DNA replication

Why did DNA evolve as genetic material?

o RNA molecules, in principle, can perform the cellular functions that are
carried out by DNA. In fact, many viruses contain RNA as the genetic
material.
o Chemically, DNA is more stable than RNA.
o Hence, during the course of evolution, DNA is preferred as a more
suitable molecule for long-term repository of genetic information.
The central dogma of life
 REPLICATION OF DNA
Replication is a process in which DNA copies itself to produce identical daughter molecules of DNA.

 Replication is semiconservative:-

Replication is known as semiconservative since half of the original DNA is conserved in the daughter DNA. The first
experimental evidence for the semiconservative DNA replication was provided by Meselson and Stahl (1958).

 Initiation of replication:-
The initiation of DNA synthesis occurs at a site called origin of replication. In case of prokaryotes, there is a single site
whereas in eukaryotes, there are multiple sites of origin.

 Replication bubbles:-

The two complementary strands of DNA separate at the site of replication to form a bubble. Multiple replication bubbles are
formed in eukaryotic DNA molecules, which is essential for a rapid replication process.
 RNA primer:-

For the synthesis of new DNA, a short fragment of RNA (about 5-50 nucleotides, variable with species) is required as a
primer. The enzyme primase (a specific RNA polymerase) in association with single-stranded binding proteins forms a
complex called primosome, and produces RNA primers. A constant synthesis and supply of RNA primers should occur on
the lagging strand of DNA. This is in contrast to the leading strand which has almost a single RNA primer.
About Replication 5'

Lagging strand 3'

Leading strand
Replication bubble θ

3'
Lagging strand
3'
5'

θ (Theta) Replication
 (Sigma) Replication
 DNA synthesis is semidiscontinuous and bidirectional:-

The replication of DNA occurs in 5’ to 3’ direction, simultaneously, on both the strands of DNA. On one strand, the leading
(continuous or forward) strand—the DNA synthesis is continuous. On the other strand, the lagging (discontinuous or
retrograde) strand—the synthesis of DNA is discontinuous. Short pieces of DNA (15-250 nucleotides) are produced on the
lagging strand. In the replication bubble, the DNA synthesis occurs in both the directions (bidirectional) from the point of origin.

 Replication fork and DNA synthesis:-

The separation of the two strands of parent DNA results in the formation of a replication fork. The active synthesis of DNA
occurs in this region. The replication fork moves along the parent DNA as the daughter DNA molecules are synthesized.

 DNA helicases : These enzymes bind to both the DNA strands at the replication fork. Helicases move along the DNA helix
and separate the strands. Their function is comparable with a zip opener. Helicases are dependent on ATP for energy
supply.

 Single-stranded DNA binding (SSB) proteins : These are also known as DNA helix-destabilizing proteins. They possess no
enzyme activity. SSB proteins bind only to single-stranded DNA (separated by helicases), keep the two strands separate
and provide the template for new DNA synthesis. It is believed that SSB proteins also protect the single-stranded DNA
degradation by nucleases.
  DNA synthesis catalysed by DNA polymerase III:-

The synthesis of a new DNA strand, catalysed by DNA polymerase III, occurs in 5’ to 3’ direction. This is antiparallel to the parent
template DNA strand. The presence of all the four deoxyribonucleoside triphosphates (dATP, dGTP, dCTP and dTTP) is an
essential prerequisite for replication to take place.

The synthesis of two new DNA strands, simultaneously, takes place in the opposite direction—one is in a direction (5’ to 3’)
towards the replication fork which is continuous, the other in a direction (5’ to 3’) away from the replication fork which is
discontinuous.
The incoming deoxyribonucleotides are added one after another, to 3’ end of the growing DNA chain. A molecule of
pyrophosphate (PPi) is removed with the addition of each nucleotide. The template DNA strand (the parent) determines the
base sequence of the newly synthesized complementary DNA.
 Polarity problem:-
The DNA strand (leading strand) with its 3’-end (3’-OH) oriented towards the fork can be elongated by sequential addition of
new nucleotides. The other DNA strand (lagging strand) with 5’-end presents some problem, as there is no DNA polymerase
enzyme (in any organism) that can catalyse the addition of nucleotides to the 5’ end (i.e. 3’ to 5’ direction) of the growing
chain. This problem however is solved by synthesizing this strand as a series of small fragments. These pieces are made in the
normal 5’ to 3’ direction, and later joined together.

 Okazaki fragments : The small fragments of the discontinuously synthesized DNA are called Okazaki fragments. These are
produced on the lagging strand of the parent DNA. Okazaki pieces are later joined to form a continuous strand of DNA.
DNA polymerase I and DNA ligase are responsible for this process.

 Proof-reading function of DNA polymerase III:-

Fidelity of replication is the most important for the very existence of an organism. Besides its 5’ to 3’ directed catalytic
function, DNA polymerase III also has a proof-reading activity. It checks the incoming nucleotides and allows only the
correctly matched bases (i.e. complementary bases) to be added to the growing DNA strand. Further, DNA polymerase edits
its mistakes (if any) and removes the wrongly placed nucleotide bases.
 Replacement of RNA primer by DNA:-

The synthesis of new DNA strand continues till it is in close proximity to RNA primer. Now the DNA polymerase I comes into
picture. It removes the RNA primer and takes its position. DNA polymerase I catalyses the synthesis of a fragment of DNA
that replaces RNA primer.

The enzyme DNA ligase catalyses the formation of a phosphodiester linkage between the DNA synthesized by DNA
polymerase III and the small fragments of DNA produced by DNA polymerase I. This process—nick sealing-requires
energy, provided by the breakdown of ATP to AMP and PPi.
  Supercoils and DNA topoisomerases:-

As the double helix of DNA separates from one side and replication proceeds, supercoils are formed at the other side. The
formation of supercoils can be better understood by comparing DNA helix with two twisted ropes tied at one end. Hold the
ropes at the tied end in a fixed position. And let your friend pull the ropes apart from the other side. The formation of
supercoils is clearly observed.

Type I DNA topoisomerase cuts the single DNA strand (nuclease activity) to overcome the problem of supercoils and then
reseals the strand (ligase activity). Type II DNA topoisomerase (also known as DNA gyrase) cuts both strands and reseals
them to overcome the problem of supercoils.
 REPLICATION IN EUKARYOTES

Replication of DNA in eukaryotes closely resembles that of prokaryotes. Certain differences, however, exist. Multiple origins
of replication is a characteristic feature of eukaryotic cell. Further, at least five distinct DNA polymerases are known in
eukaryotes. Greek letters are used to number these enzymes.

The differences in the DNA replication between bacteria and human cells, attributed to the enzymes, are successfully used in
antibacterial therapy to target pathogen (bacterial) replication and spare the host (human) cells.
 PROCESS OF REPLICATION IN EUKARYOTES

The replication on the leading (continuous) strand of DNA is rather simple, involving DNA polymerase δ and a sliding clamp
called proliferating cell nuclear antigen (PCNA). PCNA is so named as it was first detected as an antigen in the nuclei of
replicating cells. PCNA forms a ring around DNA to which DNA polymerase δ binds. Formation of this ring also requires
another factor namely replication factor C (RFC).

The replication on the lagging (discontinuous) strand in eukaryotes is more complex when compared to prokaryotes or even
the leading strand of eukaryotes.

The parental strands of DNA are separated by the enzyme helicase. A single-stranded DNA binding protein called replication
protein A (RPA) binds to the exposed single-stranded template. This strand has been opened up by the replication fork (a
previously formed Okazaki fragment with an RNA primer).

The enzyme primase forms a complex with DNA polymerase α which initiates the synthesis of Okazaki fragments. The
primase activity of pol -primase complex is capable of producing 10-bp RNA primer. The enzyme activity is then switched
from primase to DNA polymerase α which elongates the primer by the addition of 20–30 deoxyribonucleotides. Thus, by the
action of pol α-primase complex, a short stretch of DNA attached to RNA is formed. And now the complex dissociates from
the DNA.
The next step is the binding of replication factor C (RFC) to the elongated primer (short RNA-DNA). RFC serves as a clamp
loader, and catalyses the assembly of proliferating cell nuclear antigen (PCNA) molecules. The DNA polymerase δ binds to
the sliding clamp and elongates the Okazaki fragment to a final length of about 150–200 bp. By this elongation, the
replication complex approaches the RNA primer of the previous Okazaki fragment.
The RNA primer removal is carried out by a pair of enzymes namely RNase H and flap endonuclease I (FENI). This gap
created by RNA removal is filled by continued elongation of the new Okazaki fragment (carried out by polymerase δ). The
small nick that remains is finally sealed by DNA ligase.

Eukaryotic DNA is tightly bound to histones (basic proteins) to form nucleosomes which, in turn, organize into
chromosomes. During the course of replication, the chromosomes are relaxed and the nucleosomes get loosened. The DNA
strands separate for replication, and the parental histones associate with one of the parental strands. As the synthesis of
new DNA strand proceeds, histones are also produced simultaneously, on the parent strand. At the end of replication, of the
two daughter chromosomal DNAs formed, one contains the parental histones while the other has the newly synthesized
histones.
 An outline of DNA replication on the lagging strand in eukaryotes

(RPA–Replication protein A; PCNA–Proliferating cell nuclear antigen; RFC–Replication factor C; RNase H–Ribonuclease H; FENI–
Flap endonuclease I; Note : Leading strand not shown)
 TELOMERES AND TELOMERASE

There are certain difficulties in the replication of linear DNAs (or chromosomes) of eukaryotic cells. The leading strand of DNA
can be completely synthesized to the very end of its template. This is not possible with the lagging strand, since the removal of
the primer RNA leaves a small gap which cannot be filled. Consequently, the daughter chromosomes will have shortened DNA
molecules. This becomes significant after several cell cycles involving replication of chromosomes. The result is that over a
period of time, the chromosomes may lose certain essential genes and the cell dies. This is however, avoided to a large extent.

Telomeres are the special structures that prevent the continuous loss of DNA at the end of the chromosomes during the
course of replication. Thus, they protect the ends of the chromosomes, and are also responsible to prevent the chromosomes
from fusing with each other. Telomeres are many repeat sequences of six nucleotides present at the ends of eukaryotic
chromosomes. Human telomeres contain thousands of repeat TTAGGG sequences, which can be up to a length of 1500 bp.

Replication of DNA with telomeres: Formation of telomere

Telomerase is highly active in the early embryo, and after birth it is active in the reproductive and stem cells. Stem cells
divide continuously throughout the lifetime of an organism to produce new cells. These cells in turn are responsible to
tissues and organs in the functional state e.g. hematopoietic stem cells of bone marrow.

Many biologists link the process of telomere shortening with cell senescence (i.e. cell death). This is mainly based on the
observations made in the in vitro mammalian cell cultures. However, some researchers question this relation between telomere
shortening and senescence.

Cancerous cells are able to divide continuously. There is a strong evidence to suggest that the absence of senescence in cancer
cells is linked to the activation of the enzyme telomerase. Thus, telomere length is maintained throughout multiple cell
divisions. It is however, not clear whether telomerase activation is a cause or an effect of cancer. There is however, evidence to
suggest that telomerase activation is in fact the cause of certain cancers e.g. dyskeratosis congenita due to a mutation in the
gene responsible for the RNA component of telomerase.

The enzyme telomerase is an attractive target for cancer chemotherapy. The drugs have been designed to inactivate
telomerase, and consequently induce senescence in the cancer cells. This in turn prevents the rapid cell proliferation.
 SUMMARY
1. The central dogma of life revolves around the flow of information from DNA to RNA, and from there to proteins.

2. Replication is a process in which DNA copies itself to produce identical daughter molecules of DNA. DNA replication is
semiconservative, bidirectional and occurs by the formation of bubbles and forks.

3. Prokaryotic DNA syntheis is catalysed by the enzyme DNA polymerase III. This enzyme possesses proof-reading activity and
edits the mistakes that might occur during nucleotide incorporation.

4. Replication in eukaryotes (particularly on the lagging strand) is more complex and involves several factors e.g. replication
protein A, replication factor C, flap endonuclease.

5. Telomeres (repeat TTAGGG sequences) are the special structures that prevent the continuous loss of DNA at the end of
the chromosome during the course of replication.