SEPTMEBER 20TH, 2023
DNA Replication
Copying DNA to produce an identical copy
What is DNA
A naive introduction
Self-replicating material in nearly all living organisms is the main
constituent of chromosomes.
It is the carrier of genetic information.
A good introduction
DNA, standing for Deoxyribonucleic acid, is a polymer of nucleic acid
containing a ribose sugar and phosphate group backbone and a four
nitrogenous base pairs, namely, Adenine(A), Guanine(G), Thymine(T),
Cytosine(C).
It is a double helical molecule tightly coiled into the cell’s nucleus.
Each cell contains around 2 metres length of DNA, which is the same as
coiling a two km-long rope in a basketball.
Since your body contains around 10 trillion cells, DNA can reach the sun
more than 100 times.
Each strand has a backbone
made of alternating sugar
(deoxyribose) and phosphate
groups. Attached to each sugar
is one of four bases:
adenine bonds with thymine,
cytosine bonds with guanine.
source: wikipedia
Discovery of DNA: A historical overview
Friedrich Miescher: (1869)
A very intelligent, shy, and introspective man. Shy and introspective,
probably because he has had a severe hearing impairment since
childhood.
He wanted to study lymphocytes, but due to the shortage of
availability, he studied leucocytes (WBCs), which he could get from
the used bandages from the nearby hospital.
He isolated WBCs from the bandage using sodium sulphate.
Then, the WBCs were washed with warm alcohol to remove lipids and
digested the proteins present in the cytoplasm using the proteolytic
enzyme Pepsin.
However, the pepsin he used back then was not similar to what we use
nowadays.
He extracted a pig’s stomach and used the active but impure enzymes
as protein digestors.
He was left with a greyish residue, which he knew was a new substance
because it could not be protein, as proteins were already dissolved in
pepsin.
Since this new material was from the nucleus, he called it Nuclein, which
was later called nucleic acid.
Frederick Griffith: (1928)
Demonstrated the transformation principle in bacteria.
Did experiments on mice with R and S strains of Streptococcus
pneumoniae.
Avery–MacLeod–McCarty: (1944)
Identifies the DNA as the transforming principle
Erwin Chargaff: (1950)
Discovers the base pairing rules for DNA.
Adenine with Thymine (2 Hydrogen Bonds)
Cytosine with Guanine (3 Hydrogen Bonds)
Hershey-Chase: (1952)
Unequivocally established that DNA carried hereditary information.
They did experiments on bacteriophages using isotopic labelling.
Rosalind Franklin: (1953)
There is more to the fact that Watson and Crick discovered the structure
of the DNA.
Rosalind Franklin and a PhD student, Raymond Gosling, took an X-ray
diffraction photograph of a DNA double helical molecule. (The famous
Photograph 51)
Rosy was a clever student and got a scholarship to study in Cambridge
and completed a degree equivalent to the Bachelor of Science, as the
degree was not awarded to women back then.
She nevertheless earned her PhD from Cambridge in 1945 and went to
France for Post Doctoral Research. But eventually, she returned to London
and joined the King’s College, where she took the x-ray diffraction
photograph of double-helical DNA.
She was unhappy at King’s College as it was male-dominated, and wasn’t
even allowed to eat in the same mess as her male colleagues. Also, she
had some misunderstandings with senior researcher Maurice Wilkins, who
showed her unpublished photograph of DNA to Watson and Crick, and
they soon published their famous paper on DNA without crediting
Rosalind.
Rosalind, however, returned to France, to which Wilkins wrote to Watson
that the “smoke of witchcraft” was going away.
By the time Rosalind published her work in the Journal Nature, Watson and
Crick had already published their papers, and they won the Nobel Prize in
1962 for the same form.
Rosy died in 1957 at age 37 due to ovarian cancer, likely due to the
prolonged exposure to radiation without proper precautions she
underwent in attempts to photograph the DNA molecule.
She was portrayed as a “witch” by James Watson in his book “The Double
Helix”, and unlike others whom Watson criticized, she was the only one
who could not make any case in her defence as she had already died.
Structure of DNA
DNA is a polymer of nucleotides, molecules with a phosphorylated
sugar and nitrogenous base.
Sugar, joined with phosphodiester bonds, forms the backbone. The
nitrogenous bases are inside the spiral, forming hydrogen bonds with
the nitrogenous bases of the other strand.
Both the spirals are wound around each other and form a helix.
The sequence of the nitrogenous base pairs is unique to each
species. This fact is used in DNA fingerprinting and finding
evolutionary relations among different species.
DNA Replication
Millions of cells reproduce in our body daily; this needs the duplication
of the genetic material, i.e., DNA.
The duplication of DNA to form a copy of itself is called DNA
Replication.
DNA Replication is a susceptible and chronologically complex process
Mechanism of DNA Replication
Initiation and Unwinding:
During initiation, so-called initiator proteins bind to the replication origin,
a base-pair sequence of nucleotides known as oriC.
This binding triggers events that unwind the DNA double helix into two
singlestranded DNA molecules. Several groups of proteins are involved in
this unwinding. For example, the DNA helicases are responsible for
breaking the hydrogen bonds that join the complementary nucleotide
bases to each other.
Because the newly unwound single strands tend to rejoin, another
group of proteins, the single-strand-binding proteins, keep the single
strands stable until elongation begins. A third family of proteins, the
topoisomerases, reduce some of the torsional strain caused by the
unwinding of the double helix.
1
4
Primer Synthesis:
Primer synthesis marks the beginning of the actual synthesis of the
new DNA molecule. Primers are short stretches of nucleotides
(about 10 to 12 bases in length) synthesized by an RNA polymerase
enzyme called primase.
Primers are required because DNA polymerases, the enzymes
responsible for the actual addition of nucleotides to the new DNA
strand, can only add deoxyribonucleotides to the 3'-OH group of an
existing chain and cannot begin synthesis de novo(anew).
Primase, on the other hand, can add ribonucleotides de novo. Later,
after elongation is complete, the primer is removed and replaced
with DNA nucleotides
Elongation:
Finally, elongation —the addition of nucleotides to the new DNA
strand— begins after the added primer.
Synthesis of the growing strand involves adding one by one
nucleotide in the exact order specified by the original (template)
strand.
One of the critical features of the Watson-Crick DNA model is that
adenine is always paired with thymine, and cytosine is always paired
with guanine. So, for example, if the original strand reads A-G-C-T,
the new strand will read T-C-G-A.
More on DNA Polymerase:
After a primer is synthesized on a DNA strand, synthesis and
elongation can proceed in only one direction.
As previously mentioned, DNA polymerase can only add to the 3'
end, so the 5' end of the primer remains unaltered.
Consequently, synthesis proceeds immediately only along the so-
called leading strand. This immediate replication is known as
continuous replication.
The other strand (in the 5' direction from the primer) is called the
lagging strand, and replication along it is called discontinuous
replication.
The double helix has to unwind before another primer can be
synthesized further up on the lagging strand. Synthesis can then
occur from the 3' end of that new primer.
Next, the double helix unwinds a bit more, and another spurt of
replication proceeds. As a result, replication along the lagging strand
can only proceed in short, discontinuous spurts called Okazaki
Fragments, named after the married couple who discovered them.
Termination:
Termination happens when the DNA Polymerase reaches the end of
the strands.
We can easily understand that in the last section of the lagging
strand, when the RNA primer is removed, the DNA Polymerase can’t
seal the gap (because there is no primer).
So, the end of the parental strand where the last primer binds isn't
replicated.
These ends of linear DNA consist of noncoding DNA containing
repeat sequences called telomeres. As a result, a part of the
telomere is removed in every cycle of DNA Replication.
Proof Reading:
When an incorrect nucleotide is added to the growing strand,
replication is stalled because the nucleotide's exposed 3′-OH group
is in the "wrong" position.
DNA polymerase enzymes recognise this during proofreading and
replace the incorrectly inserted nucleotide to continue replication.
Enzymes like nucleases remove the wrong nucleotides, and the DNA
Polymerase fills the gaps.
Proofreading fixes about 99% of these types of errors.
Some Interesting facts
Telomeres, Hayflick Limit and Cellular Immortality:
As discussed above, DNA can’t be synthesized at the ends of linear
strands.
But this way, DNA will become shorter at each replication.
To prevent this, Telomeres come into play.
Telomeres are repetitive strings of DNA found at the ends of
chromosome pairs within diploid cells.
These strings are usually compared to the plastic ends of shoelaces
(called aglets) that keep the laces from fraying.
Telomeres provide the same protection to chromosomes, but the
telomere at the end of each chromosome pair is shortened with
each cellular division.
Hayflick Limit is the maximum number of times a cell can reproduce
before its telomere is depleted.
Apoptosis begins once the Hayflick limit is reached,
i.e., Telomere is depleted.
Another breakthrough in cellular ageing was uncovered just under
a decade later.
Telomerase is a protein found in all cells, but in normal cells, it's
turned off -- it doesn't do anything.
However, telomerase is active in abnormal cells like tumours and
germ cells.
It contains an RNA template capable of producing new telomeres on
the ends of chromosomes in ageing cells.
If active telomerase is added to normal adult cells, they replicate
long beyond their Hayflick limit.
In one study that supports this notion, researchers reported that
cells to which they introduced telomerase had replicated 20 more
times than their average life span would indicate -- and were still
dividing.
Knot Theory: Why do my earphones entangle so much and the
DNA doesn’t?
Knot Theory is a mathematical discipline of studying Knots and
their forms.
Historically, Knot Theory was considered an intellectual exercise
with no real-life applications.
But recently, Knot Theory has found potential applications in the
chemistry of extremely tough materials.
In biology, We have seen some applications recently.
Bacterial DNA is circular, and when replicated, the two daughter
DNAs are entangled and can’t separate independently. Instead,
a specialised molecule named type II topoisomerase comes
into action.
It carefully breaks some bonds and rejoins them upon
detangling the two daughter DNAs.
If we inhibit the activity of Type II topoisomerase, the Bacterial
population die.
Similar molecules are found in humans also; although we don’t
have circular DNA, our DNA is long enough to entangle.
To rescue this, Human type II Topoisomerase comes into play.
And this is why DNA, despite being so long and tightly packed,
does not entangle.
