The Meselson-Stahl experiment by Matthew Meselson and Franklin Stahl showed that DNA

replication was semiconservative.

Semiconservative replication means that the DNA double helix strand, on replication, produces
two double-stranded DNA helices. Each one has an original DNA helix strand and one new
synthenized DNA helix strand.

In 1953, James Watson and Francis Crick discovered the structure of DNA. Going by the
complementary structure of the base sequences of the DNA strands (cytosine base pairing with
guanine base, and adenine base pairing with thymine base), they proposed that during DNA
replication the old DNA strands would be used in the making of the new DNA strands, resulting
in new DNA helices containing one new strand and one old strand. That is, the DNA replication
would be semiconservative replication. At that time, there was no conclusive evidence of this.

Matthew Meselson and Franklin Stahl set out to prove this hypothesis in 1958. They considered
the semiconservative replication model as well as two other models, the conservative replication
model and the dispersive replication model. In conservative DNA replication, two entirely new
DNA strands would be produced. In dispersive DNA replication, the old and new DNA would be
interspersed with each other along each strand.

The Meselson-Stahl Experiment

To begin with, to be able to tell the old and new DNA apart, Meselson and Stahl grew several
generations of Echerichia coli bacteria in two different mediums, one in the "heavy" nitrogen
isotope 15N and the other in the "light" nitrogen isotope 14N. So, it turned out, that one bacterial
culture contained the heavy form of nitrogen and the other contained the light form of nitrogen.

Then, taking samples of each and extracting DNA into solution, the scientists mixed the two
DNA solutions together and added the mix to a CsCl (cesium chloride) solution of the same
density. This mix was then processed at high speed in an ultracentrifuge. The result was a
mixture separated by density—the "heavy" mix was denser than the "light" mix and so the
"heavy" mix sank to the bottom and the "light" mix moved up. Thus it was possible to tell the
two apart.

Next, the scientists grew several generations of E.coli in 'heavy' nitrogen medium, and extracted
a DNA sample from these bacterial cells. They called this sample "generation zero" and prepared
it for centrifugation. The result showed that the DNA in generation zero had a heavier density,
that is was in the "heavy" form.

The scientists then transferred the E.coli bacterial cells to the "light" nitrogen medium and
allowed it to grow in that. They took samples from this every 20 minutes and processed these
samples too in an ultracentrifuge.

Results
The first result, after one generation, showed that the DNA had an intermediate density rather
than heavy or light. This ruled out conservative replication, as, in that case, the result wouldn't
have shown intermediate density, but rather equal amounts of heavy and light DNA. On the other
hand, intermediate density would be possible with both semiconservative and dispersive
replication.

The second result, after two generations, showed that one part of the DNA had intermediate
density and the other part had light density. This ruled out dispersive replication as in that case
the DNA distribution would have been same between the strands and the resulting density would
have been lower than the intermediate one.

The third result, after several more generations, showed that now a larger portion of the DNA
had light density, synthenized from the first and second generations, and a smaller portion of
DNA had intermediate density, sythesized from the heavy and light DNA. This proved the
semiconservative hypothesis and was a major step in developmental biology research.

Rolling circle replication describes a process of unidirectional nucleic acid replication that can
rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids, the
genomes of bacteriophages, and the circular RNA genome of viroids. Some eukaryotic viruses
also replicate their DNA via a rolling circle mechanism.

[edit] Circular DNA replication

Rolling circle DNA replication is initiated by an initiator protein encoded by the plasmid or
bacteriophage DNA, which nicks one strand of the double-stranded, circular DNA molecule at a
site called the double-strand origin, or DSO. The initiator protein remains bound to the 5'
phosphate end of the nicked strand, and the free 3' hydroxyl end is released to serve as a primer
for DNA synthesis by DNA polymerase III. Using the unnicked strand as a template, replication
proceeds around the circular DNA molecule, displacing the nicked strand as single-stranded
DNA. Displacement of the nicked strand is carried out by a host-encoded helicase called PcrA
(the abbreviation standing for plasmid copy reduced) in the presence of the plasmid replication
initiation protein.

Continued DNA synthesis can produce multiple single-stranded linear copies of the original
DNA in a continuous head-to-tail series called a concatemer. These linear copies can be
converted to double-stranded circular molecules through the following process:

First, the initiator protein makes another nick to terminate synthesis of the first (leading) strand.
RNA polymerase and DNA polymerase III then replicate the single-stranded origin (SSO) DNA
to make another double-stranded circle. DNA polymerase I removes the primer, replacing it with
DNA, and DNA ligase joins the ends to make another molecule of double-stranded circular
DNA.

Rolling circle replication has found wide uses in academic research and biotechnology, and has
been successfully used for amplification of DNA from very small amounts of starting material.
Why do DNA circles occur? The rolling circle model for DNA replication suggests that DNA must be
circular in order to be copied completely; the basic mode of reproducing an entire genome is to copy it
from a circular template, using the circularity in an intrinsic way to guarantee that all of the genetic
information is preserved. The guarantee is enforced by copying always more than one full genome's
worth: copying the circle plus a bit. The model uses an asymmetric mode of replication and thus can
employ the E. coli DNA polymerase or analogous enzymes. The synthesis begins by opening one strand
of the original circle at a specific point. We imagine that the positive strand is opened, that the newly
exposed 5′ end is attached to the ‘membrane,’ and that a new copy of this strand is synthesized by chain
elongation of the 3′ end of the old...

Replication via theta forms is not the only method by which circular molecules can replicate
their genetic information. Another method is rolling circle replication, though it generates
linear copies of a genome rather than circular copies.

Consider a circular molecule of double-stranded DNA with a nick in one of the two
phosphodiester backbones. As long as there is a free 3' OH end, this can serve as a template for
DNA polymerase. When the 3' OH end is extended, the 5' end can be displaced in a manner
analogous to the strand displacement reaction. Synthesis on this strand is also analogous to
leading strand synthesis. The displaced strand can, in turn, serve as an template for replication
as long as a suitable primer is available. Synthesis on this strand is analogous to lagging strand
synthesis.

If synthesis continues in this manner, the consequence of this mechanism of replication can be
the production of concatemer copies of the circular molecule. As a result, multiple copies of a
genome are produced.

A rolling circle mode of replication is seen both during replication of bacteriophage lambda
where rapid production of many copies of the genome is desired, and in the replication of
bacteriophage M13 where only a single copy is produced each time.

AUTORADIOGRAPHIC DEMONSTRATION OF DNA REPLICATION (John

Cairns Experiment):

    The Semiconservative method of replication was verified photographically in 1963 by J.Cairns
who used the technique of autoradiography.  This technique makes use of the fact that
radioactive atoms expose photographic film. The visible silver grains on the film can then be
counted to provide an estimate of the quantity of radioactive material present.  Cairns grew
E.Coli bacteria in a medium containing radioactive thymine, a component of one of the DNA
nucleotides.  The radioactivity was in tritium (31H).  The DNA was then carefully extracted from
the bacteria and placed on photographic emulsion for a period of time.  The emulsion was then
developed to produce autoradiograph that was examined under the electron microscope.  Each
grain of silver represents a radioactive decay.  Interpretation of this autoradiograph reveals
several points.   

The first, known at the time, is that the E.Coli DNA is a circle.  The Second point is that
DNA is replicated while maintaining the integrity of the circle i.e., the circle does not appear to
be broken in the process of DNA replication; an intermediate theta structure is formed which is
due to the formation of replication eye.  Third, replication of the DNA seems to be occurring at
one or two moving Y-junctions in the circle Replication forks, which further supports the
Semiconservative replication.

REPLICATION IS BI-DIRECTIONAL:

    In some cases, radioactivity had not been applied to the cell until DNA replication had already
begun. In these cases, the radioactive label appeared after the theta structure had already begun
forming.  After some time, the process was stopped and the autoradiographs, Cairns found
growth to be bi-directional.  This finding has subsequently been verified by both
autoradiographic and genetic analysis.

Result:

    As the grain track pattern present on both forks with unlabeled DNA in the middle, it was
concluded that the direction of DNA synthesis is bi-directional.

    The unidirectional replication was ruled out because had it been the case, the grain tracks
should have been on only one side of the point of origin.

 
  

REIJI OKAZAKI EXPERIMENT (Semidiscontinuous Replication):

    Autoradiograph images of replicating DNA of E.Coli suggest that DNA's two antiparallel
strands are simultaneously replicated as the replicating fork advances in diametrically opposite
directions.

    All known DNA polymerases catalyze the chain growth only in 5'--> 3' direction.  Therefore,
synthesizing daughter DNA on 3’--> 5' template is done by polymerase as the 3' group is free for
the chain growth in 5'-->3' direction.  But how can polymerase catalyze replication on template
of 3'-->5'.  In this case only 5' end would be available, polymerase not use it to start the chain
growth. Reiji Okazaki’s Pulse-Chase Experiment answered this baffling question.
    Okazaki's model of Semidiscontinuous replication makes two predictions that his research
team tested experimentally.

1) Since atleast half of the newly synthesized DNA appears first as short pieces, it is necessary to
fix the short pieces of DNA by labeling them with radioactive precursors. These short bursts of
labeling with radioactive substances are called pulses.  The stopping period is called chase.

2) If we eliminate the enzyme (DNA ligase) that is responsible for Stiching together the short
pieces of DNA, we ought to too be able to see these pieces even with long pulses of DNA
precursor.

    For his model system, Okazaki chases replication of bacteriophage T4 DNA.  This had the
advantage of simplicity, as well as the availability of mutants of T4 DNA ligase.

    To test first prediction, Okazaki gave shorter and shorter pulses of tritium-labeled thymidine to
E.Coli cells that were replicating T4 DNA (as short as two seconds). Finally, he measured the
approximate size of the newly synthesized DNAs by ultracentrifugation.  It is found to be
between 7S - 11S.  Large DNA pieces sediment faster than smaller pieces toward the bottom of
the centrifuge tube.  The smaller DNA fragments found to have 1000-2000 nucleotides in
length.  With increasing pulse time, another band of tritium labeled DNA appears much nearer to
the bottom of centrifuge tube.  This is the result of attaching small, newly formed pieces of
labeled DNA too much larger.  These fragments are referred as Okazaki fragments.

     Okazaki's group performed second experiment with the T4 mutant containing a defective
DNA ligase group.  In this case, Okazaki fragments found to be present for even one minute of
pulses.  Therefore Reiji Okazaki's Pulse Chase experiment with the replicating T4m
DNA molecules showed that the DNA replication in one of the strand is occurs in a
Semidiscontinuous fashion.

 Okazaki and Semidiscontinuous Replication

After the discovery of DNA polymerase by Arthur Kornberg, the properties of the enzyme
became quite well known. One of the most critical is that DNA polymerase (in fact all known
DNA polymerases) synthesize DNA in one direction only: 5' to 3'. This fact led to a dilemma
regarding how the semiconservative model would work for a DNA molecule.

Reiji Okazaki was a brilliant experimenter who took on this problem. As an aside, Okazaki was
born near Hiroshima, Japan, in 1930. He was a teenager there at the time of the explosion of the
first of two nuclear bombs that the US dropped at the end of World War II. Reiji's scientific
career was cut short by his untimely death from cancer in 1975 at the age of 44, perhaps related
to his exposure to the fallout of that blast.

Okazaki reasoned that there were three possibilities for replicating a double-stranded DNA
molecule, shown in Figure 20.6 of your text:
The continuous model is impossible, based upon the nature of DNA polymerases (replication in
only one direction). Therefore he needed to demonstrate that one of the other two models was
actually taking place.

Any discontinuous synthesis requires that there be, at least transiently, small pieces of DNA in a
replicating structure. Okazaki decided to look for these small pieces. He employed the
ultracentrifuge to do this. This time, the separation is based upon size, so that he could see these
smaller pieces and also follow what happens to them during replication.

Let's look at the Svedberg equation again:

This time, notice that the relative motion of the molecules (the S value) is directly related to the
size (M). In fact molecules that have the same shape (same frictional coefficient, f) will separate
solely as a function of their size. This is called velocity gradient separation. In order to assure
that all his molecules had the same shape, Okazaki denatured the DNA with alkali.

His experimental strategy was to use a pulse-labeling technique. In this case, a culture of
bacterial cells infected with a bacterial virus is given radioactively labeled DNA precursor. This
is a different kind of gradient than before. In this case, using sucrose, the DNA molecules never
find their equilibrium position (sucrose solutions are much less dense than CsCl solutions) and so
the molecules are always in motion. You have to stop the ultracentrifuge at an experimentally
determined time to do the experiment. In this case, only DNA synthesis that has taken place
during the time of the pulse will produce radiolabeled molecules that can be located in the
gradient.

Some of Okazaki's data is shown in Figure 20.7 in your text:

On the left you see that at very short times of labeling (short pulses) very short pieces of DNA
are found (2 sec, 7 sec, 15 sec). However, with longer and longer times, the pieces of DNA get
increasing longer (120 sec). He then tried the same experiment with a mutant virus that was
defective in a gene called DNA ligase. We will see that this is the enzyme that joins pieces of
DNA together into larger structures. In this case (on the right) the labeled pieces of DNA
remained short, even after long times of radiolabeling.

These data suggested to Okazaki that DNA replication occurred by the synthesis of small pieces
that were later linked together by DNA ligase into larger pieces. In order to prove this, he did
what is called a pulse-chase experiment. He radiolabeled his uninfected bacterial culture for a
short time, and then followed this by adding a large excess of unlabeled precursor. This resulted
in a great decrease in the amount of radiolabel incorporated and allowed him to follow the fate of
the short pieces. One of his figures from his 1968 Cold Spring Harbor paper is shown here:
In this case a 30 second pulse (A) is followed by a 5 minute chase (B) and then by a 60 minute
chase (C). The data show that the DNA that was labeled during the 30 second pulse eventually
winds up in very large DNA, equivalent to the size of the genome of the bacterial cell in this case
(shown with a 14C label as a marker).

Okazaki concluded that DNA replication proceeds by a discontinuous mechanism. His data
actually suggested that both strands are copied discontinuously. It wasn't until he used a mutant
deficient in a particular repair process (uracil excision) that he understood that fragments of one
strand produced by this repair had nothing to do with the actual replication process. The small
fragments that can be observed during the short radiolabeling periods are called Okazaki
fragments in his honor.

Okazaki used a pulse chase type experiment to confirm discontinuous strand replication. He took
actively replicating DNA, then added "hot" tritiated nucleotides for a short pulse of about 5
seconds. During the 5 seconds the radioactive nucleotides were incorporated into the growing
DNA strands. After the pulse Okazaki chased with "cold" un-labeled nucleotides for varying
amounts of time and quickly isolated the DNA.[clarification needed] Then the DNA was centrifuged and
analyzed for radioactivity. What Okazaki found was that with short chases of about 7 to 15
seconds most of the radioactivity was found in the small fragments higher in the tube after
centrifuge. However with longer chases more radioactivity was found in the lower, larger
strands. This confirmed that during synthesis first small fragments are formed on the lagging
strand, then later these fragments are combined and incorporated into much larger strands. The
small fragments found on the lagging strand are called Okazaki fragments.

[edit] References
REPLICATION EYE:

    It is formed during   replication in both eukaryotic and prokaryotic DNA.  It is the place where
replication occurs actively. It is otherwise known as replication bubble.  Formation of the
replication eye provides the theta like structure to the circular DNA during replication in
prokaryotes. Each replication bubble found to have two replication forks, each at the corner of an
eye.

 REPLICATION FORK:

    The corner region of the replication bubble or replication eye is referred as replication fork.
Generally it is point from which the replication elongated or continued.  Bi-directional nature of
replication was identified by studying the appearance of nucleotides at the replication forks in
connection with the change in the radioactive material intensity. Each replication bubble found to
have two replication forks, each at the corner of an eye.

SEMIDISCONTINUOUS REPLICATION:

            During Replication, half of the DNA strand synthesized in fragments where as remaining
half synthesized continuously.  This is referred as semidiscontinuous replication.  This happens
only because of the nature of the DNA polymerases i.e., they can synthesize DNA by extending
3’end and they are unable to extend DNA from 5’end.  This nature of replication is proved by
Okazaki by an experiment. 

How can we experimentally prove that the DNA replication follows 2 directions from one initiation
point? In Albert's biology is mentioned an experiment with H^3 thymidine culture of cells, and then after
autoradiography we can observe the DNA replication bubble. Is this the experiment?

Use dideoxynucleotides to block formation of okazaki fragments in the lagging strand

how do dideoxynucleotides block formation of Okazaki fragments but not the leading strand?