DNA REPLICATION

DNA replication is the physiologic process by which DNA is doubled. Replication takes place
within dividing cells and allows cells to generate new genetic material (DNA) using the original
strand as a template. Generally, replication follows three stages: initiation, elongation and
termination. DNA replication is highly regulated and coordinated and involves a number key
enzymes: helicase, primase, ligase, topoisomerase and DNA polymerase. DNA replication is
considered a semi-conservative process for the following reason: One strand is formed from the
original molecule while the other is newly synthesized. The two strands of the double helix
separate and are used as template (parent strands) for formation of new strands. New strands
are formed by addition of nucleotides, one by one and then linking them together. The result is
two DNA molecules both made of one original strand and one newly synthesized strand
Initiation:
Replication cannot begin and proceed until first, the double helical strands unwind and
separate by removal of hydrogen bonds holding the strands together. DNA helicase enzyme
accomplishes the unwinding and separation of strands. It starts by disconnecting DNA strands
in a region rich in Adenine (A) and Thymine (T). Since these bases have only two hydrogen
bonds, instead of three, it is the ideal place for helicase to start unwinding strands. Unwinding
creates a replication fork that has a leading and a lagging strand, a result of the antiparallel
nature of the strands.
Helicases are enzymes that utilize ATP as source of energy in order to break down hydrogen
bonds between complementary bases allowing the two strands to be separated. ATP is used to
move the helicase along the DNA molecule.
Elongation:
DNA polymerase links nucleotides together using pre-existing strand as a template thus forming
a new strand and DNA molecule. DNA polymerase moves along DNA strand in the same
direction adding one nucleotide after another. For DNA polymerase to start synthesizing a new
strand from 5’ to 3’ direction, it requires a short RNA sequence (10 nucleotides in length) called
RNA primer. The RNA primer is generated with the help of RNA primase enzyme to initiate
placing RNA bases complementary to the parent strand bases. Multiple RNA primers are
needed for the lagging strand which is then used by DNA polymerase to begin elongation phase
of DNA replication. DNA replication is high fidelity (low chances of error) due to ability of DNA
polymerase to detect, remove and fix any errors in replication process.

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On the leading strand, DNA continues in constant motion while the lagging strand is copied in
short segments in the opposite direction since DNA polymerase only codes in 5’ to 3’ direction.
These short repetitive lagging fragments are called Okazaki fragments.
Termination:
The process of expanding new DNA strands continues until there is no more DNA template left
to replicate (at end of chromosome) or the two replications forks meet and consequently
terminate. The meeting is random and not regulated. Once the Okazaki fragments synthesis is
complete the RNA primer is no longer required and must be eliminated and replaced with a
proper DNA sequence. Two enzymes are important: Flap endonuclease 1 (FEN1) and RNase H
remove this RNA primer. The gap is filled by Polymerase delta enzyme that uses parental DNA
strand as template to add remaining bases. The enzyme DNA ligase connects the Okazaki
fragments on the lagging strand by creating phosphodiester bonds. The generation of these
bonds is the final step in DNA replication resulting in formation of two new daughter DNA
double helices.

TRANSCRIPTION
A process by which RNA copy is synthesized from the genes’ DNA base sequence. This copy is
called messenger RNA (mRNA) which carries the genes’ protein information. The sequences of
bases in a gene, in itself, does not give any observable characteristics. The function of the gene
is to specify the sequence f amino acids in a particular polypeptide. These proteins then directly
or indirectly determine observable characteristics of an individual. Transcription requires the
action of the enzyme RNA polymerase to generate a single strand RNA from one of the parent
DNA strands. The RNA polymerase can initiate RNA synthesis without a primer. Transcription
process is not as accurate as replication. This implies that there are a number of errors since
RNA polymerase lacks proof reading ability exhibited by DNA polymerase.

Initiation: To initiate transcription, RNA polymerase must first identify the gene to be
transcribed, the correct strand of the double strand DNA to be copied and then bind to the
specific sequence on the DNA called the promoter. A strand of DNA is read by RNA polymerase
in the 3’ to 5’ direction and its RNA transcript is synthesized in the 5’ to 3’ direction. The
promoter region is known as TATA box due to presence of high frequency of Adenine and
Thymine nucleobases.

Elongation: Once RNA polymerase binds to the gene to be transcribed, it separates the double
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helix and synthesizes RNA in 5’ to 3’ direction. The RNA polymerase places complementary
bases to the template strand. Instead of Thymine, the polymerase introduces a new base called
Uracil (U). This forms the elongation phase as the polymerase continues down the strand to
create complementary single stand RNA. Elongation end at the hairpin loops called termination
sequence which causes the polymerase to fall off.

Termination: Once synthesized, RNA undergoes post transcriptional modification to prevent its
degradation during the exit from nucleus to the cytoplasm from where it will be translated in to
protein. The single strand RNA receives 5’ capping by 7-methylguanosine and addition of poly-A
tail on the 3’ end. Newly synthesized RNA travels through nuclear pores to the cytoplasm in
order to be translated into proteins required by the cell.

TRANSLATION
Messenger RNA carries information required for the synthesis of proteins. Translation is the
synthesis of polypeptides with amino acid sequence determined by the base sequence of the
messenger RNA. This is the second of the two processes required to produce a specific
polypeptide and it occurs in specific organelles called ribosomes. The amino acid sequence of
polypeptides is determined by the length of mRNA according to the genetic code transcribed.
There are four different bases and twenty different amino acids. The sequence of three bases is
called a codon. Each codon codes for a specific amino acid to be added to the polypeptide.
Amino acids are carried on another type of RNA called transfer RNA (tRNA). Transfer RNA has
three base anticodons complementary to the mRNA codon for a particular amino acid.

The DNA code is a triplet code. Each triplet - a group of three bases codes for a specific amino
acid. This code determines the type of amino acids and the order in which they are joined to
make a specific protein. The sequence of amino acids in a protein determines the structure and
function. The starting region of mRNA sequence always begins with a START codon; AUG and
terminates with STOP codon. Codons of three bases on mRNA correspond to one amino acid in
a peptide. The triplet of bases on the tRNA is known as an anticodon.
Example: UGG codes for amino acid Tryptophan, GGC codes for Glycine, UCA for amino acid
serine.
process of translation: Ribosomes bind to mRNA in the cytoplasm and move along the
molecule in 5’ to 3’ direction until it reaches a START codon (AUG). The anticodons on tRNA
align opposite appropriate codons according to complementary base pairing e.g. AUG = UAC.
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Each tRNA carries a specific amino acid according to the genetic code. Ribosomes catalyze the
formation of peptide bonds between adjacent amino acids. The ribosome moves along the
mRNA molecule synthesizing a polypeptide chain until it reaches STOP codon. At this point,
translation ceases and polypeptides are released.

THE NEUROMUSCULAR JUNCTION

Introduction
The ability to move purposely is an essential feature of life. The neuromuscular junction is the
site where the central nervous system (CNS) interacts with skeletal muscle fibers and causes
them to contract. The neuromuscular junction (abbreviated NMJ) is a synaptic connection
between the terminal end of a motor neuron and a skeletal muscle. The role of NMJ is to
convert temporal sequence of action potentials in motor neurons into muscle contractions.
Clinically, NMJ is important since it’s the site of action of many pharmacological drugs as well as
focal point for many diseases characterized by impaired neuromuscular transmission. Diseases
affecting the neuromuscular junction often produce specific symptoms including muscle
weaknesses, incoordination and loss of reflexes (areflexia). Pathophysiologically, there are
three important diseases that involve the neuromuscular junction: Myasthenia Gravis (MG),
Botulism and Lambert-Eaton Syndrome (LES)

Structure of neuromuscular junction

Structurally, the neuromuscular junction consists of presynaptic nerve terminal, postsynaptic
motor endplate and the synaptic cleft.

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Figure 1: Structure of Neuromuscular Junction. Adapted from Diseases of Neuromuscular
Junction; www.researchgate.net

Presynaptic nerve terminal: A myelinated motor neuron on reaching the target muscle loses its
myelin sheath and forms a complex of branching nerve endings called nerve terminals or nerve
buttons. The nerve terminal membrane develops areas of membrane thickening called the
active zones. The active zones are rich in proteins and rows of voltage-gated calcium (Ca)
channels. Also present at the nerve terminal are membrane potassium (K) channels,
mitochondria, endoplasmic reticulum and synaptic vesicles. The synaptic vesicles (SV) store
acetylcholine (ACh), the neurotransmitter at the neuromuscular junction. The synaptic vesicles
are concentrated around the active zones. The membrane of the synaptic vesicle has two
critical proteins: synaptobrevin and synaptotagmin which are essential for fusion and docking
of synaptic vesicles at the active zone. The arrival of the active potential at the nerve terminal
stimulates the opening of voltage-gated calcium channels and subsequent influx of calcium ions
(Ca2+). Increased calcium in the nerve terminal leads to docking of synaptic vesicles at active
zones and exocytotic release of acetylcholine from synaptic vesicles into the synaptic cleft.

Synaptic cleft: also called the junctional cleft, it is a space between the nerve terminal and
plasma membrane of the target muscle. In to this cleft, acetylcholine is released before it

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interacts with nicotinic Acetylcholine receptors at the motor endplate. Also present at the
synaptic cleft is an important enzyme called acetylcholinesterase responsible for catabolism of
acetylcholine so that its effect on postsynaptic receptors is not prolonged.

Motor endplate: the motor endplate forms the post-synaptic part of the neuromuscular
junction. The target muscle plasma membrane (also called sarcolemma) thickens to form motor
endplate. The thickening of the sarcolemma forms depressions called junctional folds. The
terminal nerve endings do not penetrate the moor endplate but they fit into the junctional
folds. The junctional folds have nicotinic acetylcholine receptors. These receptors are
acetylcholine gated ion channels. Binding of acetylcholine onto these receptors opens the
channels allowing influx of sodium ions (Na +) from extracellular fluid in to the muscle
membrane. This influx changes the postsynaptic membrane potential from -90mV to -45Mv.
The decrease in membrane potential s called endplate potential. In the neuromuscular junction,
endplate potential is strong enough to generate and transmits action potential to the muscle
membrane (sarcolemma). This results in muscle contraction. To prevent sustained
depolarization and muscle contraction and to allow for repolarization, acetylcholine unbinds
form the receptors and is metabolized by acetylcholinesterase, breaking it into choline and
acetate. Choline is taken back into the nerve terminal by specific uptake protein and reused for
synthesis of acetylcholine within the nerve terminal.

References
Ratiliff W. A., SayKally N. J., Kane M. J., Citron B. A.: Neuromuscular Junction Morphology and
Gene Dysregulation in the Wobbler Model of Spinal Neurodegeneration. J. Mol Neurosci. 2018
Sep; 66 (1) 114 – 120.

Hirsch N. P. Neuromuscular junction in Health and Disease. Br J Anaesth. 2007 Jul;99(1):132–

138

Slater C. R. The Structure of Human Neuromuscular Junctions: Some unanswered molecular

questions. Int J Mol Sci. 2017 Oct;18(10)

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Discuss Myasthenia Gravis and Lambert-Eaton Syndrome
Pathophysiologically, there are three important diseases that involve the neuromuscular
junction and are characterized by impaired neuromuscular transmission: Myasthenia Gravis
(MG), Botulism and Lambert-Eaton Syndrome (LES).

Myasthenia Gravis: it is an autoimmune disease (type II hypersensitivity reaction) resulting the

production of auto-antibodies against acetylcholine receptors located at the post-synaptic
endplate. The auto-antibodies decrease the number and distribution of acetylcholine receptors
thus decreasing the effect of endogenous acetylcholine thus preventing generation,
transmission of endplate potential and muscle contraction. Consequently, muscle weakness
occurs. The effects are more severe in the extraocular muscles that generally are in constant
use and have low density of acetylcholine receptors.

Treatment of Myasthenia Gravis aims to increase the levels of acetylcholine in the synaptic cleft
through the use of acetylcholinesterase inhibitors. These prevent breakdown of acetylcholine
at the synaptic cleft and out-compete the antibodies. Examples of acetylcholinesterase
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inhibitors are Neostigmine, Edrophonium and Physostigmine. Edrophonium has a limitation of
being short acting. Thus, it’s not used for treatment but in supporting diagnosis of Myasthenia
Gravis. If muscle and extra-ocular muscle weakness diminishes on administration of
Edrophonium a positive diagnosis may be proposed. Physostigmine is also not used for
treatment because its is a lipid soluble amine that easily crosses the blood brain barrier.

Additional medical management approaches for Myasthenia Gravis include: use of steroids
(prednisone) and steroid sparing agents (azathioprine, cyclophosphamide, and tacrolimus).
Rituximab, an anti- B cell monoclonal antibody may also be used in refractory patients.
Complications of Myasthenia Gravis include acute flare-up where the patient develops acute
muscle weakness and respiratory involvement. Such acute cases may be managed with
intravenous immunoglobulins which bind to the auto-antibodies or plasmapheresis which
removes auto-antibodies from blood. Patients with respiratory difficulties may require
ventilatory support.

Lambert-Eaton Syndrome (LES): is an auto-immune condition caused by antibodies against

voltage gated calcium channels on the presynaptic membrane. This prevents calcium from
entering the nerve terminal thus, preventing docking and fusion of acetylcholine vesicles with
synaptic membrane. By preventing the release of acetylcholine into the synaptic cleft, it
prevents muscle contraction. It is notable that about 50% of LES patients also have lung
carcinomas. Smocking significantly increases the risk. The significant symptoms in LES are
general muscle weakness affecting proximal limbs and decreased tendon reflexes. Muscle
weaknesses tend to improve with continued and progressive use. This is because, with
continued limb use, there is repeated attempts at muscle contraction and calcium gradient
builds up outside the pre-synaptic calcium channel which eventually allows endogenous
calcium to outcompete the auto-antibodies. This triggers release of acetylcholine to the
synaptic cleft. 80% of LES patients complain of proximal muscle weakness in both arms and
legs. Oropharyngeal and ocular muscles are mildly affected. Autonomic symptoms such as dry
mouth, constipation, impotence in males and postural hypotension may be present.
Diagnosis of LES depends the symptoms triad: proximal muscle weakness, depressed reflexes
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and autonomic dysfunction.

References:
Behin A., Le Panse R. New Pathways and Therapeutic Targets in Autoimmune Myasthenia
Gravis. J Neuromuscul Dis. 2015;5(3):265 - 277

Young J. D., Leavitt J. A. Lambert-Eaton Myasthenic Syndrome: Ocular Signs and Symptoms. J
Neuroophthalmol. 2016 Mar;36(1):20-22

Discuss Clostridium tetani and Clostridium botulinum

The most common and clinically relevant Clostridium species are Clostridium botulinum which
causes botulism, Clostridium tetani which causes tetanus, Clostridium perfringens which causes
food poisoning, gas gangrene and necrotizing fasciitis.
Two well know species are Clostridium tetani and Clostridium botulinum responsible for
production of closely related protein neurotoxins that cause paralytic syndromes. Tetanus
neurotoxin (TeNT) and botulinum neurotoxin (BoNT) have similar amino terminal domains
consisting of zinc endopeptidase.
Neurospecific binding: from the site of production or adsorption, TeNT and BoNT diffuse in the
body fluids up to the presynaptic membrane of cholinergic nerve terminals where they bind.
Botulism and tetanus are both severe neurological zoonotic diseases. While botulism is
characterized by flaccid paralysis, tetanus consists of spastic paralysis. In severe cases of both
diseases, death occurs by respiratory depression.

Differences between Tetanus and Botulism

Description Tetanus Botulism
Causative Organism Clostridium tetani Clostridium botulinum

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 Type of disease Infection Intoxication
Mode of acquisition Wounds, operation, Home canned food, infant
unhygienic birth, abortion, botulism, adult intestinal
umbilical cord infection colonization
Etiology Bacterium infects wounds Bacterium releases
and releases tetanus botulinum neurotoxin (BoNT)
neurotoxin (TeNT) which is ingested
Signs of disease Spastic paralysis Flaccid paralysis
CNS involvement Inhibitory neurons of spinal No CNS involvement
cord
Diagnosis Clinical Serum or stool culture, ELISA
Prevention Vaccination and boosters Proper food preparation and
hygiene

Clostridium tetani:
Tetanus neurotoxin (TeNT) is another highly potent neurotoxin which binds to presynaptic
motor nerve terminal blocking release of acetylcholine and resulting in spastic paralysis. It is
also transported retrogradely to the central nervous system (CNS) where it exerts a primary
effect of blocking the release of inhibitory amino acid transmitters gamma-aminobutyric acid
(GABA) and glycine at the inhibitory neurons of the spinal cord resulting in spastic paralysis.
Muscle rigidity and spasms occur manifesting trismus (lockjaw), dysphagia, opistotonus, rigidity
and spasms of respiratory and laryngeal and abdominal muscles which may cause respiratory
failure. These manifest as tetanus symptoms. Since Botulism toxin largely remains in lower
motor neuron terminals inhibiting acetylcholine release and muscle activity. Therefore,
botulinum toxin may reduce tetanus symptoms. Trismus may be treated with botulinum toxin
injection into masseter and temporalis muscles. The muscular rigidity and spasms of tetanus
are caused by Tetanus neurotoxin (TeNT).

Clostridium botulinum
Clostridium botulinum is an anaerobic gram-positive, spore-forming rod-shaped bacterium that
produces botulinum toxin, a cause of botulism. It is ubiquitous, widely distributed as a
saprophyte in the soil, animal manure and vegetation.
Pathophysiology: The pathogenesis of Clostridium botulinum is due to production of a very
potent neurotoxin. Botulinum toxin (BT) differs from other exotoxins since it is produced
intracellularly, not secreted, and appears outside only after autolysis of the cell. It consists of a
complex mixture of proteins containing botulinum neurotoxin (BoNT) and non-toxic proteins.
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Botulinum neurotoxin consists of heavy and light chains linked by disulfide bond. It is
synthesized as an inactive single chain polypeptide which is activated by proteolytic cleavage
into a heavy and light chains. The neurotoxins exist in seven different serotypes, A to G.
Although all serotypes inhibit acetylcholine release from nerve terminals, they differ in
intracellular target proteins, characteristics and potency. The seven serotypes bind and enter
peripheral cholinergic nerve terminals from where they inhibit the release of acetylcholine
resulting in flaccid paralysis. If paralysis extends to respiratory muscles, the patient dies of
respiratory failure.

Blocking of acetylcholine release is permanent but the action is short acting as recovery occurs
in 2 to 4 months once new terminal axons generate. Spores do not produce toxins. Toxin
production therefore requires spore germination which occurs in anaerobic conditions. Spores
do not germinate in adult intestines. However, they may germinate in infant intestines.

Botulinum neurotoxin (BoNT) gets transported to the cholinergic nerve endings via blood. It
does not affect the central nervous system. However, it inhibits the release of acetylcholine
with chemical denervation of nerve fiber resulting in flaccid paralysis and atrophy. Because
chemical denervation is reversible, BoNT has temporary effects, the muscle being progressively
rennervated by nerve ending sprouting. Botulism is characterized by descending symmetric
flaccid paralysis of voluntary muscles, loss of deep tendon reflexes, constipation, respiratory
muscle paralysis leading to respiratory failure and death. Botulism toxin largely remains in
lower motor neuron terminals inhibiting acetylcholine release and muscle activity. Therefore,
botulinum toxin may reduce tetanus symptoms. Trismus may be treated with botulinum toxin
injection into masseter and temporalis muscles. During outbreaks of botulism, prophylactic
dose of antitoxin should be given intramuscular to all suspects.

References:

Durre P. Physiology and Sporulation in Clostridium. Microbiol Spectr. 2014 Aug;2(4):0010-2012

Sobel J., Rao A. K. Making the Best of the Evidence: Towards National Clinical Guidelines for
Botulism. Cli Infec Dis. 2017 Dec 27;66(S1-S3)

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