replicates the leading strand, the second catalytic
core replicates the lagging strand, and the primosome unwinds
the parental DNA molecule and synthesizes the RNA primers
needed for the discontinuous synthesis of the lagging strand.
The Replisome.
▪ This model shows how some of the key
replication proteins in bacteria illustrated in
Figure may actually be organized at the
replication fork.
▪ The DNA polymerase holoenzyme contains
three core polymerase subunits
interconnected by t subunits.
▪ The lagging strand DNA is folded into a
loop resembling the slide of a trombone,
thereby allowing the DNA polymerase
subunits on the leading and lagging
strands to remain connected even though
the two template strands are oriented with
opposite polarity in the parental DNA
molecule.
▪ The sliding clamp and one of the catalytic
subunits remain on the leading strand as
replication continues.
▪ On the lagging strand, the sliding clamp
and catalytic subunit detach as each
Okazaki fragment is completed.
▪ The clamp loader loads a sliding clamp and
catalytic subunit onto each new Okazaki
fragment as it forms after RNA priming. A
similar structure is found in eukaryotes.
 Replication Is Terminated When the Replication
Forks Meet at the Termination Sequences
❖ On the opposite side of the E. coli chromosome from oriC is a pair of
termination sequences called ter sequences. A protein known as the
termination utilization substance (Tus) binds to the ter sequences and stops
the movement of the replication forks.
❖ As shown in Figure, one of the ter sequences designated T1 prevents the
advancement of the fork moving left to right, but allows the movement of the
other fork.
❖ Alternatively, T2 prevents the advancement of the fork moving right to left,
but allows the advancement of the other fork.
❖ In any given cell, only one ter sequence is required to stop the advancement
of one replication fork, and then the other fork ends its synthesis of DNA
when it reaches the halted replication fork.
❖ In other words, DNA replication ends when oppositely advancing forks meet,
usually at T1 or T2. Finally, DNA ligase covalently links the two daughter
strands, creating two circular, double-stranded molecules.
The termination of DNA replication.
Two sites in the bacterial chromosome, shown as gray boxes, are
ter sequences designated T1 and T2. The T1 site prevents the
further advancement of the fork moving left to right, and T2
prevents the advancement of the fork moving right to left. As
shown in the inset, the binding of Tus prevents the replication
forks from proceeding past the ter sequences in a particular
direction.
❖ After DNA replication is completed, one last
problem may exist. DNA replication often
results in two intertwined DNA molecules
known as catenanes.
❖ Fortunately, catenanes are only transient
structures in DNA replication. In E. coli,
topoisomerase II introduces a temporary break
into the DNA strands and then rejoins them
after the strands have become unlocked.
❖ This allows the catenanes to be separated into
individual circular molecules.

Separation of catenanes: DNA replication can result in

two intertwined chromosomes called catenanes. These
catenanes can be separated by the action of
topoisomerase II.
 The Fidelity of DNA Replication
❖ Overall, the error rate in replication is less than one mistake per billion nucleotides.
How is this incredible accuracy achieved?
❖ DNA polymerases are very particular in pairing nucleotides with their complements
on the template strand.
❖ Errors in nucleotide selection by DNA polymerase arise only about once per
100,000 nucleotides.
❖ Most of the errors that do arise in nucleotide selection are corrected in a second
process called proofreading.
❖ When a DNA polymerase inserts an incorrect nucleotide into the growing strand,
the 3′-OH group of the mispaired nucleotide is not correctly positioned in the active
site of the DNA polymerase for accepting the next nucleotide.
❖ The incorrect positioning stalls the polymerization reaction, and the 3′→5′
exonuclease activity of DNA polymerase removes the incorrectly paired nucleotide.
❖ DNA polymerase then inserts the correct nucleotide.
❖ Together, proofreading and nucleotide selection result in an error rate of only one
in 10 million nucleotides.
Model of DNA replication
 Important Points to Remember
1. Replication is always semiconservative.
2. Replication begins at sequences called origins.
3. DNA synthesis is initiated by short segments of RNA called primers.
4. The elongation of DNA strands is always in the 5′→3′ direction.
5. New DNA is synthesized from dNTPs; in the polymerization of DNA, two
phosphate groups are cleaved from a dNTP and the resulting nucleotide is
added to the 3′-OH group of the growing nucleotide strand.
6. Replication is continuous on the leading strand and discontinuous on the
lagging strand.
7. New nucleotide strands are complementary and antiparallel to their template
strands.
8. Replication takes place at very high rates and is astonishingly accurate,
thanks to precise nucleotide selection, proofreading, and repair mechanisms.