Unit 5: DNA structure and replication

Discovery of DNA; Watson and Crick model of

DNA- structure; semiconservative replication
(Meselson & Stahl experiment); DNA replication
mechanism in E. coli (semi-discontinuous mode
and Y-fork).
 DNA, the Genetic Material
DNA, deoxyribonucleic acid, is the genetic material in our cells. It was
passed on to us from our parents and determines our characteristics.

The discovery that DNA is the genetic material was another

important milestone in molecular biology.
 DNA: Miescher to Watson and Crick – historic
perspective
• 1866- Gregor Mendel discovers the basic principles of genetics.
1869-Friedrich Miescher identifies “nuclein”. He was a Swiss Physiologist
Chemist.
• The biochemical investigation of DNA began with Friedrich Miescher.
• He carried out the first systematic chemical studies of cell nuclei.
• In 1868 Miescher isolated a phosphorus-containing substance, which he called
“nuclein,” from the nuclei of pus cells (leukocytes) obtained from discarded
surgical bandages.
• He found nuclein to consist of an acidic portion, which we know today as DNA,
and a basic portion, protein. Miescher later found a similar acidic substance in
the heads of sperm cells from salmon.
• Although he partially purified nuclein
and studied its properties, the covalent
(primary) structure of DNA was not
known with certainty until the late
1940s.
• 1928- British bacteriologist Frederick
Griffith conducted a series of
experiments using Streptococcus
pneumoniae bacteria and mice.

• Griffith wasn't trying to identify the

genetic material, but rather, trying to
develop a vaccine against pneumonia.

• In his experiments, Griffith used two

related strains of bacteria, known as R
and S.
• He also discovers one of the important
process in bacteria i.e. Transformation.
• 1944-Oswald Avery, an
immunochemist at the hospital of
the Rockefeller Institute for Medical
Research identifies DNA as the
‘Transforming Principle’.
(Transformation is the process seen in
bacteria. In this, bacteria directly
uptake and incorporate foreign
genetic material from its surroundings
through the cell membrane.)
• It was first reported in
Streptococcus pneumoniae by
Griffith in 1928.
• 1950-Erwin Chargaff discovers that composition of DNA varies between
species.
(In any ds. DNA number of G units is equal to the no. of cysteine units and
the no. of A units is equal to the number of thymine units. This discovery is
known as Chargaff’s rule.)

Some quotes from Erwin Chargaff

• Science is wonderfully equipped to answer the

question 'How?' but it gets terribly confused
when you ask the question 'Why?‘

• Molecular biology is essentially the practice of

biochemistry without a license.
• 1952-Rosalind Franklin from
photographs generated using X-
ray diffraction technique
crystallised DNA fibers.
• 1953-James Watson and
Francis Crick discovers
the double helix
structure of DNA.
 SUMMARY

DNA: Miescher to Watson and Crick – historic perspective

• 1866- Gregor Mendel discovers the basic principles of genetics.

• 1869-Friedrich Miescher identifies “nuclein”. He was a Swiss
Physiologist Chemist.
• 1928- British bacteriologist Frederick Griffith conducted a series of
experiments using Streptococcus pneumoniae bacteria and mice.
• Griffith wasn't trying to identify the genetic material, but rather, trying
to develop a vaccine against pneumonia.

• In his experiments, Griffith used two related strains of bacteria, known

as R and S.
• He also discovers one of the important process in bacteria i.e.
Transformation.
• 1950-Erwin Chargaff discovers that composition of DNA varies between
species.
(In any ds. DNA number of G units is equal to the no. of cysteine units and
the no. of A units is equal to the number of thymine units. This discovery is
known as Chargaff’s rule.)
• 1952-Rosalind Franklin from photographs generated using X-ray
diffraction technique crystallised DNA fibers.
• 1953-James Watson and Francis Crick discovers the double double helix
structure of DNA.
 STRUCTURE OF DNA
DNA is composed of nucleotides.

A nucleotide is made up of:

a sugar (deoxyribose),
a phosphate group, and
one of four nitrogenous bases: adenine (A), thymine (T), guanine (G) or cytosine (C)

C and T bases, have just one ring, are called pyrimidines, while A and G bases,
which have two rings, are called purines.

DNA nucleotides assemble in chains linked by covalent bonds, which form between
the deoxyribose sugar of one nucleotide and the phosphate group of the next.

This arrangement makes an alternating chain of deoxyribose sugar and phosphate

groups in the DNA polymer, a structure known as the sugar-phosphate backbone
• Purines are always bonded to a pyrimidine (A-T, C-G)
• Reason: size of base and number of hydrogen bonds.
• A=T 2 bonds C≡G 3 bonds
 Ribose & Deoxyribose

• Ribose and deoxyribose are 5-carbon sugars called as pentose sugar found
in RNA and DNA, respectively.
• A combination of a base and a sugar is called a nucleoside

• Deoxyribose, found in DNA, is a modified sugar, lacking one oxygen atom

(hence the name "deoxy").
This difference of one oxygen atom is important for the enzymes that
recognize DNA and RNA, because it allows these two molecules to be easily
distinguished inside organisms.
• Ribose is also called ribofuranose because of the structural relationship
to furane.

The only structural difference between ribose and deoxyribose is the loss
of an -OH group in position 2 in the ring. This difference affects secondary
structure and stability.
Nomenclature of bases, nucleosides, and nucleotides
❑ Altered or unusual bases are also found in DNA.

❑ They have roles in regulating or protecting the genetic

information

Such as methylated forms of DNA bases—

• 7-methylguanine,
• 5-methylcytosine,
• 5-hydroxymethylcytosine
Formation of nucleotide by removal of water
 N-ß-glycosidic bond
formed by removal of
water
Phosphate
group is
easterified to
the 5’ carbon

The base of a nucleotide is joined

covalently at N1 of pyrimidines and
N9 of purines in an N-ß-glycosyl
bond to the C 1 carbon of the
pentose
The phosphate group on one nucleotide links to the 3' carbon
atom on the sugar of another one. In the process, a molecule of
water is lost - another condensation reaction.
 What is a Phosphodiester bond?

• A phospodiester bond is a covalent bond in which a phosphate group joins

adjacent carbons through ester linkages.
• The bond is the result of a condensation reaction between a hydroxyl group of
two sugar groups and a phosphate group.
• The diester bond between phosphoric acid and two sugar molecules in the
DNA and RNA backbone links two nucelotides together to form oligonucleotide
polymers.
• The phosphodiester bond links a 3' carbon to a 5' carbon in DNA and RNA.
• During the reaction of two of the hydroxyl groups in phosphoric
acid with a hydroxyl group in two other molecules two ester
bonds in a phosphodiester group are formed.

• A condensation reaction in which a water molecule is lost

generates each ester bond.

• During polymerization of nucleotides to form nucleic acids, the

hydroxyl group on the phosphate group attaches to the 3’ carbon
of a sugar of one nucleotide to form an ester bond to the
phosphate of another nucleotide.

• The reaction forms a phosphodiester linkage eliminates a water

molecule.
▪ The links between the nucleotides are called phosphodiester bonds.

▪ The chemical orientation of the DNA is such that, the 3’-end has a free
hydroxyl group at the 3’-carbon of a sugar, and the 5’end has a free
phosphate group at the 5’-carbon of a sugar.

▪ Since the synthesis proceeds from the 5’ to the 3’-end, according to

convention sequences are written from in the 5’ -> 3' direction.
❑ DNA polymerases catalyze the formation of polynucleotide chains
through the addition of new nucleotides from incoming
deoxynucleoside triphosphates.

❑ The polymerase reaction needs an appropriate DNA template to take

place. Each incoming nucleoside triphosphate first forms a base pair
with a base in the template.

❑ Next, the DNA polymerase links the incoming base with the
predecessor in the chain. Therefore, DNA polymerases are template-
directed enzymes.
The two strands of the double helix are held together by base
pairing in an Antiparallel orientation.

• Double helix is held together by weak, noncovalent bonds between base

pairs. A on one chain always pairs with T on another chain and likewise
for C and G.

• The two strands have the same helical geometry but base pairing holds
them together with the opposite polarity.

i.e. the base at the 5’ end of one strand is paired with the base at the 3 ‘
end of the other strand.
Thus, DNA is composed of two strands that run alongside each other but
point in opposite directions or in antiparallel orientation.

In a double-stranded DNA molecule, the 5' end (phosphate-bearing end) of

one strand aligns with the 3' end (hydroxyl-bearing end) of its partner, and
vice versa.
This antiparallel orientation is a stereochemical
consequence of the way that A and T, and G and
C, pair with each other.
• The planes of the bases are
perpendicular to the helix axis.

• The planes of the sugars are

almost at right angles to those
of the bases.

• The diameter of helix is 20 Å

(2nm).

• The bases are 3.4 Å apart

along the helix axis and are
related by a rotation of 36˚.

• Therefore, helical structure

repeats after 10 residues on
each chain i.e. at intervals of
34 Å.
• Two chains are held together by H2 bonds between base pairs A=T
and CΞG
• The specific positioning of the bases is called base
complementarity.

• Base pairing occurs according to the Chargaff’s rule.

• Thus, composition of A is always equals to the composition of T

and composition of C is always equals to the composition of G

• As larger Purine (A or G) is always paired with a small Pyramidine (T

or C), the diameter of the helix is uniform, about 2 nm.
 Summary

The complementary base-pairing enables the base pairs to be

packed in the energetically most favorable arrangement in the
interior of the double helix.

In this arrangement, each base pair is of similar width, thus

holding the sugar-phosphate backbones an equal distance apart
along the DNA molecule.

To maximize the efficiency of base-pair packing, the two sugar-

phosphate backbones wind around each other to form a double
helix, with one complete turn every ten base pairs.

The members of each base pair can fit together within the double
helix only if the two strands of the helix are antiparallel—that is,
only if the polarity of one strand is oriented opposite to that of the
other strand
Fill in the blanks

1. The base of a nucleotide is joined covalently at ______ of

pyrimidines and ________ of purines in an N-ß-glycosyl bond to
the _______carbon of the pentose

2. The phosphodiester bond links a _____ carbon to a _____

carbon in DNA and RNA.

3. The reaction forms a phosphodiester linkage _______ a water

molecule.

4. Two nucleotides are joined via ___________

5. DNA is composed of two strands that run alongside each other

but point in _______
 Right-handed helix
• The two strands of DNA twist around each other to form a right-
handed helix.

• All helices have a handedness, which is a property that describes

how their grooves are oriented in space.

• The twisting of the DNA double helix

and the geometry of the bases
creates a wider gap (called the major
groove) and a narrower gap (called
the minor groove) that run along the
length of the molecule.

• These grooves are important binding

sites for proteins that maintain DNA
and regulate gene activity.
 Why are there a minor groove and a major
groove?
It is a simple consequence of the geometry of the base
pair.
The angle at which the two sugars protrude from the base
pairs (i.e. the angle between the glycosidic bonds) is
about 120˚ for the narrow angle or 240 ˚ for the wide
angle.
As a result, as more and more base pairs stack on top of
each other, the narrow angle between the sugars on one
edge of the base pairs generates a minor groove and the
large angle on the other edge generates a major groove.
Where do you see
glycosidic bonds ?
SUMMARY
 Types of DNA

• Most of the DNA is in the classic Watson-Crick form simply called

as B-DNA or B-form DNA.

• In certain condition ,different forms of DNAs are found to be

appeared like A-DNA, Z-DNA, C- DNA.

• This deviation in forms are based on their structural diversity.

 DIFFERENT FORMS OF DNA

• X-ray analysis of DNA crystals at atomic resolution have revealed that

DNA exhibits much more structural diversity than formally discovered.

Such variations are:

1. B-DNA: Most common ,originally deduced from X-ray diffraction of

sodium salt of DNA fibres at 92% relative humidity. Right-handed.

2. A-DNA: Originally identified by X-ray diffraction of analysis of DNA fibres

at 75% relative humidity . Right-handed.

3. C-DNA: Formed at 66% relative humidity and in the presence of Li+ and
Mg2+ ions. Right-handed.

4. Z-DNA: Left handed double helical structure winds to the left in a zig-
zag pattern observed under very high salt concentration.
Semiconservative Replication and the Meselson-Stahl Experiment
DNA replication is essential for cell division and the transmission of
genetic information. It follows a semiconservative mechanism,
where each new DNA molecule consists of one parent strand and
one newly synthesized strand.

What is Semiconservative Replication?

In semiconservative replication:
•Each original DNA strand acts as a template for the synthesis of a
complementary new strand.
•After replication, each daughter DNA molecule has one parental
strand and one newly formed strand.
Meselson-Stahl Experiment (1958)
The Meselson-Stahl experiment is a landmark study that provided definitive
evidence for semiconservative replication.
Objective
To determine whether DNA replication follows a conservative, semiconservative,
or dispersive model.
Experimental Procedure
1.Bacterial Culture:
1. Escherichia coli (E. coli) was grown in a medium containing heavy nitrogen
isotope (¹⁵N) for several generations.
2. This caused the DNA of the bacteria to be labeled with ¹⁵N, making it
denser.
2.Switch to Light Nitrogen:
1. The bacteria were then transferred to a medium containing the lighter
nitrogen isotope (¹⁴N) and allowed to replicate.
3.DNA Extraction:
1. DNA was extracted from bacterial cells at different time intervals after
replication in the ¹⁴N medium.
4.Density Gradient Centrifugation:
1. The extracted DNA was subjected to cesium chloride (CsCl) density gradient
centrifugation, which separated DNA based on its density.
Observations
1.Generation 0 (Before Shift to ¹⁴N Medium)
1. DNA showed a single band at the heavy (¹⁵N) position.
2.Generation 1 (After One Replication Cycle)
1. DNA formed a single band intermediate between ¹⁵N and ¹⁴N, indicating a hybrid
density.
2. This ruled out the conservative model, which predicted separate heavy and light
DNA bands.
3.Generation 2 (After Two Replication Cycles)
1. Two distinct bands were observed:
1. One at the hybrid (¹⁵N-¹⁴N) position.
2. One at the light (¹⁴N) position.
2. This confirmed semiconservative replication and ruled out the dispersive model,
which would predict only hybrid DNA.
Conclusion
The Meselson-Stahl experiment provided strong evidence that DNA replication is
semiconservative. Each daughter DNA molecule consists of one old (parental) strand and
one new (daughter) strand.
Significance of the Experiment
•Proved the fundamental nature of DNA replication.
•Supported Watson and Crick’s double-helix model of DNA.
•Laid the groundwork for advances in molecular biology, including genetic engineering and
understanding hereditary diseases.
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.
• The second essential substrate for DNA synthesis is a particular
arrangement of single-stranded DNA (ssDNA) and double-stranded
DNA (dsDNA) called a primer:template junction.
• 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.
• Only the primer is chemically modified during DNA synthesis.
• The template provides only the information necessary to select which
nucleotides are added.
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 Diagram of the
mechanism of DNA
synthesis.
DNA synthesis is initiated
when the 3’ -OH of the
primer mediates the
nucleophilic attack of the
alpha-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.
THE MECHANISM OF DNA POLYMERASE

DNA Polymerases Use a Single Active Site to Catalyze DNA

Synthesis
DNA polymerases are the enzymes which catalyzes DNA synthesis.

DNA Pol has two important properties:

1. kinetic proofreading property
2. steric exclusion of rNTPs from the DNA polymerase active site
THE MECHANISM OF DNA POLYMERASE contd.

DNA polymerase uses a single active site to catalyze the addition of any of
the four deoxynucleoside triphosphates (dNTPs).
• DNA polymerase exhibited catalytic flexibility by exploiting the nearly
identical geometry of the A:T and G:C base pairs.
• The DNA polymerase monitors the ability of the incoming nucleotide
to form an A:T or G:C base pair, rather than detecting the exact
nucleotide that enters the active site
• Only when a correct base pair is formed then catalysis occur.
• Incorrect base pairing leads to dramatically lower rates of nucleotide
addition because unfavorable alignment of these substrates.
• DNA Pol exhibits kinetic proofreading property, in which an enzyme
favors catalysis using one of several possible substrates by dramatically
increasing the rate of bond formation only when the correct substrate
is present.
• DNA polymerases are able to distinguish between
ribonucleoside and deoxyribonucleoside triphosphates (rNTPs
and dNTPs).
• Although rNTPs are present at approximately 10-fold higher
concentration in the cell.
• 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 as in
rNTPs.
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
DNA Polymerases Are Processive Enzymes

• Catalysis by DNA polymerase is rapid.

• DNA polymerases are capable of adding as many as 1000
nucleotides/sec to a primer strand.
• The speed of DNA synthesis is largely due to the processive nature of
DNA polymerase.
• Processivity is a characteristic of enzymes that operate on polymeric
substrates.
• In the case of DNA polymerases, the degree of processivity is defined
as the average number of nucleotides added each time the enzyme
binds a primer:template junction.
• Each DNA polymerase has a characteristic processivity that can range
from only a few nucleotides to more than 50,000 bases added per
binding event.
• Processivity is facilitated by sliding of DNA polymerases along the DNA
template.
DNA polymerases synthesize DNA in
a processive manner.
This illustration shows the difference
between a processive and a
nonprocessive DNA polymerase.
Both DNA polymerases bind the
primer:template junction. Upon
binding, the nonprocessive enzyme
adds a single dNTP to the 3’ end of
the primer and then is released from
the new primer:template junction. In
contrast, a processive DNA
polymerase adds many dNTPs each
time it binds to the template.
A completely nonprocessive DNA polymerase would add 1
bp/sec. In contrast, the fastest DNA polymerases add as many as
1000 nucleotides/sec by remaining associated with the template
for thousands of rounds of dNTP addition.
Consequently, a highly processive polymerase increases the
overall rate of DNA synthesis by as much as 1000-fold compared
with a nonprocessive enzyme.
• The incorrect nucleotide is removed by the exonuclease (an additional
nucleotide may also be removed).
• The removal of the mismatched base allows the primer:template
junction to re-form and rebind the polymerase active site, enabling
DNA synthesis to continue.

In essence, proofreading exonucleases work like a “delete key,” removing

only the most recent errors.
• The addition of a proofreading exonuclease greatly increases the
accuracy of DNA synthesis.
• On average, DNA polymerase inserts one incorrect nucleotide for every
105 nucleotides added.
• Proofreading exonucleases decrease the appearance of incorrect base
pairs to 1 in every 107 nucleotides added.
• This additional level of accuracy is provided by the postreplication
mismatch repair process.
Replication fork. (Red) Newly synthesized DNA; (green) RNA primers. The
Okazaki fragments shown are artificially short for illustrative purposes. In
the cell, Okazaki fragments can vary between 100 and 2000 bases
depending on the organism
• The resulting short fragments of new DNA formed on the lagging
strand are called Okazaki fragments and vary in length from
1000 to 2000 nucleotides in bacteria and from 100 to 400
nucleotides in eukaryotes.
• Shortly after being synthesized, Okazaki fragments are
covalently joined together to generate a continuous, intact
strand of new.
• Okazaki fragments are therefore transient intermediates in DNA
replication.
The Initiation of a New Strand of DNA Requires an RNA Primer.
All DNA polymerases require a primer with a free 3’ -OH. They cannot
initiate a new DNA strand de novo.

How, then, are new strands of DNA synthesis started?

RNA polymerases do what DNA polymerases cannot: start new RNA

chains de novo.
• Primase is a specialized RNA polymerase dedicated to making short
RNA primers (5–10 nucleotides long) on an ssDNA template.
• These primers are subsequently extended by DNA polymerase.

• Although DNA polymerases incorporate only deoxyribonucleotides into

DNA, they can initiate synthesis using either an RNA primer or a DNA
primer annealed to the DNA template.
• Both the leading and lagging strands require primase to initiate DNA
synthesis.
• The frequency of primase function on the two strands is different.
• Each leading strand requires only a single RNA primer.
• In contrast, the discontinuous synthesis of the lagging strand needed
for each Okazaki fragment.

• Unlike the RNA polymerases involved in messenger RNA (mRNA),

ribosomal RNA (rRNA), and transfer RNA (tRNA) synthesis, primase
does not require an extended DNA sequence to initiate RNA synthesis.
• Instead, primases prefer to initiate RNA synthesis using an ssDNA
template containing a particular trimer (GTA in the case of Escherichia
coli primase).

• Primase activity is increased when it associates with another protein

that acts at the replication fork called DNA helicase. This protein
unwinds the DNA at the replication fork, creating an ssDNA template
that can be acted on by primase.
RNA Primers Must Be Removed to Complete DNA Replication
• To complete DNA replication, the RNA primers used for initiation must
be removed and replaced with DNA.
• To replace the RNA primers with DNA, an enzyme called RNase H
recognizes and removes most of each RNA primer.
• This enzyme specifically degrades RNA that is base-paired with DNA
(the H in its name stands for “hybrid” in RNA:DNA hybrid).
• RNase H removes all of the RNA primer except the ribonucleotide
directly linked to the DNA end.
• This is because RNase H can only cleave bonds between two
ribonucleotides.
• The final ribonucleotide is removed by a 5’ exonuclease that degrades
RNA or DNA from their 5’ ends.
• Removal of the RNA primer leaves a gap in the dsDNA that is an ideal
substrate for DNA polymerase—a primer:template junction.
• DNA polymerase fills this gap until every nucleotide is base-paired,
leaving a DNA molecule that is complete except for a break in the
phosphodiester backbone between the 3’ -OH and 5’ -phosphate of the
repaired strand.
This “nick” in the DNA can be FIGURE: Removal of RNA
repaired by an enzyme called DNA primers from newly
ligase. synthesized DNA.
DNA ligases use high-energy co- The sequential function
factors (such as ATP) to create a of RNase H, 5’
phosphodiester bond between an exonuclease, DNA
adjacent 5’ -phosphate and 3’ -OH. polymerase, and DNA
ligase during the removal
Only after all RNA primers are of RNA primers is illus-
replaced by DNA and the
trated.
associated nicks are sealed then
DNA synthesis complete.
(Gray) DNA present
before RNA primer
removal; (green) RNA
primer; (red) the newly
synthesized DNA that
replaces the RNA primer.
DNA Replication in E. coli (Semi-Discontinuous Mode)

DNA replication in E. coli occurs through a semi-discontinuous

mechanism. This is necessary because DNA is double-stranded, with
antiparallel orientation — one strand runs in the 5' to 3' direction, while
the complementary strand runs in the opposite 3' to 5' direction.

Key Features:
•Semi-conservative: Each new double-stranded DNA molecule contains
one parental (original) strand and one newly synthesized strand.
•Bidirectional replication: Replication occurs in two directions
simultaneously from the origin of replication (OriC) to create a replication
bubble.
•Semi-discontinuous: One strand is synthesized continuously, while the
other is synthesized in fragments.
DNA helicases
• DNA helicases, catalyze the separation of the two strands of duplex DNA.
• These enzymes bind to and move directionally along ssDNA using the
energy from ATP hydrolysis.
• DNA helicases are hexameric proteins that assume the shape of a ring.
• These ring-shaped protein complexes encircle one of the two single
strands at the replication fork adjacent to the single-stranded:double-
stranded junction.
• Like DNA polymerases, DNA helicases act processively, because they
encircle the DNA.
• Each time they associate with substrate, they unwind multiple base pairs
of DNA.
• Each DNA helicase moves along ssDNA in a defined direction.
• This property is referred to as the polarity of the DNA helicase.
• DNA helicases can have a polarity of either 5’-3’ or 3’ - 5’.
• This direction is always defined according to the strand of DNA bound (or
encircled for a ring-shaped helicase), rather than the strand that is displaced.
• In the case of a DNA helicase that functions on the lagging-strand template of the
replication fork, the polarity is 5’ -3’ to allow the DNA helicase to proceed toward
the duplex region of the replication fork.
DNA helicases separate the two
strands of the double helix.
When ATP is added to a DNA
helicase bound to ssDNA, the
helicase moves with a defined
polarity on the ssDNA.

In the instance illustrated, the DNA

helicase has a 5’ -3’ polarity. This
polarity means that the DNA
helicase would be bound to the
lagging-strand template at the
replication fork.
Single-Stranded DNA-Binding Proteins Stabilize ssDNA before Replication

• After the DNA helicase has passed, the newly generated ssDNA must
remain free of base pairing until it can be used as a template for DNA
synthesis.
• To stabilize the separated strands, ssDNA-binding proteins (SSBs) rapidly
bind to the separated strands.
• Binding of one SSB promotes the binding of another SSB to the
immediately adjacent ssDNA.
• This is called cooperative binding and occurs because SSB molecules
bound to immediately adjacent regions of ssDNA also bind to each other.
• Cooperative binding ensures that ssDNA is rapidly coated by SSB as it
emerges from the DNA helicase. (Cooperative binding is a property of
many DNA-binding proteins.
• Once coated with SSBs, ssDNA is held in an elongated state that
facilitates its use as a template for DNA or RNA primer synthesis.
• SSBs interact with ssDNA in a sequence-independent manner.
• SSBs primarily contact ssDNA through electrostatic interactions with the
phosphate backbone and stacking interactions with the DNA bases.
Binding of single-stranded binding protein (SSB) to DNA.
(a)A limiting amount of SSBs is bound to four of the nine ssDNA
molecules shown.
(b)As more SSBs bind to DNA, they preferentially bind adjacent to
previously bound SSB molecules. Only after SSBs have completely
coated the initially bound ssDNA molecules does binding occur on
other molecules. Note that when ssDNA is coated with SSBs, it
assumes a more extended conformation that inhibits the formation
of intramolecular base pairs.
Topoisomerases Remove Supercoils Produced by DNA Unwinding at the
Replication Fork

• As the strands of DNA are separated at the replication fork, the dsDNA
in front of the fork becomes increasingly positively supercoiled.
• The supercoils are removed by topoisomerases that act on the
unreplicated dsDNA in front of the replication fork.
• If the DNA strands remain unbroken, there can be no reduction in
linking number (the number of times the two DNA strands are
intertwined) to accommodate this unwinding of the DNA duplex.
• These enzymes do this by breaking either one or both strands of the
DNA without letting go of the DNA and passing the same number of
DNA strands through the break.
• This action relieves the accumulation of supercoils.
• In this way, topoisomerases act as a “swivelase” that prevents the
accumulation of positive supercoils ahead of the replication fork.
Fig. Action of topoisomerase at the replication fork.
As positive supercoils accumulate in front of the
replication fork, topoisomerases rapidly remove
them.
In this diagram, the action of Topo II removes the
positive supercoil induced by a replication fork.
By passing one part of the unreplicated dsDNA
through a double-stranded break in a nearby
unreplicated region, the positive supercoils can be
removed.
This change would reduce the linking number by
two and thus would only have to occur once every
20 bp replicated.
Although the action of a type II topoisomerase is
illustrated here, type I topoisomerases can also
remove the positive supercoils generated by a
replication fork.
Both DNA helicase and topoisomerase perform their functions
without permanently altering the chemical structure of DNA or
synthesizing any new molecule.
• DNA helicase breaks only the hydrogen bonds that hold the two
strands of DNA together without breaking any covalent bonds.
• Although topoisomerases break one or two of the targeted
DNA’s covalent bonds, each bond broken is precisely re-formed
before the topoisomerase releases the DNA.
• Instead of altering the chemical structure of DNA, the action of
these enzymes results in a DNA molecule with an altered
conformation.
DNA Polymerases Are Specialized for Different Roles in the Cell

Activities and Functions of DNA Polymerases

DNA Polymerases contd.
E. coli has at least five DNA polymerases that are distinguished by their
• enzymatic properties,
• subunit composition, and
• Abundance
DNA polymerase III (DNA Pol III) is the primary enzyme involved in the
replication of the chromosome.
• DNA Pol III is highly processive.
• DNA Pol III is part of a larger complex that confers very high
processivity—a complex known as the DNA Pol III holoenzyme.
• In contrast, DNA polymerase I (DNA Pol I) is specialized for the removal
of the RNA primers that are used to initiate DNA synthesis.
• This DNA polymerase has a 5’ exonuclease to remove RNA or DNA
immediately upstream of the site of DNA synthesis.
• Unlike DNA Pol III holoenzyme, DNA Pol I is not highly processive,
adding only 20–100 nucleotides per binding event.
• The 5’ exonuclease of DNA Pol I can remove the RNA–DNA linkage that
is resistant to RNase H.
• When DNA Pol I completes its function, only a nick is present in the
DNA.
• As both DNA Pol I and DNA Pol III are involved in DNA replication,
both of these enzymes must be highly accurate. Thus, both proteins
include an associated proofreading exonuclease.
• The remaining three DNA polymerases in E. coli are specialized for
DNA repair and lack proofreading activities.
DNA SYNTHESIS AT THE REPLICATION FORK (more
detailed)

• Both leading and lagging strands are synthesized

simultaneously at the replication fork.
• To coordinate this, multiple DNA polymerases function
at the replication fork.
• In E. coli, the coordinate action of these polymerases is
facilitated by physically linking them together in a
large multiprotein complex called the “DNA Pol III
holoenzyme”. Composition of the
• Holoenzyme is a common name for a multiprotein DNA Pol III holoenzyme
complex in which a core enzyme activity is associated
with additional components that enhance function.
• The DNA Pol III holoenzyme includes:
• Three copies of the “core” DNA Pol III enzyme and
• One copy of the sliding clamp loader. The sliding
clamp holder includes three copies of the τ protein,
each of which interacts with one DNA Pol III core.
• Replisome - The combination of all of the proteins that function at the
replication fork is referred to as the replisome.
INITIATION OF DNA REPLICATION

The specific sites at which DNA unwinding and initiation of replication

occur are called origins of replication.
• Depending on the organism, there may be one or as many as thousands
of origins per chromosome.

A replicon is defined as the molecule of DNA or a region of DNA that

replicates from a single origin of replication.

For example, a single chromosome is found in E. coli cells which has only
one origin of replication, so, the entire chromosome is a single replicon.

In contrast, the presence of multiple origins of replication divides each

eukaryotic chromosome into multiple replicons—one for each origin of
replication.
• Initiator protein is the only sequence-specific DNA binding protein involved in
the initiation of replication.
• The remaining proteins required for replication initiation do not bind to a DNA
sequence specifically.
• In E. coli initiator protein is DnaA

oriC - single replicator for E. coli chromosomal replication where E. coli initiator
DnaA binds.
Mechanism at the Y-Fork
The Y-shaped structure formed during DNA replication is called the
replication fork. It includes essential processes involving several enzymes.
1. Initiation
•DNA replication begins at a specific origin site, OriC, recognized by the
DnaA protein.
•Helicase (DnaB) unwinds the double helix by breaking hydrogen bonds
between bases, forming the replication fork.
•Single-strand binding proteins (SSBs) stabilize the unwound DNA strands.
2. Leading Strand Synthesis (Continuous Mode)
•The leading strand is synthesized in the 5' to 3' direction by DNA
polymerase III.
•Since DNA polymerase can only synthesize in this direction, it proceeds
continuously along the template strand.
3. Lagging Strand Synthesis (Discontinuous Mode)
•The lagging strand, being antiparallel, cannot be synthesized continuously.
•Instead, it is synthesized in short fragments called Okazaki fragments.
•RNA primers are laid down by primase, providing a 3' hydroxyl group for
DNA polymerase III to extend.
•DNA polymerase III synthesizes each Okazaki fragment in the 5' to 3'
direction.
4. Fragment Joining
•DNA polymerase I removes RNA primers and replaces them with DNA
nucleotides.
•DNA ligase seals the gaps between Okazaki fragments by forming
phosphodiester bonds, creating a continuous strand.
5. Termination
•Replication stops at specific termination sequences (ter sites) with the
help of the Tus protein, which blocks helicase activity.
Enzymes Involved in E. coli DNA Replication