BIOCHEMISTRY
DNA REPLICATION
BY:
H.S.POPANIYA
Introduction
DNA carries genetic information from generation to generation.
It is responsible to preserve the identity of the species over
millions of years.
DNA may be regarded as Reserve bank of genetic information or
memory bank.
DNA is more stable than RNA. DNA is more suitable molecule for
long-term repository of genetic information.
The biological information flows from DNA to RNA, & from
there to proteins.
This is central dogma of life.
DNA in a cell must be duplicated (replicated), maintained &
passed down accurately to the daughter cells.
Replication of DNA
DNA is the genetic material. When the cell divides, the daughter
cells receive an identical copy of genetic information from the
parent cell.
Replication is a process in which DNA copies itself to produce
identical daughter molecules of DNA with high fidelity.
Replication is semiconservative:
The parent DNA has two strands complementary to each other.
Both the strands undergo simultaneous replication to produce two
daughter molecules.
Each one of the newly synthesized DNA has one-half of the parental
DNA (one strand from original) & one half of new DNA.
This is known as semiconservative replication - “half of the original
DNA is conserved in the daughter DNA”.
Initiation of Replication
The initiation of DNA synthesis occurs at a site called origin of
replication.
In prokaryotes, only one site, where as in eukaryotes, there are
multiple sites of origin. These sites mostly consist of a short
sequence of A-T base pairs.
A specific protein called dna A binds with the site of origin for
replication. This causes the double-stranded DNA to separate.
Replication Bubbles
Two complementary strands of DNA separate at the site of
replication to form a bubble.
Multiple replication bubbles are 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 (5-50
nucleotides) is required as a primer.
The enzyme primase (a specific RNA polymerase) in association with
single stranded binding proteins (SSBP) forms a complex called
primosome & produces RNA primers.
A constant synthesis & supply of RNA primers should occur on the
lagging strand of DNA.
On leading strand only one RNA primer is required.
DNA Synthesis Is Semidiscontinuous
And Bidirectional
The replication of DNA occurs in 5' to 3' direction, simultaneously,
on both strands of DNA.
Leading strand (continuous or forward):
The DNA synthesis is continuous.
Lagging strand (discontinuous):
The DNA synthesis is discontinuous, short pieces of DNA (15-250
nucleotides) are produced on lagging strand.
Replication fork
The separation of two strands of parent DNA results in the
formation of 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
DNA helicases bind to both the DNA strands at the replication
fork.
Helicases move along the DNA helix & separate the strands.
Their function is comparable with a zip opener.
Helicases are dependent on ATP for energy supply.
Single Stranded Binding (SSB) Proteins
Also called helix-destabilizing proteins. SSB proteins bind only to
single-stranded DNA.
They bind cooperatively the binding of one molecule of SSB
protein makes it easier for additional molecules of SSB protein to
bind tightly to the DNA strand.
These are not enzymes. These will provide single-stranded
template required by polymerases & also protects the DNA from
nucleases that degrades single stranded DNA.
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'
to 5' direction & they synthesize the new DNA strands in the 5' to
3' (anti parallel) direction.
The two newly synthesized nucleotide chains must grow in
opposite in the directions one in the 5' to 3' direction toward the
replication fork & one in the 5' to 3' direction away from the
replication fork.
Leading strand:
The strand that is being copied in the direction of the advancing
replication fork is called the leading strand & is synthesized
continuously.
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 joined to become a single, continuous strand.
This is called as lagging strand
DNA Polymerase III
Synthesis of a new DNA strand, catalysed by DNA polymerase III,
occurs in 5'-3' direction. This is antiparallel to the parent template
DNA strand.
The presence of all the four deoxyribonucleoside triphosphates
(dATP, dGTP, dCTP & 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'-3') towards
the replication fork which is continuous (Leading strand).
The other in a direction (5'- 3') away from the replication fork
which is discontinuous (Lagging strand).
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.
Chain Elongation
Prokaryotic & eukaryotic DNA polymerases elongate a new DNA
strand by adding deoxyribonucleotides, one at a time, to the 3'-end
of the growing chain.
The sequence of nucleotides that are added is dictated by the base
sequence of the template strand, with which the incoming
nucleotides are paired.
Okazaki Fragment
The small fragments of the discontinuously synthesized DNA are
called Okazaki pieces.
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 & 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'-3' directed catalytic function, DNA
polymerase III also has a proof-reading activity.
It checks the incoming nucleotides & allows only the correctly
matched bases to be added to the growing DNA strand.
DNA polymerase edits its mistakes & removes the wrongly placed
nucleotide bases.
For example, if the template base is cytosine & the enzyme
mistakenly inserts an adenine instead a guanine into the new chain,
the 3' to 5' exonuclease removes the misplaced nucleotide.
The 5' to 3' polymerase replaces it with the correct nucleotide
containing guanine.
Replacement of RNA Primer by DNA
The synthesis of new DNA strand continues till it is in close
proximity to RNA primer.
DNA polymerase I removes the RNA primer & takes its position.
DNA polymerase I catalyses the synthesis (5'-3' direction) 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 & the small fragments of DNA produced by DNA
polymerase I.
This process-nick sealing-requires energy, provided by the
breakdown of ATP. DNA polymerase II participates in the DNA
repair process.
Replication in Eukaryotes
Replication of DNA in eukaryotes closely resembles that of
prokaryotes. Certain differences exist.
Multiple origins of replication is a characteristic feature of
eukaryotic cell. Five distinct DNA polymerases are known in
eukaryotes.
DNA Polymerase
DNA polymerase α is responsible for the synthesis of RNA
primer for both the leading & lagging strands of DNA.
DNA polymerase β is involved in the repair of DNA.
Its function is comparable with DNA polymerase I found in
prokaryotes.
DNA polymerase γ participates in the replication of mitochondrial
DNA.
DNA polymerase δ is responsible for the replication on the
leading strand of DNA. It also possesses proof-reading activity.
DNA polymerase ε is involved in DNA synthesis on the lagging
strand & proofreading function
Thank You…
