1

Interestingly, both strands of a double-stranded DNA molecule carry the same information.
They can be regarded as the positive and negative of the same image.
image This means that
each single strand can be used to recreate an identical double stranded molecule. Watson
and Crick immediately realized that this could be the way in which genetic information is
transmitted from one mother cell to two daughter cells.

2
From Watson and Crick's paper, it was fairly clear that theoretically, DNA could be
duplicated by separating the strands,
strands and making a new daughter strand for each old one. one
But how could one show it? In principle, there are three possible ways for the replication of
DNA:
Conservative replication: The parent DNA molecule (double stranded) is inherited to one
daughter cell, and the two daughter strands together go into the other daughter cell.
Semiconservative replication: The parent DNA molecule gives rise to two daughter
molecules. Two new duplexes are made which each consist of one parent and one
daughter strand.
A third option (not depicted here) would be dispersive replication: The parent DNA
molecule and the newly synthesized strands are truly mixed. This model was discarded as
the most unlikely one.

3
The Meselson-Stahl experiment, one of the most beautiful and elegant in the history of
biology showed that DNA replication indeed happens according to the semiconservative
biology,
model.
The experiment uses the techniques of isotope labeling (to get DNA of different densities)
and centrifugation on a density gradient.

4
Ultracentrifugation: Centrifugation with very high velocity, resulting in very large
sedimentation forces (several hundred thousand times gravity).
gravity)
The technique can be used to separate DNA molecules of different densities.

5
Ultracentrifugation requires specific equipment allowing to generate and withstand the
high centrifugal forces (up to several hundred thousand times gravity).
gravity)

6
Analytical Ultracentrifugation uses special rotors with "windows" through which one can
monitor the movement of DNA during the centrifugation process.
process

7
In an analytical ultracentrifuge, the rotor has a quartz window, and one can observe the
sedimentation of the contents of the test tubes.
tubes If the substance one wants to look at is
colorless, such as DNA, one can measure the refractive index to see its concentration. In
the case of DNA its characteristic absorbance at 260 nm may be used.

8
The image shows that after a long time of centrifugation,
centrifugation DNA will form a band in the
window of the rotor. The position of the band depends on the density of the DNA
molecules. It exactly matches the density of the CsCl gradient at that particular position.
DNA is detected here not by its color but by its refractive properties.
In the scheme on the right, two compounds with different densities are being separated
by this method. Note that the gradient has the highest density at the bottom of the tube.
Compounds with higher densities are thus found below compounds with lower density.

9
On a density gradient, DNA with incorporated different isotopes of an
element can be distinguished clearly as they form sharp bands at
different p
positions. In the Meselson-Stahl experiment
p two isotopes
p of
Nitrogen are used (14N, the natural nitrogen, and 15N, a rare isotope
which makes DNA more dense).

10
The Meselson-Stahl experiment:
1) Bacterial
B t i l cellsll are grown in
i a medium
di containing
t i i 15N (th heavy
(the h i t
isotope).
) 15N i
is
incorporated into the DNA.
2) The cells are transferred into a medium containing 14N.
3) Samples of cells after different time points since transfer into 14N containing medium are
collected, DNA is isolated, and samples are centrifuged on a density gradient.
If one knows the time it takes the bacteria to divide once (approx. 20 min) one can
correlate individual n mber of generations after 15N/14N transfer
indi id al time points to the number

11
The results show that at 0 generations (= directly after transfer to new medium) all DNA is
15N labeled, after 1 generation all DNA has an intermediate density (between the 14N and
15N band), and after 2 generations half the DNA localizes to the position at 14N and half the

DNA has an intermediate density (between 14N and 15N).

12
The scheme depicts the conclusions drawn from the experiment: 15N-DNA (the parent
molecule) is hatched. Due to the semiconservative replication mechanism all DNA of the
first generation consists of one "heavy" strand (15N) and one "light" strand (14N) giving rise
to a DNA double helix of intermediate density. In the second generation, only two of the
DNA double helices still contain the parental strand while two double helices consist of 14N-
DNA only. The latter gives rise to a band of lower density as compared to both, parental
((heavy)
ea y) a
and
d first
st ge
generation
e at o ((intermediate
te ed ate de
density)
s ty) DNA.

13
14
The Meselson-Stahl experiment:
1) Bacterial
B t i l cellsll are grown in
i a medium
di containing
t i i 15N (th heavy
(the h i t
isotope).
) 15N i
is
incorporated into the DNA.
2) The cells are transferred into a medium containing 14N.
3) Samples of cells after different time points since transfer into 14N containing medium are
collected, DNA is isolated, and samples are centrifuged on a density gradient.
4) Results after centrifugation for samples observed after different incubation times on 14N
medi m (from left to right):
medium nlabeled control sample (14N only),
right) unlabeled onl ) parental DNA,
DNA first and
second generation DNA.

15
When a circular bacterial chromosome replicates, there is only one initiation point for the
replication This is called the "origin
replication. origin of replication
replication". From the origin DNA replication
proceeds in two directions.

16
Based on its appearance, the regions where the parental DNA strands (orange) become
separated and new complementary strands (red) are being synthesized,
synthesized are called a
"replication forks". Since replication simultaneously proceeds in opposite directions, two
replication forks are formed from one origin of replication.

17
In Eukaryotes (such as the fruit fly Drosophila) replication is initiated in many different
places at the same time.
time This speeds up the replication process as compared to only
having one origin of replication.

18
The enzyme DNA polymerase is the enzyme that catalyses DNA replication. The image
shows the "holoenzyme'"
holoenzyme i.e.,
i e the entire complex of proteins that participates in DNA
replication. Only the -subunits contain the DNA Polymerase activity. Since two DNS
strands need to be replicated simultaneously, there are two polymerase subunits.
The first DNA polymerase (DNA Pol. I) was identified by Arthur Kornberg in 1956. About 15
years later, one of his sons identified DNA Polymerase III from E.coli. Prokaryotic and
Eukaryotic Enzymes are quite similar, so we can use the E.coli protein as a representative.
Besides the -subunit which is responsible
p for DNA p
polymerisation,
y the
-subunits acts as
"clamps" to hold the polymerase on the DNA strand. The -subunit acts as a "clamp
loader" for one of the strands and the - and -subunits are responsible for correcting
errors in a process called proofreading.

19
Chemically, the elongation of any DNA strand always takes place from 5' to 3' end. New
nucleotide monomers are added as deoxynucleoside triphosphates (in the example it is a
dATP). The monomer gets attacked by the 3' hydroxyl group of the previous nucleotide,
the 3' nucleotide of the DNA strand. This attack targets the -phosphorus atom of the
monomer. The nucleotide monomer is a molecule with a high energy bond, breaking of
which supplies the energy necessary for DNA synthesis. Thus, the monomer is the
activated reactant. The new nucleotide becomes attached to the DNA strand through a
phosphodiester bond.
The diphosphate
Th di h h t (=pyrophosphate)
( h h t ) that
th t is
i liberated
lib t d from
f th monomer during
the d i this
thi reaction
ti
becomes hydrolyzed quickly by a cytosolic enzyme. This provides further energy and
makes sure that DNA synthesis is not easily reversible in cells.
How is elongation then coupled to the correct base pairing during replication?

20
During replication, the elongation of the new DNA strand still occurs from 5' to 3' end.
However the nucleotide monomer (here: dATP) first forms hydrogen bonds with the base
However,
of the template strand. After correct pairing of complementary bases, the phosphodiester
bond is formed.
DNA elongation depends on two things: 1) a DNA template (the template strand) and 2) a
short stretch of nucleotides ("primer") with a free 3' OH-group. The DNA polymerase III
enzyme cannot "start from scratch" and fuse two single nucleotides.

21
After formation of the phosphodiester bond, the newly added nucleotide again has a free 3'
hydroxyl group and the next nucleotide may be added.
added

22
This is the structure of the DNA Polymerase catalytic -subunit (so it is NOT the
holoenzyme) If you take your right hand and look at your palm,
holoenzyme). palm you already get an idea of
how DNA polymerase "holds" the DNA molecule during replication.

23
DNA polymerase III (The core enzyme of the DNA polymerase complex found in E. coli)
has a structure similar to structure of a hand in which the DNA molecule is held.
held
Note that the enzyme uses a single DNA strand as the so called "template" (orange) and
synthesizes the new, complementary strand (red) by adding individual nucleotides. These
are Deoxy-Nucleoside Triphosphates (dNTPs) and they are added to the 3' end of the
newly synthesized strand (here referred to as "primer strand").

24
DNA polymerase III is one of the fastest enzymes we know. It makes, however mistakes
(approximately 1/100000 -1/1000000 nucleotides added).
added)
To further minimize this error rate, DNA polymerase has a proofreading mechanism that
makes sure that the number of errors in DNA replication is very low. The proofreading is
based on the shape of a base-pair when it is properly formed. When hydrogen bonds
between the two bases form correctly, the base-pair adopts a certain compact geometry,
which is not achieved by pyrimidine/pyrimidine or purine/purine base-pairs (i.e. not C/G or
A/T).
The shape of the base pair is read out by hydrogen bonds to the minor groove of the new
base pair.
If a wrong base has been added, the so called "exonuclease acivity" of DNA polymerase III
cleaves off the last nucleotide from the 3' end. We say it has 3'5' exonuclease activity.
The remaining error rate (i.e. not correcting a wrong base pairing) is 1%. Hence the overall
error rate is reduced to about 1x10-7- 10-8.

25
Upon nucleotide binding, the DNA polymerase undergoes a conformational change
forming a tight pocket into which a correct base-pair fits but incorrect base pair does not.
not
This means that if the shape of the product is not correct the product gets rejected (the
incorrect nucleotide dissociates).
Another proof-reading step follows through activity of 3' to 5' exonuclease (nuclease that
can only work at the outer parts of a molecule and in the 3' to 5' end direction). An
incorrectly added nucleotide can be cut off and a new nucleotide monomer may come in.
This is why the nucleotide monomer is activated as a triphosphate. In case of an error, a
new monomer comes in i andd nott the
th entire
ti DNA polymer
l i lost:
is l t After
Aft cutting
tti off
ff the
th
incorrect nucleotide, the DNA polymer bears a free 3' OH-group to which another
nucleotide can be added.

26
Knowing that DNA synthesis always proceeds in the 5'-3' direction, we can now take a
closer look at the replication forks again.
again

27
For DNA polymerase to access a DNA strand the DNA double helix has to unwind. Such
part of the DNA molecule is called a replication fork.
fork
As DNA replication is directional, the two replicating DNA strands are not the same. Only
the strand that replicates in the direction of the fork movement can be synthesized
continuously and is called the "leading strand". The strand that replicates in the direction
opposite to the fork movement is called "lagging strand". If the replication fork moves
farther (i.e. opens to the left on this image) the lagging strand cannot be elongated in the
same direction.
The solution is to start synthesizing a new piece of DNA.

28
Using radioactive H-isotopes to mark newly synthesized DNA, it could be shown that only
one of the strands (the leading strand) was indeed synthesized continuously (giving long
DNA fragments) while the other, the "lagging" strand consisted of many smaller fragments.
The latter are called Okazaki Fragments in honor of the scientist who discovered them.

29
The leading strand replicates continuously, the lagging strand replicates fragment by
fragment On the lagging strand the replication takes place from centre of the replication
fragment.
fork (where the unwound DNA strands come together) until it reaches a double stranded
piece of DNA. Each new Okazaki fragment needs to be primed (remember that DNA
polymerase needs a primer and cannot start with single nucleotides!). An RNA polymerase
(called "primase") takes on the role of a primer, as it can start RNA synthesis from
individual RNA nucleotides (de novo synthesis). Due to the structural similarities of RNA
and DNA, DNA polymerase III can then take over and add DNA nucleotides to the RNA
primers Note: the Okazaki fragments are initiated from RNA primers.
primers. primers

30
The RNA primers are removed and replaced with Deoxynucleotides by DNA polymerase I
(also an exonuclease,
exonuclease but it removes nucleotides from the 5 5' end.
end It is a 55' to 3
3' end
exonuclease). The Okazaki fragments are fused (ligated) together by another enzyme,
DNA ligase. The ligation is energy dependent (requires energy) as energy is needed to join
the DNA fragments.

31
While the leading strand is continuously synthesized, part of the lagging strand remains
single-stranded until a new Okazaki fragment can be generated.
generated To avoid the formation of
hairpins by folding back of the lagging strand, single strand binding proteins (SSB, name!)
bind to the DNA. SSB keeps the single strands of the DNA separated by preventing them
from folding onto themselves (like loops in RNA).
Binding of SSB does not only prevent the single strand from folding back onto itself, but it
also straightens it out, thus providing an optimal template for DNA polymerases.

32
The replisome
Th li i a protein
is t i complex
l that
th t consists
i t off the
th DNA polymerase
l h l
holoenzyme, th
the
primosome and additional proteins. The Primosome contains helicase, the enzyme that
unwinds the DNA in the replication fork; it also contains primase for the generation of the
RNA primers for the Okazaki fragments. Once replication has been initiated, DNA primase
does not have to be continuously associated with DNA polymerase, but it is needed for
adding a new primer for each Okazaki fragment to be generated in the lagging strand.

33
The replication machinery is a huge complex that can be viewed by electron microscopy
(image B,
B left).
left) While one cannot distinguish individual parts of the replisome the looping of
the lagging strand as well as parental DNA and the leading strand can be identified
(schematic depiction in Fig. C, right).

34
Thi figure
This fi i taken
is t k from
f a 2008 Nature
N t publication.
bli ti It gives
i a depiction
d i ti off how
h th DNA-
the DNA
Polymerase Holoenzyme together with the Primosome (Helicase and primase) binds to
DNA with the different subunits. Except for the SSB protein and the proofreading subunits
you will see the same subunits in the animation.

35
36
37
38
39
40
41
42
43
44
45
46