DNA Replication

Arif Rafid
B.Pharm (JU), MS in Chemistry (LU, Texas)

Lecturer, Department of Pharmacy

Southeast University
DNA Replication
Naturally occurring DNA exists in many forms. Single- and double-
stranded DNAs are known, and both can exist in linear and circular
forms. As a result, it is difficult to generalize about all possible
cases of DNA replication.
Since many DNAs are double-stranded, it is convenient to present
some general features of the replication of double-stranded DNA
that apply to both linear and circular DNA. It is also more
convenient to start the discussion with replication of prokaryotic
DNA, especially of that of E. coli, and then compare it with the
replication of eukaryotic DNA.
The process by which one double-helical DNA molecule is
duplicated to produce two such double-stranded molecules is
complex.
The cell faces three important challenges in carrying out the
necessary steps.
• The first challenge is separating the two DNA strands. The two
strands of DNA are wound around each other in such a way
that they must be unwound if they are to be separated. the cell
also must protect the unwound portions of DNA from the
action of nucleases that preferentially attack single-stranded
DNA.
• The second task involves synthesizing of DNA from the 5' to
the 3' end. Two antiparallel strands must be synthesized in the
same direction on antiparallel templates. In other words, the
template has one 5' → 3' strand and one 3' → 5‘ strand, as does
the newly synthesized DNA.
• The third task is guarding against errors in replication, ensuring
that the correct base is added to the growing polynucleotide
chain.
DNA replication is “semiconservative”

Figure 1: Labeling pattern of

15N strands in
semiconservative replication.
G0 indicates original strands;
G1 indicates new strands after
the first generation; G2
indicates new strands after the
second generation.
Figure 2: Experimental
evidence for semiconservative
replication. Heavy DNA
labeled with 15N forms a band
at the bottom of the tube, and
light DNA with 14N forms a
band at the top. DNA that
forms a band at an
intermediate position has one
heavy strand and one light
strand.
Direction of Replication
During replication, the DNA double helix unwinds at a specific
point called the origin of replication (OriC in E. coli). New
polynucleotide chains are synthesized using each of the exposed
strands as a template.
It has been established that DNA synthesis is bidirectional in most
organisms, with the exception of a few viruses and plasmids. Both
new polynucleotide chains are synthesized in the 5' → 3' direction.
Figure 3: Replication in prokaryotes and eukaryotes
Semidiscontinuous DNA Replication
All synthesis of nucleotide chains occurs in the 5' → 3' direction
from the perspective of the chain being synthesized. This is due to
the nature of the reaction of DNA synthesis. The last nucleotide
added to a growing chain has a 3'-hydroxyl on the sugar. The
incoming nucleotide has a 5'-triphosphate on its sugar.
The 3'-hydroxyl group at the end of the growing chain is a
nucleophile. It attacks the phosphorus adjacent to the sugar in the
nucleotide to be added to the growing chain, leading to the
elimination of the pyrophosphate and the formation of a new
phosphodiester bond.
Figure 4: The addition of a
nucleotide to a growing DNA
chain. The 3'-hydroxyl group at
the end of the growing DNA
chain is a nucleophile. It
attacks at the phosphorus
adjacent to the sugar in the
nucleotide, which is added to
the growing chain.
Pyrophosphate is eliminated,
and a new phosphodiester
bond is formed.
How can replication proceed along the DNA if the
two strands are going in opposite directions?
The problem is solved by different modes of polymerization for
the two growing strands. One newly formed strand (the leading
strand) is formed continuously from its 5' end to its 3' end at the
replication fork on the exposed 3' to 5' template strand.
The other strand (the lagging strand) is formed
semidiscontinuously in small fragments (sometimes called the
Okazaki fragments), typically 1000 to 2000 nucleotides long). The
fragments of the lagging strand are then linked together by an
enzyme called DNA ligase.
Figure 5: The semidiscontinuous model for DNA replication. Newly synthesized DNA is shown in red.
Because DNA polymerases only polymerize nucleotides 5’→3’ both strands must be synthesized in the
5’→3’ direction. Thus, the copy of the parental 3’→5’ strand is synthesized continuously; this newly made
strand is designated the leading strand.
Problem
The following two compounds have been widely used in the
treatment of AIDS. Can you propose a mechanism based on your
knowledge so far?

Figure 6: 3'-azido-3'-
deoxythymidine, AZT
(Zidovudine); and 2'-
3'- dideoxyinosine,
DDI (Didanosine)
DNA Polymerases
DNA polymerases are the group of enzymes responsible for
catalyzing DNA replication and repair.
At least five DNA polymerases are present in E. coli. All of these
polymerases add nucleotides to a growing polynucleotide chain
but have different roles in the overall replication process.
Polymerase I consists of a single polypeptide chain, but
polymerases II and III are multisubunit proteins that share some
common subunits.
Polymerase II is not required for replication; rather, it is strictly a
repair enzyme. Recently, two more polymerases, Pol IV and Pol V,
were discovered. They, too, are repair enzymes, and are involved
in a unique repair mechanism called the SOS response.
Requirement for RNA Primer
If DNA polymerases are added to a single-stranded DNA template
with all the deoxyribonucleoside triphosphates (dNTP) necessary
to make a strand of DNA, no reaction occurs. It was discovered
that DNA polymerases cannot catalyze de novo synthesis.
DNA polymerases require the presence of a primer, a short
oligonucleotide strand to which the growing polynucleotide chain
is covalently attached in the early stages of replication.
In essence, DNA polymerases must have a nucleotide with a free
3'-hydroxyl already in place so that they can add the first
nucleotide as part of the growing chain. In natural replication,
this primer is RNA.
DNA polymerase reaction requires all four deoxyribonucleoside
triphosphates (dNTP) i.e. dTTP, dATP, dGTP, and dCTP. Mg2+ and a
DNA template are also necessary. Because of the requirement for
an RNA primer, all four ribonucleoside triphosphates (NTPs) i.e.
ATP, UTP, GTP, and CTP - are needed as well; they are
incorporated into the primer.
Exonuclease Activity of DNA Polymerases Is
Required for Proofreading and DNA Repair
It is now known that DNA polymerase I has a specialized function
in replication – repairing and “patching” DNA—and that DNA
polymerase III is the enzyme primarily responsible for the
polymerization of the newly formed DNA strand.
The major function of DNA polymerases II, IV, and V is as repair
enzymes.
The exonuclease activities are part of the proofreading-and-repair
functions of DNA polymerases, a process by which incorrect
nucleotides are removed from the polynucleotide so that the
correct nucleotides can be incorporated.
The 3' → 5' exonuclease activity, which all three polymerases
possess, is part of the proofreading function; incorrect nucleotides
are removed in the course of replication and are replaced by the
correct ones. Proofreading is done one nucleotide at a time.
The 5' → 3‘ exonuclease activity clears away short stretches of
nucleotides during repair, usually involving several nucleotides at a
time. This is also how the RNA primers are removed.
Proteins Required for DNA Replication
DNA replication is carried out in all organisms by a multiprotein
complex called the replisome. In bacteria, the replisome consists
of 13 different proteins that work together to synthesize DNA on
the leading and lagging strands.
Supercoiling and Replication
The prokaryotic DNA is negatively supercoiled in its natural state;
however, opening the helix during replication would introduce
positive supercoils ahead of the replication fork.
If the replication fork continued to move, the torsional strain of
the positive supercoils would eventually make further replication
impossible.
DNA gyrase fights these positive supercoils by putting negative
supercoils ahead of the replication fork.

A helix- destabilizing protein, called a helicase, promotes

unwinding by binding at the replication fork. (DNA helicase is the
enzyme that unwinds the DNA double helix by breaking the
hydrogen bonds down the center of the strand.) A number of
helicases are known, including the dnaB protein and the rep
protein.
Figure 7: General features of a replication fork
Single-stranded DNA is protected
long enough for replication by SSBs
Single-stranded regions of DNA are very susceptible to
degradation by nucleases.
The single-strand binding protein (SSB), stabilizes the single-
stranded regions by binding tightly to these portions of the
molecule.
The presence of this DNA-binding protein protects the single-
stranded regions from hydrolysis by the nucleases.
The Primase Reaction
One of the great surprises in studies of DNA replication was the
discovery that RNA serves as a primer in DNA replication. In
retrospect, it is not surprising at all, because RNA can be formed
de novo without a primer, even though DNA synthesis requires a
primer – a phenomena that supports the RNA World Hypothesis.

It is the enzyme primase, which is a type of RNA polymerase, is

responsible for copying a short stretch of the DNA template
strand to produce the RNA primer sequence.
Synthesis and Ligation
The synthesis of two new strands of DNA is begun by DNA
polymerase III. The newly formed DNA is linked to the 3'-hydroxyl
of the RNA primer, and synthesis proceeds from the 5' end to the
3' end on both the leading and the lagging strands.

As the replication fork moves, the RNA primer is removed by DNA

polymerase I, using its exonuclease activity. The primer is replaced
by deoxyribonucleotides, also by DNA polymerase I using its
polymerase activity.
How many DNA polymerases actually
participate in replication process?
For decades the dogma has been that the replisome contains two
molecules of Pol III, one for the leading strand and one for the
lagging strand.
While this was intuitive, a couple of studies from 2010–2012 have
shown that the stoichiometry is actually that three Pol III enzymes
are involved, as shown in Figure 7.
By labeling specific molecules at the replisome with fluorescent
markers and watching the location of bound proteins, researchers
were able to show that three molecules are bound at or near the
replisome.
By comparing locations of Pol III and single-stranded binding
proteins, they concluded that a new Pol III is used for each Okazaki
fragment, and that the replisome has one Pol III dedicated to
leading strand synthesis and two dedicated to lagging strand
synthesis.
Long Story Short
“Each of your cells knows how to replicate DNA, yet
you have to spend hours of studies to understand it.”

References & Further Studies

 Biochemistry by Campbell, Farrell, and McDougal, 9th Ed.