DNA Replication
Meslelson-Stahl Experiment
Three hypotheses had been previously proposed for the method of replication of DNA.
In the semiconservative hypothesis, proposed by Watson and Crick, the two strands of a DNA
molecule separate during replication. Each strand then acts as a template for synthesis of a
new strand.
The conservative hypothesis proposed that the entire DNA molecule acted as a template for
the synthesis of an entirely
new one. According to this
model, histone proteins bind
to the DNA, revolving the
strand and exposing the
nucleotide
bases
(which
normally line the interior)
for hydrogen bonding.
The dispersive hypothesis is
exemplified by a model
proposed by Max Delbrück,
which attempts to solve the problem of unwinding the two strands of the double helix by a
mechanism that breaks the DNA backbone every 10 nucleotides or so, untwists the molecule,
and attaches the old strand to the end of the newly synthesized one. This would synthesize the
DNA in short pieces alternating from one strand to the other.
Each of these three models makes a different prediction about the distribution of the "old"
DNA in molecules formed after replication. In the conservative hypothesis, after replication,
one molecule is the entirely conserved "old" molecule, and the other is all newly synthesized
DNA. The semiconservative hypothesis predicts that each molecule after replication will
contain one old and one new strand. The dispersive model predicts that each strand of each
new molecule will contain a mixture of old and new DNA.
The Meselson-Stahl Experiment
The Meselson–Stahl experiment was an experiment by Matthew Meselson and Franklin
Stahl in 1958 which supported the hypothesis that DNA replication was semiconservative. In
semiconservative replication, when the double stranded DNA helix is replicated each of the
two new double-stranded DNA helixes consisted of one strand from the original helix and
one newly synthesized. It has been called "the most beautiful experiment in biology.
Nitrogen is a major constituent of DNA. 14N is by far the most abundant isotope of nitrogen,
but DNA with the heavier (but non-radioactive) 15N isotope is also functional.
E. coli were grown for several generations in a medium with 15N. When DNA is extracted
from these cells and centrifuged on a salt density gradient, the DNA separates out at the point
at which its density equals that of the salt solution. The DNA of the cells grown in 15N
medium had a higher density than cells grown in normal 14N medium. After that, E. coli cells
with only 15N in their DNA were transferred to a 14N medium and were allowed to divide; the
progress of cell division was monitored by microscopic cell counts and by colony assay.
DNA was extracted periodically and was compared to pure 14N DNA and 15N DNA. After
one replication, the DNA was found to have intermediate density. Since conservative
replication would result in equal amounts of DNA of the higher and lower densities (but no
DNA of an intermediate density), conservative replication was excluded. However, this result
was consistent with both semiconservative and dispersive replication. Semiconservative
replication would result in double-stranded DNA with one strand of 15N DNA, and one of 14N
DNA, while dispersive replication would result in double-stranded DNA with both strands
having mixtures of 15N and 14N DNA, either of which would have appeared as DNA of an
intermediate density.
The authors continued to sample cells as replication continued. DNA from cells after two
replications had been completed was found to consist of equal amounts of DNA with two
different densities, one corresponding to the intermediate density of DNA of cells grown for
only one division in 14N medium, the other corresponding to DNA from cells grown
exclusively in 14N medium. This was inconsistent with dispersive replication, which would
have resulted in a single density, lower than the intermediate density of the one-generation
cells, but still higher than cells grown only in 14N DNA medium, as the original 15N DNA
would have been split evenly among all DNA strands. The result was consistent with the
semiconservative replication hypothesis.

Generation 0 is grown in the heavy (15N) Nitrogen. This is represented by the blue strands.

It is then moved to a mixture of light (14N) Nitrogen – red strand. Now when the DNA
replicates the new strand will be contain the 14N. This means that the old strand contains
15N and the new strand contains 14N.

The next two generations are both kept in light (14N) nitrogen and so as more DNA
replicates, the new strands are all made of 14N – the red strands.
DNA Replication Mechanism in Prokaryotes
Watson and Crick first reasoned that complementary base pairing provides the basis of
fidelity in DNA replication; that is, that each base in the template strand dictates the
complementary base in the new strand. However, we now know that the process of DNA
replication is very complex and requires the participation of many different components.
Let’s examine each of these components and see how they fit together to produce our current
picture of DNA synthesis in E. coli, the best-studied cellular replication system. In the
preceding section, we introduced the concept of the replication fork. Figure 1 gives a detailed
Figure 1: DNA replication fork.
schematic view of fork movement during DNA replication; we can refer to this illustration as
we consider each component of the process.
DNA Polymerases:
In the late 1950s, Arthur Kornberg successfully identified and purified the first DNA
polymerase, an enzyme that catalyzes the replication reaction.
This
reaction
works
only
with
the
triphosphate forms of the nucleotides (such
as deoxyadenosine triphosphate, or dATP).
The total amount of DNA at the end of the
reaction can be as much as 20 times the
amount of original input DNA, so most of
the DNA present at the end must be
progeny DNA. Figure 2 depicts the chainelongation
reaction,
or polymerization reaction, catalyzed by
DNA polymerases. We now know that there
are three DNA polymerases in E. coli. The
first enzyme that
Kornberg
purified
is
Figure 2: Chain elongation reaction
catalyzed by DNA polymerase
enzyme has three activities, which appear to be located in different parts of the molecule:
called DNA
polymerase I or pol
I. This
1. a polymerase activity, which catalyzes chain growth in the 5′ → 3′ direction;
2. a 3′ → 5′ exonuclease activity, which removes mismatched bases; and
3. A 5′ → 3′ exonuclease activity, which degrades double-stranded DNA.
Subsequently, two additional polymerases, pol II and pol III, were identified in E. coli. Pol II
may repair damaged DNA, although no particular role has been assigned to this enzyme. Pol
III, together with pol I, has a role in the replication of E. coli DNA. The complete complex,
or holoenzyme, of pol III contains at least 20 different polypeptide subunits, although the
catalytic “core” consists of only three subunits, alpha (α), epsilon (ϵ), and theta (θ).
DNA
polymerase
III
holoenzyme is
the
primary enzyme complex
involved
in prokaryotic DNA replication. It was discovered by Thomas Kornberg (son of Arthur
Kornberg) and Malcolm Gefter in 1970. The complex has high processivity (i.e. the number
of nucleotides added per binding event) and, specifically referring to the replication of
the E.coli genome, works in conjunction with four other DNA polymerases (Pol I, Pol II, Pol
IV, and Pol V). Being the primary holoenzyme involved in replication activity, the DNA Pol
III holoenzyme also has proofreading capabilities that correct replication mistakes by means
of exonuclease activity working 3'→5'. DNA Pol III is a component of the replisome, which
is located at the replication fork.
The replisome is composed of the following:

2 DNA
Pol
III
enzymes,
each
comprising α, ε and θ subunits. (It has been
proven that there is a third copy of Pol III at the
replisome.[1])

the α subunit (encoded by the dnaE gene)
has the polymerase activity.

the ε subunit (dnaQ) has 3'→5' exonuclease
activity.

the θ subunit (holE) stimulates the ε
subunit's proofreading.

2 β units (dnaN) which act as sliding DNA
clamps, they keep the polymerase bound to the
DNA.

2 τ units (dnaX) which acts to dimerize two of the core enzymes (α, ε, and θ subunits).

1 γ unit (also dnaX) which acts as a clamp loader for the lagging strand Okazaki
fragments, helping the two β subunits to form a unit and bind to DNA. The γ unit is made
up of 5 γ subunits which include 3 γ subunits, 1 δ subunit (holA), and 1 δ' subunit (holB).
The δ is involved in copying of the lagging strand.
Χ (holC) and Ψ (holD) which form a 1:1 complex and bind to γ or τ.
Activity
DNA polymerase III synthesizes base pairs at a rate of around 1000 nucleotides per
second.[3] DNA Pol III activity begins after strand separation at the origin of replication.
Because DNA synthesis cannot start de novo, an RNA primer, complementary to part of the
single-stranded DNA, is synthesized by primase (an RNA polymerase)
Prokaryotic Origin of Replication
E. coli replication begins from a fixed origin but then proceeds bidirectionally (with moving
forks at both ends of the replicating piece), as shown in Figure 3, ending at a site called
the terminus. The unique origin is termed oriC is 245 bp long and has several components,
as illustrated in Figure 8-23. First, there is a side-by-side, or tandem, set of 13-bp sequences,
which are nearly identical. There is also a set of binding sites for a protein, the DnaA protein.
An initial step in DNA synthesis is the unwinding of the DNA at the origin in response to
binding of the DnaA protein. The consequences of bidirectional replication can be seen
in Figure 8-24, which gives a larger view of DNA replication.
Priming DNA synthesis
DNA polymerases can extend a chain but
cannot start a chain. Therefore, as already
mentioned, DNA synthesis must first be
initiated
with
short oligonucleotide,
a primer,
that
or
generates
a
segment of duplex DNA. The primer in
DNAreplication can be seen in Figure 3 (see
also Figure
8-20). RNA primers
are
synthesized either by RNA polymeraseor by
an enzyme termed primase.
Primase
synthesizes a short (approximately 30 bp
long) stretch of RNA complementary to a
specific region of the chromosome. The RNA chain is then extended with DNA by DNA
polymerase. E. coli primase forms a complex with the template DNA, and additional
proteins, such as DnaB, DnaT, PriA, Pri B, and Pri C. The entire complex is termed
a primosome.
Leading strand and Lagging Strand
DNA polymerases synthesize new chains only in the 5′ → 3′ direction and therefore, because
of the antiparallel nature of the DNA molecule, move in a 3′ → 5′ direction on
the template strand. The consequence of this polarity is that while one new strand,
the leading strand, is synthesized continuously, the other, the lagging strand, must be
synthesized in short, discontinuous segments, as can be seen in Figure 4. The addition of
nucleotides
along
the
template
for
the lagging strand must proceed toward the
template’s
5′
end
(because replication always moves
along
the template in a 3′ → 5′ direction so that
the new strand can grow 5′ → 3′). Thus,
the new strand must grow in a direction
opposite
that
of
the
movement
of
the replication fork. As fork movement
exposes a new section of lagging-strand
template, a new lagging-strand fragment is
begun and proceeds away from the fork
until it is stopped by the preceding
fragment. In E. coli, pol III carries out
Figure 4: Initiation
of DNA synthesis by
an RNA primer
most of the DNA synthesis on both
strands, and pol I fills in the gaps left in the
lagging strand, which are then sealed by
the enzyme DNA ligase. DNA ligases join broken pieces of DNA by catalyzing the
formation of a phosphodiester bond between the 5′ phosphate end of a hydrogenbonded nucleotide and an adjacent 3′ OH group. It is the only enzyme that can seal DNA
chains. Figure 5 shows the lagging-strand synthesis and gap repair in detail. The primers for
the discontinuous synthesis on the lagging strand are synthesized by primase (step a). The
primers are extended by DNA polymerase (step b) to yield DNA fragments that were first
detected
by
Reiji
Okazaki
and
are
termed Okazaki
fragments. The
5′ →
3′ exonucleaseactivity of pol I removes the primers (step c) and fills in the gaps with DNA,
which are sealed by DNA ligase (step d). One proposed mechanism that allows the same
dimeric holoenzyme molecule to participate in both leading- and lagging-strand synthesis is
shown in Figure 8-31. Here, the looping of the template for the lagging strand allows a single
pol III dimer to generate both daughter strands. After approximately 1000 base pairs, pol III
will release the segment of lagging-strand duplex and allow a new loop to be formed.
Helicases and Topoisomerases
Helicases are enzymes that disrupt the hydrogen bonds that hold the two DNA strands
together in a double helix. Hydrolysis of ATP drives the reaction. Among E. coli helicases
are the DnaB protein and the Rep protein. The Rep protein may help to unwind the double
helix ahead of the polymerase (refer to Figure 8-20). The unwound DNA is stabilized by
theexplain why replication is in the 5′ → 3′ direction. As we saw earlier, new bases are added
when the 3′ OH on the terminal deoxyribose of the growing strand attacks the high-energy
phosphate of the nucleotide triphosphate that is being added (see Figure 8-21). Chain growth
is thus 5′ → 3′. It is conceivable that replication could be in the 3′ → 5′ direction, the 5′
triphosphate at the bottom would be the last base on the chain, and the 3′ OH that attacks it
would be on the free nucleotide triphosphate about to be added to the strand). However, if
replication were in this direction, there would be exonuclease excisions at the 5′ end of the
strand. When a mismatched base was removed, a 5′ OH would be left at the end of the
growing strand. The 3′ OH of an incoming nucleotide triphosphate would thus be facing this
5′ OH instead of the high-energy 5′ triphosphate necessary for bond formation. No bond
would form and strand growth would stop. Therefore, replication is not in the 3′ → 5′
direction.
Figure 6: The 3′ → 5′ exonuclease action
of DNA polymerase III
Figure 5: The overall structure of a growing fork (top) and steps in the synthesis of the lagging
strand. (From H. Lodish, D. Baltimore, A. Berk, S. L. Zipursky, P. Matsudaira, and J.
Darnell, Molecular Cell Biology, 3d ed. Copyright © 1995 by Scientific American Books, Inc.)
In E. coli, Tus (Terminus utilization substance) is a protein that binds to terminator
sequences. Tus binds to 10 closely related sites encoded in the chromosome know as Ter
sites. The bound Tus protein effectively halts DNA polymerase movement. Ter sites are
designated TerA, TerB, TerC…… TerJ. Finally topoisomerase IV unlinks the two circular
DNA duplexes. The multiple Ter sites in the chromosome are oriented such that the two
oppositely moving replication forks are both stalled in the desired termination region.