Three models of DNA replication.

1. In conservative replication, the entire double-stranded DNA molecule serves as a template for a whole
new molecule of DNA, and the original DNA molecule is fully conserved during replication. With
conservative replication, after one round of replication, 50% of the molecules would consist entirely of
the original DNA and 50% would consist entirely of new DNA. After a second round of replication, 25%
of the molecules would consist entirely of the original DNA and 75% would consist entirely of new DNA.
With each additional round of replication, the proportion of molecules with new DNA would increase,
although the number of molecules with the original DNA would remain constant.
2. In dispersive replication it was believed that both nucleotide strands break down (disperse) into
fragments, which serve as templates for the synthesis of new DNA fragments, and then somehow
reassemble into two complete DNA molecules. In this model, each resulting DNA molecule is interspersed
with fragments of old and new DNA; none of the original molecule is conserved. Dispersive replication
would always produce hybrid molecules, containing some original and some new DNA, but the
proportion of new DNA within the molecules would increase with each replication event.
3. Semiconservative replication is intermediate between these two models; the two nucleotide strands
unwind and each serves as a template for a new DNA molecule. With semiconservative replication, one
round of replication would produce two hybrid molecules, each consisting of half original DNA and half
new DNA. After a second round of replication, half the molecules would be hybrid, and the other half
would consist of new DNA only. Additional rounds of replication would produce more and more
molecules consisting entirely of new DNA, and a few hybrid molecules would persist.

Meselson and Stahl’s Experiment

Done by Matthew Meselson and Franklin Stahl to determine which of the three models of replication
applied to E. coli cells. They designed an experiment using two isotopes of nitrogen, 14N (the common form)
and 15N (a rare, heavy form). The Steps of the experiment were as follows;
1. They grew a culture of E. coli in a medium that contained 15N as the sole nitrogen source for many
generations.
2. They took a sample of these bacteria and switched the rest of the bacteria to a medium that contained
only 14N.
3. Took additional samples of bacteria over the next few cellular generations, and examined their DNA, it
was assumed that the DNA that was synthesized before the change in medium contained 15N and
would be relatively heavy, whereas any DNA synthesized after the switch contained 14N and would be
relatively light.
4. The heavy (15N laden) DNA and the light (14N-containing) DNA were differentiated with the use of
equilibrium density gradient centrifugation (Centrifugation of a heavy salt solution and a substance of
unknown density, for long periods, to create a gradient of density within the tube, high density at the
bottom and low density at the top. The molecules of lower density (Unknown sample) rise and heavy
molecules sink.
5. Results
a. DNA from bacteria grown only on medium containing 15N produced a single band at the
position expected of DNA containing only 15N.
b. 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--> Disproved
Conservative model.
c. 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

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 containing only 14N. samples taken after additional rounds of replication produced the same
two bands, and the band representing light DNA became progressively stronger
6. Conclusion→ DNA replication occurs Semi-conservatively.

A) The Mechanism of Bacterial DNA Replication

Replicon:-
Is the portion of the genome that contains an origin of replication and that is replicated as a single
unit.
Direction of replication:-
DNA Synthesis Proceeds in a 5’ to 3’ Direction and Is Semi-discontinuous. A new strand of DNA is
always synthesizedin the 5’ to 3’ direction, with the free 3’ OH as the pointat which the DNA is
elongated.Because the twoDNA strands are antiparallel, the strand serving as thetemplate is read
from its 3’ end toward its 5’ end.
Continuous and discontinuous replication:-
During replication one strand is synthesized continuously and the other discontinuously.The
continuous strand, or leading strand, is the one in which 5’→3’ synthesis proceeds in the same
direction as replication fork movement. The discontinuous strand, or lagging strand, is the one in
which 5’→3’ synthesis proceeds in the direction oppositeto the direction of fork movement. Okazaki
fragments range in length from a few hundred to a few thousand nucleotides, depending on the cell
type.

Basic features of DNA Replication in Bcteriay

Each of the two progeny DNA moleculesconsists of one new strand and one old strand and hence
DNAreplication is said to be semiconservative.
• Template:- DNA ploymerases require an template
• Primer:- the polymerases require a primer,A primer is a strand segment (complementary to
the template) with a free 3’-hydroxyl group to which a nucleotide can be added; the free 3’
end of the primer is called the primer terminus.
• Replication rate: 750 to 1,000 base pairs per second
• Highly Specific/Accurate: Error probability →one in 10-9 or 10 -1o per base pair replicated,
due to Hydrogen bond complementarity, gross geometry ascertained by Pol I and through
3’→5’ proof reading of all DNA polymerase enzymes.
• Bacterial DNA Replication Initiates from a Single Origin of Replication
• Replication fork: the place at which the DNA helix is unwound and individual strands are
replicated
• When the replication forks move around the circular chromosomes observed in most
bacteria, a structure shaped like the Greek letter theta is formed.
• Because the bacterial chromosome is a single replicon, the forksmeet on the other side and
two separate chromosomes are released.

COMPONENTS REQUIRED FOR REPLICATION IN BACTERIAL CELL

Component Function

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Initiator protein (DnaA) Binds to origin and separates strands of DNA to initiate replication
DNA helicase (DnaB) Unwinds DNA at replication fork
Single-strand-binding Attach to single-stranded DNA and prevent re-annealing
Proteins
DNA gyrase Moves ahead of the replication fork, making and re-sealing breaks in the
double-helical DNA to release torque that builds up as a result
ofunwinding at the replication fork
DnaC Helicase loader, helps direct Helicase to DNA template
DNA primase Synthesizes short RNA primers to provide a 3’-OH group for attachment
of DNA nucleotides
DNA polymerase III Elongates a new nucleotide strand from the 3’-OH group provided by the
primer
DNA polymerase I Removes RNA primers and replaces them with DNA
DNA ligase Joins Okazaki fragments by sealing nicks in the sugar–phosphate
backbone of newly synthesized DNA
Tus Termination of replication
Topoisomerase IV Separation of chromosomes upon completion of replication

STEPS INVOLVED IN BACTERIAL DNA REPLICATION

Bacterial DNA replication takes place in three stages: initiation, elongation, andtermination.

1. Initiation
The circular chromosome of E. coli has a single replication origin (oriC). The minimal sequence
requiredfor oriC to function consists of 245 bp that contain several critical sites. At least nine different
enzymes or proteins participate in the initiation phase of replication. A single complex of four to five
DnaA protein molecules (Initiator proteins)bind to the four 9 bp repeats in the origin oriC then
recognizes and successively denatures the DNA in the region of the three 13 bp repeats, which are
rich in A=T pairs and cause a short section of DNA to unwind. This process requires ATP and the
bacterial histone like protein HU. The DnaC protein then loads the DnaB protein onto the unwound
region. Two ring shaped hexamers of DnaB, one loaded onto each DNAstrand, act as helicases,
unwinding the DNA bi-directionally and creating two potential replication forks. Many molecules of
SSB bind cooperatively to single stranded DNA, stabilizing the separated strands and preventing
renaturation while gyrase relieves the topological stress produced by the DnaB helicase

2. Elongation

Primers:
All DNA polymerases require a nucleotide with a 3’-OH group to which a new nucleotide can be
added. An enzyme complex called primosome involving primase (DnaG/RNA Polymerase) and its
accessory proteins, synthesizes short stretches of nucleotide(about 10–12 nucleotides long) primers
to get DNA replication started. 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 in 5’→3’ direction.The single primer on the leading strand is probably synthesized by the
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primase–helicase complex on the template of the lagging strand of the other replication fork, at the
opposite end of the replication bubble.

DNA polymerase III holoenzyme complex :

After DNA is unwound and a primer has been added, DNA polymerase III holoenzyme complex
elongates the polynucleotidestrand by catalyzing DNA polymerization. DNA polymerase III
holoenzyme is a multifunctional enzyme composed of 10 different proteins. Its polymerization and
proofreading activities resideinits α and ε (epsilon) subunits, respectively. The θ subunit associates
with α and ε to form a core polymerase, which can polymerize DNA but with limited processivity
(average number of nucleotides added before a polymerase dissociates).
Within the complex are two core enzymes (linked by T2γδδ’-clamp loading complex), each of which
binds to one strand of DNA and is responsible for catalyzing DNA synthesis and proofreading the
product to ensure fidelity of replication. Associated with each core enzyme is a subunit called the
betaclamp. The β clamp tethers a core enzyme to the DNA strand. The clamp loader at the center of
the holoenzyme is responsible for loading the β clamp onto DNA.

Because there are two core enzymes, both strands are produced by asingle asymmetric DNA
polymerase III dimer, which is accomplished by looping the DNA of the lagging strand bringing
together the two points of polymerization. One of the protein subunits of the DNApolymerase III core
enzyme (ε subunit) has 3‘ to 5' exonuclease activity. This activity enables the polymerase core enzyme
tocheck each newly incorporated base to see that it forms stable hydrogen bonds.
• When synthesis of an Okazaki fragment has been completed, replication halts, and the core
subunits of DNA polymerase III dissociate from their β sliding clamp (and from the
completed Okazaki fragment) and associate with the new clamp this initiates synthesis of
a new Okazaki fragment.
• After most of the lagging strand has been synthesized by the formation of Okazaki
fragments, DNA polymerase I removes the RNA primers through 5’-3’ exonuclease activity
and the adjacent 3'-0H from the deoxy-ribonucleotide is used to fill the gap between
Okazaki fragments.
• Finally, the Okazaki fragments are joined by the enzyme DNA ligase, which forms a
phosphodiester bond between the 3 '-OH of the growing strand and the 5 '-phosphate of an
Okazaki fragment.
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 associated with
replication; the other three have specialized functions in DNA repair.

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 Characteristics Of DNA Polymerases in E.coli

5’-3’ 3’-5’ 5’-3’

DNA Polymerase Polymerization Exonuclease Exonuclease Function
I Yes Yes Yes Removes and replaces primers
II Yes Yes No DNA repair; restarts replication after damaged DNA
Halts synthesis
III Yes Yes No Elongates DNA
IV Yes No No DNA repair
V Yes No No DNA repair; translation
DNA synthesis

3. Termination
In some DNA molecules, replication is terminated whenever two replication forks meet. In others,
specific termination sequences (Ter- containing multiple copies of a 20 bp sequence) block further
replication. A termination protein, called Tus(terminus utilization substance) in E. coli, binds to
these sequences. Tus blocks the movement of helicase, thus stalling the replication fork and
preventing further DNA replication.The final fewhundred base pairs of DNA between these large
proteincomplexes are then replicated (by an as yet unknown mechanism), completing two
topologically interlinked(catenated) circular chromosomes leading to problems such as: -
• Catenanes: -formation of interlocked chromosomes. Catenanes are produced when
topoisomerases break and rejoin DNA strands to ease supercoiling ahead of the
replication fork. Solved by the action of other topoisomerases topoisomerase IV (a
type II topoisomerase) that break both strands of one molecule, pass the other DNA
molecule through the break, and then rejoin the strands
• Dimerized chromosome:- two chromosomes joined together to form a single
chromosome twice as long. Solved by recombinase enzymes (e.g., XerCD in E. coli)
catalyze an intra-molecular cross-over that separates the two chromosomes

B) DNA Replication in Eukaryotic Cells

DNA molecules in eukaryotic cells are considerably larger and are organized into complex
nucleoprotein structures known as chromatin. The essential features of DNA replication are the same
in eukaryotes and prokaryotes, and many of the protein complexes are functionally and structurally
conserved. However some variations do exist, extensively studied in yeast, they are as follows;
• The rate of replication fork movement in eukaryotes is~50 nucleotides/s
• Origins of replication are called autonomously replicating sequences (ARS) or replicators,
usually span a length of 150bp and contain several essential conserved sequences.
• DNA replication in Eukaryotes is effected by multiple replicons (each with a separate
replicator, Yeast has about 400 replicators distributed among its 16 chromosomes).
• DNA replication is initiated by a multiple subunit protein, the origin recognition complex
(ORC), which binds to several sequences within the replicator.
• The Licensing of DNA Replication :
o In Eukaryotes minichromosome maintenance proteins (MCM2 to MCM7) must bind
to the DNA for replication to initiate at an origin. After replication has begun at an
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 origin, a protein called Geminin prevents MCM from binding to DNA and reinitiating
replication at that origin. At the end of mitosis, Geminin is degraded, allowing MCM to
bind once again to DNA and relicense the origin.
• A ring-shaped replicative helicase complex, analogous to the bacterial DnaB helicase.
• RPA (replication protein A) is a eukaryotic single-stranded DNA–binding protein
• RFC (replication factor C) is a clamp loader which facilitates the assembly of active
replication complexes. (They are structurally similar to bacterial clamp-loading (γ) complex.
• The Polymerase enzymes
o DNA polymerase α : a multisubunit enzyme with one subunit having a primase
activity and the largest subunit containing the polymerization activity, however, this
polymerase has no proofreading 3’→5’ exonuclease activity. DNA polymerase α is
believed to function only in the synthesis of short primers (containing either RNA or
DNA) for Okazaki fragments on the lagging strand.
o DNA Polymerase δ : comparable to the dimeric bacterial DNA polymerase III, it is the
major subunit that Assists DNA polymerase α in the replication of nuclear
chromosomes, it has a 3’→5’ proofreading exonuclease activity and is believed to carry
out both leading and lagging strand synthesis. It is associated with and stimulated by
a protein called proliferating cell nuclear antigen (PCNA), structurally and functionally
similar to β subunit of E. coli DNA polymerase III, which forms a circular clamp that
greatly enhances the processivity of the polymerase.
o DNA polymerase ε: Believed to play a similar role to DNA polymerase I, such as
removing the primers of Okazaki fragments on the lagging strand or DNA repair.
Recent research shows that DNA polymerase ε replicates the leading strand.
• Replication of Telomeres: The linear chromosomes of eukaryotes present additional
challenges in DNA replication, and are not readily replicated by cellular DNA polymerases
o Telomeres, generally consist of many tandem copies of a short oligonucleotide
sequences, TxGy in one strand and CyAx in the complementary strand (Values of x and
y between 1 to 4). And the TG strand being longer than its complement, leaving a
region of single-stranded DNA of up to a fewhundred nucleotides at the 3’ end.
o Without a special mechanism for replicating the ends, chromosomes would be
shortened in each cell generation. The enzyme telomerase solves this problem by
adding telomeres to chromosome ends.