DNA REPLICATION

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Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini
 1. DNA Replication
 In both prokaryotes and eukaryotes, DNA replication
occurs as a prelude to cell division. This DNA replication
phase is called the S (synthesis) phase. The two daughter
DNA molecules formed from replication eventually
become chromosomes in their own right in the daughter
cells.
 As with all phenomena that involve nucleic acids, the
basic machinery of DNA replication depends on
complementarity of DNA molecules and on the ability of
proteins to form specific interactions with DNA of specific
sequences.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini
2. The model of Watson and Crick
The model of DNA replication proposed by Watson and
Crick is based on the hydrogen-bonded specificity of the
base pairs. Complementary strands are shown in different
colors. The fact that new strands can grow only in the
5’-to-3’ direction adds complexities to the detailed
mechanism of replication.
If this model is correct, then each daughter molecule
should contain one parental nucleotide chain and one
newly synthesized nucleotide chain. This prediction has
been tested in both prokaryotes and eukaryotes. A little
thought shows that there are at least three different ways
in which a parental DNA molecule might be related to the
daughter molecules. These hypothetical modes are called
semiconservative (the Watson-Crick model), conservative,
and dispersive

Genetica per Scienze Naturali

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3. Three alternative patterns for DNA replication
In semiconservative replication,
each daughter duplex contains one
parental and one newly synthesized
strand. However, in conservative
replication, one daughter duplex
consists of two newly synthesized
strands, and the parent duplex is
conserved. Dispersive replication
results in daughter duplexes that
consist of strands containing only
segments of parental DNA and
newly synthesized DNA

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 4. The Meselson-Stahl experiment
 In 1958, Matthew Meselson and Franklin Stahl set out to distinguish among the
three models. They grew E. coli cells in a medium containing the heavy isotope of
nitrogen 15N rather than the normal light (14N) form. This isotope was inserted into
the nitrogen bases, which then were incorporated into newly synthesized DNA
strands.

 After many cell divisions in 15N, the

DNA of the cells were well labeled
with the heavy isotope. The cells were
then removed from the 15N medium
and put into a 14N medium; after one
and two cell divisions, samples were
taken. DNA was extracted from the
cells in each of these samples and put
into a solution of cesium chloride
(CsCl) in an ultracentrifuge.

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 5. Centrifugation of DNA
in a cesium chloride (CsCl) gradient
 If cesium chloride is spun in a centrifuge at tremendously high speeds (50,000 rpm)
for many hours, the cesium and chloride ions tend to be pushed by centrifugal force
toward the bottom of the tube. Ultimately, a gradient of Cs+ and Cl ions is
established in the tube, with the highest ion concentration at the bottom.
 Molecules of DNA in the solution also are pushed toward the bottom by centrifugal
force. But, as they travel down the tube, they encounter the increasing salt
concentration, which tends to push them back up owing to the buoyancy of DNA
(its tendency to float). Thus, the DNA finally "settles" at some point in the tube
where the centrifugal forces just balance the buoyancy of the molecules in the
cesium chloride gradient.
 The buoyancy of DNA depends on its density (which in turn depends on the ratio of
GC to AT base pairs). The presence of the heavier isotope of nitrogen changes the
buoyant density of DNA. The DNA extracted from cells grown for several
generations on 15N medium can be readily distinguished from the DNA of cells
grown on 14N medium by the equilibrium position reached in a cesium chloride
gradient. Such samples are commonly called heavy and light DNA, respectively.

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6. The proof of the semiconservative model
Meselson and Stahl found that, one generation
after the heavy cells were moved to 14N
medium, the DNA formed a single band of an
intermediate density between the densities of the
heavy and light controls. After two generations
in 14N medium, the DNA formed two bands: one
at the intermediate position, the other at the light
position.
This result would be expected from the
semiconservative mode of replication; in fact,
the result is compatible with only this mode if
the experiment begins with chromosomes
composed of individual double helices

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 7. Harlequin chromosomes
 With the use of a more modern staining technique, it is now possible to visualize the
semiconservative replication of chromosomes at mitosis. In this procedure, the
chromosomes go through two rounds of replication in the presence of
bromodeoxyuridine (BUdR), which replaces thymidine in the newly synthesized
DNA. The chromosomes are then stained with Giemsa stain, producing the
appearance shown. (The light blue lines represent the BUdR-substituted strands.)

Genetica per Scienze Naturali

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8. Visualizing sister chromatids
If cells dividing in culture are treated with
BrdU during S phase, the cells are fooled
into incorporating it — instead of
thymidine — into their DNA.
One of the properties of the resulting
DNA is that it fails to take up stain in a
normal way.
When cells are allowed to duplicate their
chromosomes once in BrdU, the
chromosome that appear at the next
metaphase stain normally.
However, when the cells duplicate their
chromosomes a second time in BrdU, one
of the sister chromatids that appears at
the next metaphase stains normally, while
its sister chromatid does not.

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 9. 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).

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 10. DNA polymerases in E. coli
 We now know that there are three DNA polymerases in E. coli. The
first enzyme that Kornberg purified is called DNA polymerase I or
pol I. This enzyme has three activities, which appear to be located in
different parts of the molecule:
 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. Pol III, together
with pol I, has a role in the replication of E. coli DNA

Genetica per Scienze Naturali

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 11. DNA replication fork
The complete complex, or holoenzyme, of pol III contains at least 20 different
polypeptide subunits, although the catalytic "core" consists of only three subunits.
The pol III complex will complete the replication of single-stranded DNA if there is
at least a short segment of duplex already present.

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 12. Prokaryotic origins of replication
 E. coli replication begins from a fixed origin, termed oriC, but then proceeds
bidirectionally (with moving forks at both ends of the replicating piece). It is 245 bp
long and has several components. 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.

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13. A replicating E. coli chromosome
The DNA has been labeled with 3H-deoxythymidine, and
the radioactivity has been detected by overlaying the
replicating chromosome with photographic emulsion. The
autoradiograph shows that the E. coli chromosome has
two replication forks.

Although there seem to be

two bubbles of replication,
actually the point where the
two smaller bubbles meet is
actually just where two
strands of DNA are laying
across one another

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 14. Eukaryotic origins of replication
 Bacteria such as E. coli usually require a 40-minute
replication-division cycle, but, in eukaryotes, the cycle can
vary from 1.4 hours in yeast to 24 hours in cultured animal
cells and may last from 100 to 200 hours in some cells.
 Eukaryotes have to solve the problem of coordinating the
replication of more than one chromosome, as well as
replicating the complex structure of the chromosome itself.
 In eukaryotes, replication proceeds from multiple points of
origin.
 Experiments in yeast indicate the existence of about 400
replication origins distributed among the 17 chromosomes,
and in humans there are estimated to be more than 10,000
growing forks
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 15. Replication bubbles in the fruit fly

Electron micrograph of
replicating DNA in the
embryo of the fruit fly
D. melanogaster

At least 20 different bubbles, therefore with at least 40 different replication forks, can
be observed in this electron micrograph (and accompanying drawn representation of
the electron micrograph.) The large number of replication origins in eukaryotic
chromosomes vs. E. coli's one, enables the slower replication apparatus to copy the
larger eukaryotic genome in approximately the same amount of time as the prokaryotic
genome is replicated Genetica per Scienze Naturali
a.a. 08-09 prof S. Presciuttini
16. Replication bubbles

Electron micrograph of
DNA extracted from
rapidly dividing nuclei
of early D. Melanogaster
embryos. The arrows
mark replication
bubbles; the diameters of
DNA chain in both arms
of these bubbles indicate
that they are double-
stranded.

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17. Priming DNA synthesis
DNA polymerases can extend a chain but cannot
start a chain. Therefore, DNA synthesis must first
be initiated with a primer, or short oligonucleotide,
that generates a segment of duplex DNA.
RNA primers are synthesized either by RNA
polymerase or 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, Pri A, Pri B, and Pri C. The entire
complex is termed a primosome.

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18. 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. 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.

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 19. Discontinuous synthesis
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 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 hydrogen-bonded nucleotide
and an adjacent 3’ OH group. It is the only enzyme that can seal DNA chains.

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20. Steps in DNA synthesis
a) The primers for the
discontinuous synthesis on the
lagging strand are synthesized
by primase.
b) The primers are extended by
DNA polymerase to yield DNA
fragments that were first
detected by Reiji Okazaki and
are termed Okazaki
fragments.
c) The 5’ → 3’ exonuclease
activity of pol I removes the
primers and fills in the gaps
with DNA,
d) which are sealed by DNA
ligase.

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21. A comprehensive view of the replication fork

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 22. Other DNA-modifying enzymes
 Helicases are enzymes that disrupt the hydrogen bonds that hold the two DNA
strands together in a double helix. 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. The unwound DNA is stabilized by the single-stranded binding
(SSB) protein, which binds to the single-stranded DNA and retards reformation of
the duplex.
 The action of helicases during DNA replication generates twists in the circular
DNA that need to be removed to allow replication to continue. Circular DNA can be
twisted and coiled, much like the extra coils that can be introduced into a rubber
band.
 This supercoiling can be created or relaxed by enzymes termed topoisomerases.
There are two basic types of isomerases. Type I enzymes induce a single-stranded
break into the DNA duplex. Type II enzymes cause a break in both strands. In E.
coli, topo I and topo III are examples of type I enzymes, whereas gyrase is an
example of a type II enzyme.

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 23. The action of topoisomerases
 Untwisting of the DNA strands to open the replication fork causes
extra twisting at other regions, and the supercoiling releases the strain
of the extra twisting. During replication, gyrase is needed to remove
positive supercoils ahead of the replication fork

Swivel function of topoisomerase during replication. Extra-twisted

(positively supercoiled) regions accumulate ahead of the fork as the
parental strands separate for replication. A topoisomerase is required
to remove these regions, acting as a swivel to allow extensive
replication. Genetica per Scienze Naturali
a.a. 08-09 prof S. Presciuttini