PART II - MOLECULAR BIOLOGY
Module VII Nature of Genetic Materials
DNA REPLICATION
Genetic information present in double stranded DNA molecule is transmitted from
one cell to another cell at the time of mitosis and from parent to progeny by faithful
replication of parental DNA molecules. DNA molecule is coiled and twisted and has
enormous size. This imposes several restrictions on DNA replication. DNA molecule must be
uncoiled and the two strands must be separated for the replication process.
The main role of replication is to duplicate the base sequence of parent DNA
molecule. The two strands have complementary base pairing. Adenine of one strand pairs
with thymine of the opposite strand and guanine pairs with cytosine. This specific
complementary base pairing provides the mechanism for the replication.
The two strands uncoil and permanently separate from each other. Each strand
functions as a template for the new complementary daughter strand. The base sequence of
parent or old strand directs the base sequence of new or daughter strand. If there is adenine in
the parent or old strand, complementary thymine will be added to the new strand. Similarly, if
there is cytosine in the parent strand, complementary guanine will be copied into the new
daughter strand. Maintenance of integrity of genetic information is the main feature of
replication.
Mode of Replication
Watson and Crick proposed that each strand of double helical DNA serves as template
for synthesis of its complementary strand. Depending on whether the old strands (the
template strands) are conserved in the original double helix or not, the possible modes of
replication are
A. Conservative replication: In the conservative replication the two newly synthesized strands
come together to form a helix and the parental strands reassociate. This allows to conserve
the original helix.
B. Semiconservative replication:
Involves strand separation of parental DNA. After
replication in the two DNA duplexes, one new strand and one old strand associates.
C. Dispersive replication: In the dispersive replication the parental DNA is broken down into
smaller fragments. They are dispersed into two new double helixes following replication.
Hence each strand of duplex DNA consists of both old and new DNA. This mode of
replication cannot produce exact replicas.
DNA Replication is Semi-Conservative:
Watson and Crick model suggested that DNA replication is semi-conservative. It
implies that half of the DNA is conserved. Only one new strand is synthesized, the other
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strand is the original DNA strand (template) that is retained. Each parental DNA strand
serves as a template for one new complementary strand.
The new strand is hydrogen bonded to its parental template strand and forms double
helix. Each of these strands of the double helix contains one original parental strand and one
newly formed strand.
Meselson and Stahl Experiment:
Mathew Meselson and Franklin Stahl proved experimentally that parental strands of a
helix are distributed equally between the two daughter molecules. They made use of the
heavy isotope 15 N as a tag to differentially label the parental strands. E. coli was grown in a
medium containing 15 N labelled ammonium chloride (NH4 Cl). The heavy isotope of nitrogen
(15N) contains one more neutron than the naturally occurring 14 N isotope unlike unstable
radioactive isotopes, 15 N is stable.
In this way both strands of DNA molecules were labelled with radioactive heavy
isotope 15 N in their purines and pyrimidines. Therefore both strands were heavy or HH DNA.
The bacteria were then transferred into a medium containing the common non-radioactive
nitrogen 14 N, which is a light medium. It was found that after one cell division daughter
molecules had one 15 N strand the other 14 N strand. So this is a hybrid molecule, a heavy light
of HL DNA.
After the second cell division, out of four molecules, two DNA molecules contained
14 14
N N (LL). The other two were hybrid molecules 15 N 14 N (HL). This proves that during
replication, one parent strand is conserved and the other new strand is synthesized. Thus
DNA replication is a semi-conservative process.
Basic requirements for DNA Synthesis
A. Substrates: Deoxynucleoside triphosphates (dNTPs) are the substrate for DNA synthesis.
The four types of deoxynucleoside triphosphate are deoxy adenosine triphosphate (dATP),
deoxy guanosine triphosphate (dGTP), deoxycytidine triphposphate (dCTP) and
deoxythymidine triphosphate (dTTP). During the polymerization of nucleotides α and ß
phosphates are hydrolysed to get the energy.
B. Template: DNA replication cannot occur without a template. A macromolecular pattern
for the synthesis of an informational macromolecule is called template. The template guides
the addition of appropriate complementary nucleotide to the newly synthesized DNA strand
according to Watson – Crick base pairing. In semiconservative replication each strand of
parental DNA serves as template.
C. Primer: A primer is a segment of short nucleotides which is complementary to the
template, with a free 3‘ – hydroxyl group to which nucleotides can be added. So that
nucleotides are polymerized during DNA synthesis in 5‘ → 3‗direction. Most of the primers
are RNA primers. For starting DNA synthesis primer is required. This is because DNA
polymerases can only add nucleotides to a preexisting strand. Primers are generally
oligonucleotides if RNA synthesized by primases. Primer will not be found in the final DNA.
This is removed by polymerase I.
D. Proteins: Many enzymatic and nonenzymatic proteins are required for translation. They
are polymerases, helicases, topoisomerases, ligases, SSB proteins etc.
(i) DNA polymerases: Arthur Kornberg in 1955 characterized the DNA polymerase from E.
coli cells. Later E. coli cell was found to contain five types DNA polymerases. They are I, II,
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III, IV and V. More than 90% of the DNA polymerase activity observed in E. coli extracts
can be accounted for DNA polymerase I. It polymerises nucleotides specifically in 5‗ → 3‗
direction. The 3‗ →5‗ exonuclease activity remove wrong paired nucleotide added in the
replication. The activity is called proof reading. DNA polymerase II is involved in DNA
repair. DNA polymerase III is the principal replication enzyme of E. coli. NA polymerases
IV and V, identified in 1999 and are involved in an unusual form of DNA repair.
(ii) Helicases: The DNA synthesis process requires the separation of two parental strands.
This is carried out by the enzyme helicase (Dna B protein). They move along the DNA and
separate the stands using chemical energy from ATP.
(iii) Topoisomerases: The strand separation by helicases created topological stress in the
helical DNA, which is relieved by the action of topoisomerases. DNA topoisomerase II also
called DNA gyrase plays mojor role, not only removing supercoils but also in decatenating
DNA.
(iv) Primases: Primers are short segments of nucleotides, generally of RNA, to initiate
replication. These are synthesized by primases. Ultimately the RNA primers are removed and
replaced by DNA by the DNA polymerase I with its 5‗→ 3‗ exonuclease activity. After
removal of RNA primer the gap is filled with DNA.
(v) DNA ligases: The nicks in the polymerizing DNA back bone are sealed by the DNA
ligases. The nicks in okazaki fragments of lagging stand, the nick on circular DNA after
leading strand synthesis and the nick after primer removal, all are sealed yb ligases. The
energy required for sealing is obtained by the hydrolysis of NAD+ or ATP.
(vi) SSB proteins: Single strand binding (SSB) proteins binds as tetramer to the separated
DNA strands, to stabilize them. SSB proteins maintain DNA in unpaired form. The SSB
proteins are stripped off from DNA just before it can be replicated by polymerase.
The Replication Process
The replication of the DNA double helix is a complex process and it takes place in
five interlocking steps.
1. Opening up the DNA double helix. The very stable DNA double helix must be opened up
and its strands separated from each other for semiconservative replication to occur.
Stage one: Initiating replication. The binding of initiator proteins to the replication origin
starts an intricate series of interactions that opens the helix.
Stage two: Unwinding the duplex. After initiation, ―unwinding‖ enzymes called helicases
bind to and move along one strand, shouldering aside the other strand as they go.
Stage three: Stabilizing the single strands. The unwound portion of the DNA double helix is
stabilized by single-strand binding protein, which binds to the exposed single strands,
protecting them from cleavage and preventing them from rewinding.
Stage four: Relieving the torque generated by unwinding. For replication to proceed at 1000
nucleotides per second, the parental helix ahead of the replication fork must rotate 100
revolutions per second. To relieve the resulting twisting, called torque, enzymes known as
topisomerases—or, more informally, gyrases— cleave a strand of the helix, allow it to swivel
around the intact strand, and then reseal the broken strand.
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2. Building a primer. New DNA cannot be synthesized on the exposed templates until a
primer is constructed, as DNA polymerases require 3′ primers to initiate replication. The
necessary primer is a short stretch of RNA, added by a specialized RNA polymerase called
primase in a multisubunit complex informally called a primosome. .
3. Assembling complementary strands. Next, the dimeric DNA polymerase III then binds
to the replication fork. Synthesis of two strands is different. The strand synthesized in 5‗→ 3‗
direction is called leading strand and strand appear to be synthesized in 3‗→5‗ direction is
called lagging strand. While the leading strand complexes with one half of the polymerase
dimer, the lagging strand is thought to loop around and complex with the other half of the
polymerase dimer. Moving in concert down the parental double helix, DNA polymerase III
catalyzes the formation of complementary sequences on each of the two single strands at the
same time.
4. Removing the primer. The lagging strand synthesis is not a continuous process. It occurs
in short fragments called Okazaki fragments of 1-2 kb long. Each fragment synthesis need the
synthesis needs the synthesis of RNA primer by primase followed by binding of DNA
polymerase III to elongate the fragment in 5‗→ 3‗. The elongation of okazaki fragment is
completed when it encounters the 5‗–end of the previous fragment. The DNA polymerase I
removes the RNA primer and add deoxy nucleotides, and fills in the gap, as well as any gaps
between Okazaki fragments.
5. Joining the Okazaki fragments. After any gaps between Okazaki fragments are filled in,
the enzyme DNA ligase joins the fragments to the lagging strand.
Finally the two replication forks of the circular E. coli chromosome met at a region
called terminus region. This region contains 20 bp sequences called - ter sequences. A protein
called Tus (Terminus utilization substance) binds to Ter sequences. This complex terminated
the replication of E. coli. Sometimes the completely replicated two DNA circles are
topologically linked.
The two DNAs linked topologically are called catenanes. The two double stranded
DNAs are separated by topoisomerase II and IV. The separation of two DNA helices in
catenanes is called decatenation.
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