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Theme 4 Module 2 Script

Slide 1:
Theme 4, Module 2: Replication YOUR SUMMARY NOTES:

Slide 2:
In this module we will:
 Examine the early research that proposed
and showed evidence for the manner in
which DNA is replicated
 We will also identify how DNA is
replicated into identical daughter strands
from parental template strands and how a
complex of proteins are important during
the replication process
 We will also compare eukaryotic and
prokaryotic replication mechanisms
 And finally, we will understand the
importance of telomere length
conservation

Slide 3:
Unit 1: Transmission of information

Slide 4:
DNA is the macromolecule that determines the
characteristics of the cell. We know from the
early pioneering work of Francis Crick, James
Watson and Rosalind Franklin, that the molecule
exists in a helical structure with purines paired
with pyrimidines along the entire DNA helix.
While the hydrogen bonds that form between
base pairing of nucleotides renders the DNA
molecule a certain stability, researchers came to
suspect very early on that there must be a
copying mechanism for this genetic material. In
fact, Watson and Crick concluded their 1953
Nature paper that proposes the structure of DNA
with the statement, “It has not escaped our notice
that the specific pairing we have postulated
immediately suggests a copying mechanism for
genetic material.” While Watson and Crick
proposed the characteristic helical structure of
DNA, they also proposed through this concluding
statement that the base-pairing in DNA should
allow for a mechanism by which the genetic
information in DNA could be copied. They were
certainly on to something!
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Theme 4 Module 2 Script

Slide 5:
The knowledge that there could be a copying YOUR SUMMARY NOTES:
mechanism of DNA initiated a scientific revolution
that has led to many advances in cellular and
molecular biology research. In a follow-up 1954
paper by Watson and Crick, they proposed a
hypothesis for how DNA replicates. Specifically,
Watson and Crick proposed that DNA consists of
a pair of template chains which are
complementary to each other, and that prior to
replication, the hydrogen bonds are broken
between these complementary strands, which
allows for unwinding and separation. By saying
that a strand is complementary to another, what
is meant is that each strand contains the
information necessary to reconstruct the other.
Watson and Crick believed that when a cell
copies its DNA, that each strand serves as a
template for the ordering of new nucleotides
(according to the base-pairing rules) into a new
complementary strand. The end results is that,
while replication of DNA would begin with one
parent DNA double helix, that by the end, there
would be two new helices, with the new double
stranded DNA being an exact copy of the
“parental” molecule. This model of DNA
replication predicted by Watson and Crick
proposed that when a DNA double helix
replicates, that each of the two daughter DNA
molecules would have one old strand, from the
parental molecule and one newly made strand.
This model is known as the semiconservative
model of DNA replication and went many years
without testing.

Slide 6:
Checkpoint question #1
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Theme 4 Module 2 Script

Slide 7:
Unit 2: Evidence for the semiconservative model YOUR SUMMARY NOTES:

Slide 8:
In addition to the semiconservative model of DNA
replication that was proposed by Watson and
Crick, researchers imagined other mechanisms of
replication. Some hypothesized that perhaps
DNA replicated in a more of a conservative
fashion, with the two complementary parental
strands somehow coming back together after
replication. Other researchers imagined that
perhaps DNA replication is more dispersive, with
all four strands somehow combining into a
mixture of old and new DNA strands. It was the
research of Matthew Meselson and Franklin Stahl
that conclusively demonstrated that DNA
replicates in a semiconservative manner. In the
late 1950s, Meselson and Stahl cultured E. coli
bacterial cells for many generations in a medium
that contained the nucleotide precursors with the
radioactively labelled heavy isotope of nitrogen
(15N). Subsequently, they transferred the
bacteria into a medium that contained 14N, the
lighter isotope. From this point onwards, every
new strand of replicated DNA would be built
containing 14N rather than the 15N isotopes.

Slide 9:
Throughout their experiment, Meselson and Stahl
extracted DNA samples from the growing
bacteria and then centrifuged each DNA sample
through a solution that will separate the DNA
based on the differing densities of the
radioactively labelled molecules. Incorporation of
the 15N isotope makes the DNA “heavier” than
that with the 14N isotope so it ends up near the
bottom of the tube.
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Theme 4 Module 2 Script

Slide 10:
Meselsen and Stahl identified that the DNA from YOUR SUMMARY NOTES:
bacteria that had been growing in the media
containing the 15N isotope had only one distinct
band. In contrast, it was observed that after
transfer into and then one round of replication in
the medium containing 14N isotope, that the DNA
also appeared as a single band, but with lower
density and so was positioned higher than the
original 15N band in the centrifugation gradient.
Therefore, this band must contain some hybrid of
the 14N and 15N DNA. Based on these results,
Meselsen and Stahl rejected the conservative
model of DNA replication since there was no
individual distinct band that correlated with the
“heavier” 15N DNA.

Meselsen and Stahl were then left with two

possible models to test, the semiconservative
model, and the dispersive model of DNA
replication. To test these models, Meselsen and
Stahl allowed the bacteria to grow and divide for
many generations in the 14N-containing media
after transfer from the 15-N containing media.
What they found was that following many rounds
of bacterial replication, that the extracted DNA
could be separated into 2 distinct bands. One in
the position that contained only 14N, and another
in the position where the hybrid DNA containing
15N and 14N would be expected.

Slide 11:
These results were found to be consistent with
the fact that with DNA replication, each new
double helix is made up of one old strand and a
new strand. This was the evidence that was
needed to support what is now widely known, that
DNA is replicated in a semiconservative fashion
in the cell.
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Theme 4 Module 2 Script

Slide 12:
Beyond Meselsen and Stahl’s work, researchers
began to question whether DNA replicated in the YOUR SUMMARY NOTES:
same manner in eukaryotes as it appeared to
replicate in prokaryotic bacteria. The complexity
and size of the eukaryotic genome made the
experiments with labeled isotopes difficult, but
researchers have since then been able to label
individual nucleotides with fluorescent labels.
This allowed researchers to examine replication
of eukaryotic DNA in media containing
fluorescent nucleotides and follow entire strands
of eukaryotic DNA under the microscope. It was
found that following rounds of replication of DNA
and cell division in media containing fluorescent
nucleotides, the chromosomes of eukaryotic cells
could contain hybrid and fully labelled
nucleotides. This leads to observed faintly and
darkly fluorescing strands of labeled DNA even
within one chromosome. These results provided
further support for the semiconservative model of
DNA replication, and indeed it was shown that
this mechanism of replication is similar in
prokaryotes and eukaryotes.

Slide 13:
Checkpoint question #2

Slide 14:
Unit 3: Replicating DNA

Slide 15:
DNA replication begins in the S-phase of the cell
cycle at specific regions along DNA called the
origins of replication. While both eukaryotes and
prokaryotes replicate their DNA in a
semiconservative manner, the organization of the
genome leads to different ways to initiate
replication. Let us consider replication in
prokaryotes which begins at a single origin of
replication, after which replication continues
around the circular chromosome from this one
initiation site.
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Theme 4 Module 2 Script

continues, there are important differences in the

manner in which each parent strand is replicated.

Slide 16:
The process of DNA replication is similar to that YOUR SUMMARY NOTES:
of transcription. Specifically, during DNA
replication, the template strand is copied from the
3’ end to the 5’ end and produces a daughter
strand that elongates in a 5’ to 3’ direction.
During the process of DNA replication, each
incoming complementary nucleotide engages in a
hydrogen bond with the nucleotide on the
template strand and interacts with the 3’ hydroxyl
of the existing polymer that is forming on the new
daughter strand. A phosphodiester bond forms
between the growing daughter strand and the
new incoming nucleotide, allowing it to become
part of the DNA molecule backbone on the
daughter strand (and in the process producing a
pyrophosphate). But how does DNA replication
begin?

Slide 17:
Since the two complementary strands in a DNA
molecule run in an antiparallel fashion, both
strands can serve as simultaneous templates
from which DNA can be replicated. The
unwinding of the DNA double helix results in the
separation of the parental strands at regions
called replication forks within the origins of
replication. The initiation of replication requires
that a short stretch of an RNA molecule or a
primer (that is usually 5-10 nucleotides long) be
synthesized and base pair with the template DNA
strands. The primer is required since the
enzymatic machinery that elongates a new
daughter strand can only do so from an existing
piece of DNA or RNA. As elongation progresses,
the polymerization of each newly replicated
daughter strand is catalyzed by the DNA
polymerase enzyme in a 5’ to 3’ direction. It is
this enzyme that synthesizes a replicated DNA
strand from the primers that anneal to the
template strand. As the process of replication
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Theme 4 Module 2 Script

Slide 18:
Due to the chemical nature of DNA
polymerization by DNA polymerase being limited YOUR SUMMARY NOTES:
to elongation in a 5’ to 3’ direction, replication of
one daughter strand is continuous from the
primer and is often referred to as the leading
strand. On the leading strand, only one primer is
required, and the DNA polymerase is able to
continuously add nucleotides to the new daughter
strand as the replication fork progresses. In
contrast, the other antiparallel parent strand has
discontinuous (or fragmented) replication occur.
The produced daughter strand is referred to as
the lagging strand.

Since DNA polymerase can only add nucleotides

to the 3’ end of a polymerizing DNA molecule,
replication from this strand requires that DNA
polymerase replicate the DNA in a direction that
is away from the replication fork. The end result
of replication and production of the lagging strand
is that the lagging strand will contain segments or
fragments of DNA. These are often referred to as
Okazaki fragments, named after Reiji Okazaki,
the scientist who discovered them in E. coli.
While only one primer is required for the
synthesis of the leading daughter strand, each
Okazaki fragment on the lagging strand is formed
by separate primers. After DNA polymerase
forms an Okazaki fragment, another DNA
polymerase is able to replace the RNA primer
sequences of all daughter strands with DNA
nucleotides. In addition to DNA polymerase,
many other proteins have been found to serve
important roles during the DNA replication
process.
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Theme 4 Module 2 Script

enzyme that is responsible for removing the RNA

primer after DNA replication and replacing those
short sequences with DNA nucleotides. Research
has shown that much of the process of replication
is similar between prokaryotes and eukaryotes,
however it has been found that eukaryotes
utilized a different set of DNA polymerases.

Slide 19:
Various proteins that participate in DNA
replication form a single large complex called the YOUR SUMMARY NOTES:
replication complex. During initiation, DNA
helicase enzymes are able to bind to the parental
DNA strands at the origin of replication and
initiate the unwinding of the DNA double helix.
This is accomplished because DNA helicase is
able to break the hydrogen bonds between
complementary nucleotide base pairs. Since the
newly unwound DNA can have the tendency to
rejoin, single-stranded binding proteins bind to
and stabilize each parental strand until elongation
can begin. A third family of proteins, the
topoisomerases, are able to bind upstream of the
replication fork and minimize the torsional strain
that is brought about from the unwinding that
occurs at the replication fork. These proteins
serve as initiator proteins that trigger the process
of unwinding at the origin of replication. The
process of primer synthesis will follow and
actually marks the beginning of the synthesis of
the new daughter DNA molecules.

Slide 20:
An RNA polymerase enzyme by the name of
RNA primase synthesizes the short RNA
stretches of nucleotides which are
complementary to the parental strands from
which DNA polymerase can then elongate from.
While many types of DNA polymerase have been
discovered, it has been found that DNA
polymerase III does most of the elongation work
in prokaryotes, while DNA polymerase I is the
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of nucleotides in a DNA molecule were indicative

of a copying mechanism for genetic material,
however the specific manner in which DNA is
replicated from a parent template strand cannot
be solely attributed to the specificity of base
pairing. In reality although errors in DNA
replication are rare, they certainly can occur.
This can occur during the initial pairing between
incoming nucleotides and those of the template
strand. However, the cell has an innate
proofreading mechanism. During DNA
replication, DNA polymerases are able to
proofread each added nucleotide relative to the
template strand, as each DNA nucleotide is
added to the growing daughter strand. If an
incorrect nucleotide pairing is detected, DNA
polymerase removes the incorrect nucleotide, the
correct nucleotide is added, and then replication
can continue by the DNA polymerase. While
there is a high degree of accuracy with which
DNA polymerase exudes its proofreading
capabilities, mismatched nucleotides can
sometimes be evaded by this proofreading
Slide 21: mechanism. Sometimes other enzymes can help
While synthesis of the replicated leading and with the correction of replication errors.
lagging daughter strands will occur YOUR SUMMARY NOTES:
simultaneously, the lagging strand is aptly named
so due to the delay in synthesis that is brought
about relative to the leading strand. Each new
fragment of the lagging strand cannot be
replicated until enough of the template DNA is
revealed at the replication fork. The lagging
strand also requires some post-replication
processing. Since prokaryotes have one origin of
replication, during the replication of their circular
DNA, the excised primer is replaced by specific
DNA nucleotides and there is no gap in the newly
synthesized DNA. In eukaryotes, this is not the
case. The replacement of the RNA primer with
DNA nucleotides leaves a sugar phosphate
backbone at the 3’ end with a free phosphate
backbone. This is prevalent along the Okazaki
fragments of the lagging strand. An enzyme by
the name of DNA ligase is able to join the 3’ end
of a fragment to an adjacent DNA nucleotide by
catalyzing the phosphodiester bond formation
along this region of the DNA backbone and as a
result, joining adjacent replicated Okazaki
fragments together.

Slide 22:
Watson and Crick certainly predicted that the
inherent organization and base-pairing properties
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Theme 4 Module 2 Script

Slide 26:
While DNA replication in eukaryotes leads to a
leading strand that replicates the whole template
strand, that is not the case with the lagging
strand. In spite of the impressive capabilities of
DNA polymerase, the DNA replication machinery
has no way to complete the 5’ ends of daughter
strands when the DNA is linear, not a circle as in
prokaryotes. If for example an RNA primer on
the lagging strand is bound to the very end of a
template strand, once that RNA primer is
removed, it cannot be replaced with DNA
nucleotides because there is no 3’ end available
for nucleotide addition and phosphodiester bond
formation.

Slide 23:
Checkpoint question #3 YOUR SUMMARY NOTES:
Slide 24:
Unit 4: Replicating eukaryotic chromosomes

Slide 25:
As we have seen, replication in prokaryotes
begins at one origin of replication, after which
replication continues around the circular
chromosome from this one initiation site. In
contrast, eukaryotic replication originates at many
origins of replication along the linear
chromosomes. Despite these differences in the
number of origins of replication, prokaryotic and
eukaryotic cells share many common features
during replication. For example, both require a
primer for initiation of replication to occur,
elongation is always in a 5’to 3’ direction, and
specialized proteins are utilized within a
replication complex which allows for the
replication of daughter strands from parental DNA
template strands and finally, both prokaryotes
and eukaryotes have a leading strand and
lagging strand.
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Theme 4 Module 2 Script

repeats of G-T rich sequences. This non-coding

repetitive sequence of nucleotides serves as a
buffer zone so that coding genes within
chromosomes are protected. However,
telomeres do become shorter during successive
rounds of replication. One can therefore imagine
that telomeric DNA would be shorter in the
dividing cells of older individuals. This is certainly
the case for most somatic cells in eukaryotes.
But what does this mean for cells whose genome
must pretty much remain unchanged from an
organism to its offspring over many generations?
It would most certainly be a problem if the
chromosomes of germ cells became shorter with
each cell cycle. If that were the case, this would
lead to the production of gametes with missing
information. This would also be a problem in
embryonic stem cells that must go through many
many rounds of replication to produce the fully
developed organism. However, telomere
shortening does not occur in gametes or in stem
cells. What is unique about these types of cells is
that they have a special telomerase enzyme
which catalyzes the lengthening of telomeres in
these eukaryotic cells.

Slide 27: YOUR SUMMARY NOTES:

As a result, shorter and shorter DNA molecules
are produced with uneven ends with repeated
rounds of DNA replication. While this is not a
problem in prokaryotes due to their circular
chromosome, it would certainly pose a problem
within linear eukaryotic chromosomes. However,
this shortening does not generally pose a
problem in eukaryotic cells. How is it then that
the ends of linear eukaryotic chromosomes, and
any genes that might be located at the ends, are
protected from being eroded away with
successive rounds of DNA replication?

Slide 28:
The ends of linear eukaryotic chromosomes
contain regions called telomeres. These are
special nucleotides sequences that are mainly
made up of repetitions of one short nucleotide
sequence. For example, human telomeres
contain the characteristic six-nucleotide
sequence TTAGGG repeated between hundreds
to thousands of times, leading to many tandem
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ends may play during the process of aging and

even cancer progression.

Slide 30:
Checkpoint question #4

Slide 31:
In this module we have seen that:
 DNA is replicated in a semiconservative
manner in both prokaryotes and
eukaryotes.
 We have also seen how the different
structure of prokaryotic and eukaryotic
genomes leads to the formation of one
origin of replication in prokaryotes, while
eukaryotes can have multiple origins of
replication along their linear
chromosomes.
 In addition, we have seen that DNA
replication requires essential protein
replication machinery that are needed for
the synthesis of leading and lagging
daughter strands from the parental DNA
template strands
 Finally, we have learned of the
importance of telomere length
conservation in cells whose genome must
be passed on intact to subsequent
generations and as result, telomere
lengths are generally conserved in germ
cells and stem cells.

Slide 29:
Telomerase is a specific type of reverse
transcriptase. This enzyme is able to synthesize YOUR SUMMARY NOTES:
DNA from an RNA template. In fact, the
telomerase itself is a ribonucleoprotein that
contains the RNA template as part of the complex
itself. The RNA template of the telomerase
serves a very important role in elongating the
linear chromosomes of stem cells and germ cells.
To elongate the telomere regions, telomerase
binds to the tail of the telomere and subsequently
catalyzes the extension of the template strand by
adding telomere repeats. Once telomerase has
extended the template DNA strand, primase,
DNA polymerase and ligase are able to go back
and complete the daughter strand replication
from the remainder of the template strand.
Regulation of telomere length is an active area of
current research, and new findings continue to
emerge as to the role that these chromosomal
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Theme 4 Module 2 Script