2. DNA replication
• When the two strands of the DNA double helix are separated,
each can serve as a template for the replication of a new
complementary strand. This produces two daughter
molecules, each of which contains two DNA strands with an
antiparallel orientation.
• This process is called semiconservative replication because,
although the parental duplex is separated into two halves
(and, therefore, is not “conserved” as an entity), each of the
individual parental strands remains intact in one of the two
new duplexes.
2
4. Main components of DNA
replication
• DNA replication is catalyzed by DNA polymerases
• DNA Polymerases: DNA polymerases are enzymes that carry out all
forms of DNA replication. DNA polymerase synthesizes a new strand of
DNA by extending the 3’ end of an existing nucleotide chain, adding new
nucleotides complementary to the template strand.
• In prokaryotes e.g. bacteria:
There are three species of DNA polymerase
1-DNA polymerase I:
It is responsible for gap filling between the fragments of DNA by nick
translation of the RNA primers used during DNA replication. It is also
involved in DNA repair.
2-DNA polymerase II :
DNA polymerase II is activated in response to DNA damage (SOS
response). It has proofreading and DNA repair function.
3-DNA polymerase III:
It catalyzes most replication of DNA. This enzyme is 900 kDa in size, and is
composed of several subunits (they form so called holoenzyme). It plays a
role in DNA repair.
4
5. • In eukaryotes e.g. human cell
• Replication occurs during S phase of cell cycle(synthesis phase
of interphase).There are 5 species of DNA polymerase:
Polymerase Alpha (α), beta (β), gamma (ɣ), delta (δ) and
Epsilon (ε).
Probable Roles of Some Eukaryotic DNA Polymerases
DNA Polymerase α Priming of replication of both
strands
DNA Polymerase δ Elongation of lagging strand
DNA Polymerase ε Elongation of leading strand
DNA Polymerase β DNA repair
DNA Polymerase ɣ Replication of mitochondrial
DNA
5
6. Steps of DNA synthesis
A. Separation of the two complementary DNA strands:
• They must first separate over a small region, because the
polymerases use only ssDNA as a template.
• In prokaryotic organisms, DNA replication begins at a single,
unique nucleotide sequence, a site called the origin of
replication.
• In eukaryotes, replication begins at multiple sites along the
DNA helix. Having multiple origins of replication provides a
mechanism for rapidly replicating the great length of
eukaryotic DNA molecules.
6
7. Steps in prokaryotic DNA
synthesis
B. Formation of the replication fork:
• As the two strands unwind and separate, synthesis occurs at
two replication forks that move away from the origin in
opposite directions (bidirectionally), generating a replication
bubble. The term “replication fork” derives from the Y-shaped
structure in which the tines of the fork represent the
separated strand.
7
8. Origin of replication
• Replication always starts at specific locations on
the DNA, which are called origins of replication
and are recognized by their sequence. The
starting points of replication have specific
nucleotide sequences that are recognized by
enzymes and proteins responsible for initiation
of DNA replication.
8
10. Steps in prokaryotic DNA
synthesis
1. Proteins required for DNA strand separation:
Initiation of DNA replication requires the recognition of the origin by a
group of proteins that form the prepriming complex.
a. DnaA protein:
DnaA protein binds to specific nucleotide sequences (DnaA boxes)
within the origin of replication, causing the short, tandemly arranged
(one after the other) AT-rich regions in the origin to melt. Melting is
adenosine triphosphate (ATP) dependent, and results in strand
separation with the formation of localized regions of ssDNA.
b. DNA helicases:
These enzymes bind to ssDNA near the replication fork and then move
into the neighboring double-stranded region, forcing the strands apart
(in effect, unwinding the double helix). Helicases require energy
provided by ATP. 10
11. Steps in prokaryotic DNA
synthesis
c. Single-stranded DNA-binding protein:
This protein binds to the ssDNA generated by helicases. These proteins
not only keep the two strands of DNA separated in the area of the
replication origin, thus providing the single-stranded template required
by polymerases, but also protect the DNA from nucleases that degrade
ssDNA.
11
12. Steps in prokaryotic DNA
synthesis
2. Solving the problem of supercoils:
• As the two strands of the double helix are separated, a problem is
encountered, namely, the appearance of positive supercoils in the
region of DNA ahead of the replication fork as a result of
overwinding, and negative supercoils in the region behind the fork
as a result of underwinding. The accumulating positive supercoils
interfere with further unwinding of the double helix.
• DNA topoisomerases are responsible for removing supercoils in
the helix by transiently cleaving one or both of the DNA strands.
a. Type I DNA topoisomerases:
These enzymes cut and reseal one strand of the double helix.
b. Type II DNA topoisomerases (DNA gyrase found in bacteria and
plants):
These enzymes bind tightly to the DNA double helix and make
transient breaks in both strands to pass through the break and finally,
reseals the break.
12
13. Steps in prokaryotic DNA
synthesis
C. Direction of DNA replication
• The DNA polymerases responsible for copying the DNA templates
are only able to “read” the parental nucleotide sequences in the
3¯→5¯ direction, and they synthesize the new DNA strands only in
the 5¯→3¯ (antiparallel) direction. Therefore, beginning with one
parental double helix, the two newly synthesized stretches of
nucleotide chains must grow in opposite directions, one in the
5¯→3¯ direction toward the replication fork and one in the 5¯→3¯
direction away from the replication fork.
• This feat is accomplished by a slightly different mechanism on each
strand.
1. Leading strand
2. Lagging strand
13
14. Steps in prokaryotic DNA
synthesis
1. Leading strand:
The strand that is being copied in the direction of the advancing
replication fork and is synthesized continuously.
2. Lagging strand:
The strand that is being copied in the direction away from the
replication fork is synthesized discontinuously, with small
fragments of DNA being copied near the replication fork. These
short stretches of discontinuous DNA, termed Okazaki
fragments, are eventually joined (ligated) to become a single,
continuous strand.
14
15. Steps in prokaryotic DNA
synthesis
D. RNA primer
DNA polymerases cannot initiate synthesis of a
complementary strand of DNA on a totally
single-stranded template. Rather, they require
an RNA primer, which is a short, double-
stranded region consisting of RNA base-paired
to the DNA template, with a free hydroxyl
group on the 3¯ end of the RNA strand. This
hydroxyl group serves as the first acceptor of a
deoxynucleotide by action of a DNA
polymerase.
15
16. Steps in prokaryotic DNA
synthesis
1. Primase :
• It is a DNA- dependent RNA polymerase that
synthesizes short RNA primers (5-200
bases) with the help of DNA binding protein
(primosome).
• RNA primer complementary and anti-parallel
to the DNA is synthesized by primase .
• The DNA polymerase III requires a primer to
elongate at the beginning of replication .
• The primer has a free 3¯ -OH to accept
deoxy-ribonucleotides polymerized by DNA
polymerase III.
16
17. Steps in prokaryotic DNA
synthesis
2. Primosome:
The addition of primase converts the
prepriming complex of proteins required for
DNA strand separation to a primosome.
The primosome makes the RNA primer
required for leading strand synthesis and
initiates Okazaki fragment formation in lagging
strand synthesis. As with DNA synthesis, the
direction of synthesis of the primer is 5¯→3¯. 17
19. Steps in prokaryotic DNA
synthesis
E. Chain elongation
1. Using the free 3' -OH group of the RNA primer as the acceptor of the
first nucleotide, DNA polymerase III begins to add subsequent
nucleotides.
2. Chain elongation occurs as DNA polymerase III moves along the
template strand, substrate nucleotides pair with the template
according to the pairing rule i.e. A is paired with T and G is paired
with C. Thus, the daughter strand will be complementary to the
parent strand.
3. DNA polymerase III can catalyze chain growth only in the 5' to 3'
direction i.e. the new strand runs in 5' to 3' direction, while template
strand runs in 3' to 5' direction. Therefore the 2 daughter chains
must grow in opposite directions, one towards replication fork
(leading strand) and the other away from it (lagging strand).
19
20. Steps in prokaryotic DNA
synthesis
F. Excision of RNA primers and their replacement by DNA
DNA polymerase III continues to synthesize DNA on the lagging
strand until it is blocked by proximity to an RNA primer. When this
occurs, the RNA is excised and the gap filled by DNA polymerase
I.
5¯→3 ¯ Exonuclease activity:
DNA polymerase I also has a 5¯→3¯ exonuclease activity that is
able to hydrolytically remove the RNA primer.
DNA polymerase I has the following functions:
1. It Removes the RNA nucleotides (5¯→3¯ exonuclease
activity).
2. Replaces it with deoxyribonucleotides, synthesizing DNA in
the 5¯→3¯ direction (5 →3 polymerase activity).
3. Proofreads the new chain using its 3¯→5¯ exonuclease
activity to remove errors.
20
21. Steps in prokaryotic DNA
synthesis
G. DNA ligase
• The final phosphodiester linkage between the 5¯ phosphate
group on the DNA chain synthesized by DNA polymerase III
and the 3¯ hydroxyl group on the chain made by DNA
polymerase I is catalyzed by DNA ligase. The joining of these
two stretches of DNA requires energy.
21
22. Steps in Eukaryotic DNA
synthesis
The process of eukaryotic DNA replication closely follows that of
prokaryotic DNA synthesis. Some differences, such as
1. The multiple origins of replication in eukaryotic cells versus
single origins of replication in prokaryotes.
2. RNA primers are removed by RNase H and flap endonuclease-1
(FEN1) rather than by a DNA polymerase.
DNA replication occurs during the S (synthesis) phase.
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