REPLICATION OF DNA

In humans, the synthesis of a new strand of DNA occurs at the rate of about 3000
nucleotides per minute. In bacteria, about 30,000 nucleotides are added to a nascent
DNA chain per minute. Clearly, the cellular machinery responsible for DNA
replication must work very fast, but, even more importantly, it must work with great
accuracy. Indeed, the fidelity of DNA replication is amazing, with an average of only
one mistake per billion nucleotides incorporated after synthesis and the correction of
mistakes during and immediately after replication.
Semiconservative DNA Replication
DNA replication is the process of duplicating ds DNA. Experiments of Meselson and
Stahl showed that parental DNA strand could function as a template-a single strand
of DNA that specifies the nucleotide sequence of a new complementary strand.
During DNA replication:
• One parental ds DNA molecule is converted to 2 identical daughter DNA
molecules.
• When replication begins, the 2 strands of parental DNA are untwisted and
unwound (the two strands separated).
• The separation exposes the unpaired bases on both strand.
• Each parental strand could direct the synthesis of a new complementary
strand because of the specific base pairing.
This DNA replication model is known as the semiconservative model, because each
progeny molecule retains (“conserves”) one of the parental strands.

Requirements of Replication
Although the process of replication includes many components, they can be
combined into three major groups:
1. a template consisting of single-stranded DNA,
2. raw materials (deoxyribonucleoside triphosphates (dNTPs), each consisting of a
deoxyribose sugar and a base (a nucleoside) attached to three phosphate groups) to
be assembled into a new nucleotide strand, and
3. enzymes and other proteins that “read” the template and assemble the substrates
into a DNA molecule.
Mechanism of Replication
Replication takes place in four stages: initiation, unwinding, elongation, and
termination.
1. Initiation
The circular chromosome of E. coli has a single replication origin (oriC). The minimal
sequence required for oriC to function consists of 245 bp that contain several critical
sites. An initiator protein (known as DnaA in E. coli) binds to oriC and causes a
short section of DNA to unwind.
An AT-rich region and DnaA box
sequences are found within oriC. DNA
replication is initiated by the binding of
DnaA proteins to sequences within
the origin known as DnaA box
sequences. The DnaA box
sequences serve as recognition sites
for the binding of the DnaA proteins.
2. Unwinding
This binding causes the DNA to
twist and the puts torque on the
nearby AT-rich region to
denature and form a replication
bubble
3. AT base pairs are held together
by only 2 H bonds and CG base
pairs are held together by 3 H
bonds
 4. Therefore, AT-rich regions of DNA denature more easily than CG-rich regions
of DNA
Following separation of the AT-rich region, the DnaA proteins, with the help of the
DnaC protein, recruit DNA helicase proteins to this site. DNA helicase is also known
as DnaB protein.
When a DNA helicase encounters a double-stranded region, it breaks the hydrogen
bonds between the two strands, thereby generating two single strands. Two DNA
helicases begin strand separation within the oriC region and continue to separate the
DNA strands beyond the origin. These proteins use the energy from ATP hydrolysis
to catalyze the separation of the double stranded parental DNA. The process of
separation of double-stranded DNA to two single strands is called DNA denaturation
or DNA melting.
Single-strand DNA-binding (SSB) proteins bind to each single-stranded DNA,
stabilizing them and preventing them from reforming double-stranded DNA by
complementary base pairing (a process called reannealing).They also prevent the
formation of secondary structures such as hairpin. This unwinding generates
replication bubble. The point of unwinding where the 2 ss separate from the
dsDNA is called replication fork. The action of DNA helicases promotes the
movement of two replication forks outward from oriC in opposite directions. This
initiates the replication of the bacterial chromosome in both directions, an event
termed bidirectional replication.
DNA Gyrase Another protein essential for the unwinding process is the enzyme
DNA gyrase, a topoisomerase. Topoisomerases control the supercoiling of DNA. In
replication, DNA gyrase reduces the torsional strain (torque) that builds up ahead of
the replication fork as a result of unwinding. It reduces torque by making a double-
stranded break in one segment of the DNA helix, passing another segment of the
helix through the break, and then resealing the broken ends of the DNA. This action
removes a twist in the DNA and reduces the supercoiling.
A group of antibiotics called 4-quinolones kill bacteria by binding to DNA gyrase and
inhibiting its action. The inhibition of DNA gyrase results in the cessation of DNA
synthesis and bacterial growth. Many bacteria have acquired resistance to
quinolones through mutations in the gene for DNA gyrase.
5. Elongation
During the elongation phase of replication, single-stranded DNA is used as a
template for the synthesis of DNA. This process requires a series of enzymes.
Synthesis of primers each DNA helicase recruits the enzyme DNA primase
(encoded by the dnaG gene), forming a complex called the primosome. DNA
primase is important in DNA replication because DNA polymerases cannot initiate
the synthesis of a DNA strand; they can add nucleotides only to a preexisting strand.
All DNA polymerases require a nucleotide with a 3′-OH group to which a new
nucleotide can be added. Because of this requirement, DNA polymerases require a
primer which provides an existing 3′-OH group—to initiate the replication process.
An enzyme called primase synthesizes short stretches of nucleotides, or primers, to
get DNA replication started. Primase synthesizes a short stretch of RNA nucleotides
(about 10–12 nucleotides long) using DNA strand as template which provides a 3′-
OH group to which DNA polymerase can attach new DNA nucleotides. (Because
primase is an RNA polymerase, it does not require a free 3′-OH group to which new
nucleotides can be added to synthesise primer).
DNA synthesis by DNA polymerases After DNA is unwound and a primer has
been added, DNA polymerases elongate the new polynucleotide strand by adding
new nucleotides to the free 3’OH end provided by the primer. The best-studied
polymerases are those of E. coli, which has at least five different DNA polymerases.
DNA polymerase III is a large multiprotein complex that acts as the main workhorse
of replication. DNA polymerase III synthesizes nucleotide strands by adding new
nucleotides to the 3′ end of a growing DNA molecule. This enzyme has two
enzymatic activities. Its 5’→3’ polymerase activity allows it to add new nucleotides in
the 5’→3’direction. Its 3’→5’ exonuclease activity allows it to remove nucleotides in
the 3’→5’ direction, enabling it to correct errors. If a nucleotide having an incorrect
base is inserted into the growing DNA molecule, DNA polymerase III uses its 3’→5’
exonuclease activity to back up and remove the incorrect nucleotide. It then resumes
its 5’→3’ polymerase activity. These two functions together allow DNA polymerase III
to efficiently and accurately synthesize new DNA molecules.
DNA polymerases can synthesize DNA only in the 5’-to-3’ direction, hence, DNA is
made in opposite directions on the two template strands.
The new strand being made in the same direction as the movement of the replication
fork is the leading strand. Hence, the template strand that is exposed in 3’-5’
direction allows the new strand to be synthesized continuously in the 5’-3’ direction.
This strand which undergoes continuous replication is called the leading strand. The
other template strand is exposed in the 5’-3’ direction and its replication is more
complicated. This strand called lagging strand is made discontinuously in the
direction opposite that of the movement of the replication fork in short fragments
called okazaki fragments. The synthesis of each okazaki fragment (1000-2000bp)
requires a RNA primer to initiate the replication process and DNA polymerase III
then extends the primer by adding new nucleotides to its 3’ end.

In the leading strand, a single primer is made at the origin of replication. In the
lagging strand, multiple primers are made. Helicase untwists more DNA, causing
the replication fork to move along the chromosome. DNA gyrase (a form of
topoisomerase) relaxes the tension produced in the DNA ahead of the replication
fork. This tension is considerable because the replication fork rotates at about 3,000
rpm.
 6. Replication Is Terminated When the Replication Forks Meet at the Termination
Sequences
On the opposite side of the E. coli chromosome
from oriC is a pair of termination sequences
called ter sequences. A protein known as the
termination utilization substance (Tus) binds to the
ter sequences and stops the movement of the
replication forks. In any given cell, only one ter
sequence is required to stop the advancement of
one replication fork, and then the other fork ends its
synthesis of DNA when it reaches the halted
replication fork. In other words, DNA replication ends when oppositely advancing
forks meet, usually at T1 or T2.

DNA Ligase
Each new nucleotide added to the growing chain provides the 3’-oh gp needed for
the next DNA nucleotide to be added. This process continues as long as the
template is available. DNA polymerase I follows DNA polymerase III and, using its
5’→3’ exonuclease activity, removes the RNA primer. It then uses its 5’→3’
polymerase activity to replace the RNA nucleotides with DNA nucleotides. As a
result a single stranded nick is left between the two DNA fragments. The nicks
between okazaki fragments are then sealed by
DNA ligase enzyme. The DNA ligase catalyzes
the formation of a phosphodiester bond between
the 3’-OH and the 5’–phosphate groups on
either side of a nick, sealing the nick.
 Rolling-circle replication
1. Another model for replication is rolling circle, which takes place in some
bacteriophages, including ΦX174 and λ phages and in the F plasmids.
2. Rolling circle replication begins with a nick (single-stranded break) at the
origin of replication done by endonuclease enzyme. This creates a 3’-OH
group and a 5’- phosphate group.
3. New nucleotides are added to the 3’ end of the broken strand, with the inner
(unbroken) strand used as a template. As new nucleotides are added to the 3’
end, the 5’ end of the broken strand is displaced from the template, rolling out
like thread being pulled off a spool. The 3’ end grows around the circle, giving
rise to the name rolling-circle model.
4. The 5’ end continues to be displaced as the circle “rolls”, and is protected by
SSBs until discontinuous DNA synthesis makes it a dsDNA again.
 DNA POLYMERASE
All DNA polymerases from prokaryotes and eukaryotes catalyze the polymerization
of nucleotide precursors (dNTPs) into a DNA chain.
The reaction has three main features:
1. At the growing end of the DNA chain, DNA polymerase catalyses the formation of
a phosphodiester bond between the 3’-OH group of the deoxyribose on the last
nucleotide of already existing nucleotide chain and the 5’-phosphate of the dNTP
precursor. The energy for the formation of the phosphodiester bond comes from the
release of two of three phosphates from the dNTP.
2. At each step in lengthening the new DNA chain, DNA polymerase finds the correct
precursor dNTP that can form a complementary base pair with the nucleotide on the
template strand of DNA. Nucleotides are added rapidly—850 per second in E. coli
and 60–90 per second in human tissue culture cells.
The process does not occur with 100% accuracy, but the error frequency is
extremely low.
3. The direction of synthesis of the new DNA chain is only from 5’-to-3’.
One of the best understood systems of DNA replication is that of E. coli. For several
years after the discovery of DNA polymerase I, scientists believed that it was the
only DNA replication enzyme in E. coli. However, genetic studies disproved that
hypothesis. Scientists have now identified a total of five DNA polymerases, DNA Pol
I–V. Functionally, DNA Pol I and DNA Pol III are polymerases necessary for
replication, and
DNA Pol I, DNA Pol II, DNA Pol IV, and DNA Pol V are polymerases involved in DNA
repair.
The core DNA polymerase III contains the catalytic functions of the enzyme and
consists of three polypeptides: alpha, epsilon and theta. The complete DNA Pol III
enzyme, called the DNA Pol III holoenzyme, contains an additional six different
polypeptides.
Both DNA Pol I and DNA Pol III replicate DNA in the 5’-to-3’ direction. Both enzymes
also have 3’-to-5’ exonuclease activity, meaning that they can remove nucleotides
from the end of a DNA chain. This enzyme activity is used in error correction in a
proofreading mechanism. That is, if an incorrect base is inserted by DNA polymerase
(an event that occurs at a frequency of about 10 -6 for both DNA polymerase I and
DNA polymerase III, meaning that one base in a million is incorrect), in many cases
the error is recognized immediately by the enzyme. By a process resembling using a
backspace or delete key on a computer keyboard, the enzyme’s 3’-to-5’ exonuclease
activity excises the incorrect nucleotide from the new strand. Then, the DNA
polymerase resumes forward movement and inserts the correct nucleotide. With this
proofreading, the frequency of replication errors by DNA polymerase I or III is
reduced
to less than10-9.
DNA Pol I also has 5’-to-3’ exonuclease activity and can remove either DNA or RNA
nucleotides from the end of a nucleic acid strand. This activity is important in DNA
replication and is used to remove primers.