Dna replication notes
DNA replication or DNA synthesis is the process of copying
double-stranded DNA molecule.
It is the process of copying genetic material.
A DNA molecule is a long polymer built from nucleotides.
Both strands in DNA run anti-parallel to each other and are
complementary to one another.
One of the DNA strands is built in the 5′ → 3′ direction, while the
complementary strand is built in the 3′ → 5′ direction (5′ and 3’
each mark one end of a strand).
The nucleotides which make up DNA are referred to by their
bases and when bonded are referred to as base pairs.
Adenine pairs with Thymine and Cytosine pairs with Guanine.
A strand running in the 5’→ 3′ direction that has Adenine will
pair with base Thymine on the complementary strand running in
3’→ 5′ direction.
As DNA strands are antiparallel and complementary, each strand
can serve as a template for the reproduction of the opposite strand.
The template strand is preserved in its entirety and the new strand is assembled from
This process is called semiconservative replication.
Ideally, the two resulting strands are identical, although in reality there are always errors,
though proofreading and error-checking mechanisms exist to ensure a very high level of
In a cell, DNA replication is obligatory prior to cell division.
Prokaryotes persistently replicate their DNA.
In eukaryotes, timings are highly regulated and this occurs during the S phase of the cell cycle,
preceding mitosis or meiosis I.
The Replication Fork
The replication fork is a structure which forms when DNA is replicating itself.
It is created through the action of helicase, which breaks the hydrogen bonds holding the two
DNA strands together.
The resulting structure has two branching “prongs”, each one made up of a single strand of
In the first step, the double helix shown above in blue is unwound by a helicase.
Next, a molecule of DNA polymerase III shown in green binds to one strand of the DNA.
It moves along the strand, using it as a template for assembling a leading strand shown above
in red of nucleotides and reforming a double helix.
A DNA polymerase I molecule (also green) is used to bind to the other template strand
(lagging strand) as the double helix opens.
This molecule must synthesize discontinuous segments of polynucleotides (called Okazaki
Another enzyme, DNA ligase shown in violet, then stitches these together into the lagging
DNA replication begins with the “unzipping” of the parent molecule as the hydrogen bonds
between the base pairs are broken.
Once exposed, the sequence of bases on each of the separated strands serves as a template to
guide the insertion of a complementary set of bases on the strand being synthesized.
The new strands are assembled from deoxynucleoside triphosphates.
Each incoming nucleotide is covalently linked to the “free” 3′ carbon atom on the pentose as
the second and third phosphates are removed together as a molecule of pyrophosphate (PPi).
The nucleotides are assembled in the order that complements the order of bases on the
strand serving as the template.
Thus each C on the template guides the insertion of a G on the new strand, each G a C, and so
When the process is complete, two DNA molecules have been formed identical to each other
and to the parent molecule.
Lagging Strand Synthesis
In DNA replication, the lagging strand is the DNA strand at the opposite side of the replication
fork from the leading strand.
It goes from 3′ to 5′ (these numbers indicate the position of the molecule in respect to the
carbon atoms it contains).
When helicase unwinds DNA, two single stranded regions of DNA (the “replication fork”)
form. DNA polymerase cannot build a strand in the 3′- 5′ direction.
Thus, the strand complementary to the 5′->3′ template strand (known as the lagging strand) is
synthesized in short segments known as Okazaki fragments.
On the lagging strand, primase builds an RNA primer in short bursts.
DNA polymerase is then able to use the free 3′ OH group on the RNA primer to synthesize DNA
in the 5′->3′ direction.
The RNA fragments are then removed and new deoxyribonucleotides are added to fill the
gaps where the RNA was present.
DNA ligase is then able to ligate the deoxyribonucleotides together, completing the synthesis
of the lagging strand.
Leading Strand Synthesis
The leading strand is defined as the DNA strand that is synthesized in the 5′->3′ direction in a
On this strand, DNA polymerase is able to synthesize DNA using the free 3′ OH group donated
by a single RNA primer (multiple RNA primers are not used) and continuous synthesis occurs
in the direction in which the replication fork is moving.
Proteins of DNA Replication (Enzymes)
DNA exists in the nucleus as a condensed, compact structure.
To prepare DNA for replication, a series of proteins aid in the unwinding and separation of the
double-stranded DNA molecule.
These proteins are required because DNA must be single-stranded before replication can
1. DNA Helicases
These proteins bind to the double stranded DNA and stimulate the separation of the two
2. DNA single-stranded binding proteins
These proteins bind to the DNA as a tetramer and stabilize the single-stranded structure that
is generated by the action of the helicases.
Replication is 100 times faster when these proteins are attached to the single-stranded DNA.
3. DNA Gyrase
This enzyme catalyzes the formation of negative supercoils that is thought to aid with the
In addition to these proteins, several other enzymes are involved in bacterial DNA replication.
4. DNA Polymerase
DNA Polymerase I (Pol I) was the first enzyme discovered with polymerase activity, and it is
the best characterized enzyme.
Although this was the first enzyme to be discovered that had the required polymerase
activities, it is not the primary enzyme involved with bacterial DNA replication.
That enzyme is DNA Polymerase III (Pol III).
Three activities are associated with DNA polymerase I;
5′ to 3′ elongation (polymerase activity)
3′ to 5′ exonuclease (proof-reading activity)
5′ to 3′ exonuclease (repair activity)
The second two activities of DNA Pol I are important for replication, but DNA Polymerase III
(Pol III) is the enzyme that performs the 5′-3′ polymerase function.
The requirement for a free 3′ hydroxyl group is fulfilled by the RNA primers that are
synthesized at the initiation sites by these enzymes.
6. DNA Ligase
Nicks occur in the developing molecule because the RNA primer is removed and synthesis
proceeds in a discontinuous manner on the lagging strand. The final replication product does
not have any nicks because DNA ligase forms a covalent phosphodiester linkage between 3’hydroxyl and 5′-phosphate groups.