Molecular biology

Molecular Biology is the field of biology that studies the composition, structure and interactions of
cellular molecules – such as nucleic acids and proteins – that carry out the biological processes essential
for the cell’s functions and maintenance.

Replication, in the general sense, is to create a copy or a duplicate. Thus, in biology, replication
is commonly associated with DNA (DNA replication) where the DNA is copied prior to cell
division. 

DNA COPIES ITSELF TO YIELD 2 IDENTICAL COPIES OF ITSELF.

DNA REPLICATION ENSURES THE CONTINUITY OF CELL CHARACTERISTICS WITH EACH CELL DIVISION.

WHY DOES THE DNA NEED TO COPY ITSELF?

- BEFORE THE CELL DIVIDES DURING MITOSIS. IT HAS TO CREATE A COPY OF ITS DNA.
- THIS ENSURE THAT BOTH RESULTING DAUGHTER CELLS. HAS DNA THAT IS IDENTICAL TO
PARENT CELLS.

AT WHAT POINT IN THE CELL CYLCE DOES DNA REPLICATION HAPPEN?

- DNA IS COPIED IN THE S PHASE.

- S = SYNTHESIS OF TO MAKE

REVIEW:

CELL DIVISION

CELL CYCLE

 DNA replication occurs in the S phase of the cell cycle. As a matter of fact, the S-
phase of the cell cycle is named so because during the S-phase synthesis of
DNA is taking place inside the cell at this time.

Mitosis is behind the increase in your height, the repair of damaged or injured body
tissues, the increase in size of organs as you mature, and the replacement of dead or worn-out
cells. With mitosis, your body makes sure that the cells it produces are identical with old parent
cells.
For genetic consistency

Occurs only in body cells

Takes place throughout the organisms’ lifetime

Involved in asexual reproduction (growth and repair)

MEIOSIS - This is important so that the basic characteristics of a certain species will be
maintained constant through successive generations.
For genetic continuity

Occurs only in reproductive cells or gametes

Takes place only during the reproductive years of the organism

Involved in sexual reproduction (production of gametes and providing genetic variation in
organisms)

In biology, all life will cease to exist without replication. Biological replication is the essential
force in biology that sustains all life on earth whether it is unicellular or multicellular. At any
given point, millions of replications are taking place inside all organisms.
Almost every cell contains its own DNA.
As the cells divide, it is equally necessary for this parent molecule to divide.
The very purpose of replication is to ensure that every daughter cell is functionally independent. 
The role of replication in Genetics is to distribute all the genetic information equally between the
two daughter cells.

The two strands run antiparallel to each other. Each DNA strand has a 5’ end & a 3’ end. The 5’
end of one strand comes next to the 3’ end of the other strand.

The two strands of the DNA are held together with the help of hydrogen bonds that are
formed between the nucleobases.

The DNA follows a rule of base pairing: –

 Adenine always pairs with Thymine forming 2 hydrogen bonds.

 Guanine always pairs with Cytosine forming 3 hydrogen bonds. 
Note the two hydrogen bonds between adenine (purine) and thymine (pyrimidine) and three
hydrogen bonds between guanine (purine) and cytosine (pyrimidine). 
Five steps of DNA replication:

Step 1: Identification of the origin of replication (ori)

Since prokaryotic cells typically have only a single, circular chromosome, they can replicate
faster than eukaryotic cells.

Eukaryotic chromosomes are much larger, so multiple origins are needed to replicate the
entire chromosome in a short amount of time.
Prokaryotes contain a single, double-stranded circular chromosome. Eukaryotes contain
double-stranded linear DNA molecules packaged into chromosomes.

It is the point where the DNA replication begins. Right next to ori there is an AT-rich
portion of DNA to which ori binding proteins bind. The ori binding proteins unwind the
DNA at ori.
The unwound DNA is stabilized by the single-strand binding proteins (SSB) that prevent
the reannealing of the DNA.

origin sequences commonly have high AT-content.

The promoter region contains a high concentration of adenine and thymine bases. Since
adenine and thymine only share two double bonds between them, RNA polymerase will
expend less energy in opening up the double helix at this point

Step 2: The unwinding of the DNA

The further unwinding of the DNA is carried out by the enzyme helicase. As the helicase
enzyme is acting, supercoiling occurs ahead of the replication fork and is taken care of
by the enzyme topoisomerase.
Step 3: Formation of the replication fork
With the unwinding of the DNA, a replication bubble is formed. Each half of this
replication bubble is referred to as a replication fork.

Step 4: Actual DNA synthesis

Initially, an enzyme called primase forms a 10-nucleotide long RNA primer over which
the DNA polymerase III adds further nucleotides. Later, the primer is removed by DNA
polymerase I, and the gap thus created is also filled by it.
DNA Polymerase
It is the most important enzyme responsible for the replication of DNA.

- This enzyme complex reads the template DNA in the 3’ to 5’ direction and
keeps adding the complementary base pairs in the 5’ to 3’ direction.
This continuous addition of complementary base pairs produces a new daughter strand
complementary to the template strand. The DNA polymerase III requires an RNA primer
to start.

 Both strands of the DNA act as a template.

 The direction of synthesis of a new strand is always from 5’ to 3’ direction.
However, the reading of the template strand occurs from 3’ to 5’ direction.

Step 5: Termination of DNA replication

After the DNA has replicated, specialized termination sequences of the DNA known
as ter help in the termination of the DNA replication process. Ter sequence is bound by
the tus proteins also known as terminator utilization sequence. Tus proteins act as anti-
helicase and stop the further replication of DNA.
- DNA replication is semiconservative in nature – the semiconservative nature of the
DNA was put forward by Meselson and Stahl. It means that each of the new daughter
strands contains one original strand from the parent DNA molecule.

- Semi discontinuous nature of the DNA replication (See Figure 6) – one strand of the DNA is
synthesized in a continuous fashion which is known as the leading strand whereas the other
strand is synthesized in a discontinuous fashion, it is called the lagging strand.
- On the lagging strand, continuous reading of the template strand by DNA polymerase is not
possible, hence the strand gets replicated in multiple small fragments known as Okazaki
fragments. These Okazaki fragments are then joined together with the enzyme DNA ligase.

3'→5' exonuclease activity for proofreading

The basic mechanisms of DNA replication are similar across

organisms. In this article, we'll focus on DNA replication as it takes
place in the bacterium E. coli, but the mechanisms of replication are
similar in humans and other eukaryotes

The result of DNA replication is two DNA molecules consisting of one new and one old
chain of nucleotides. This is why DNA replication is described as semi-conservative, half of
the chain is part of the original DNA molecule, half is brand new.
Cells need to copy their DNA very quickly, and with very few errors (or
risk problems such as cancer). To do so, they use a variety of
enzymes and proteins, which work together to make sure DNA
replication is performed smoothly and accurately
1. Helicase
- move the replication forks forward by "unwinding" the DNA (breaking
the hydrogen bonds between the nitrogenous base pairs).

2. DNA polymerase
How, then, does DNA polymerase add the first nucleotide at a new
replication fork?

Alone, it can't! The problem is solved with the help of an enzyme

called primase. 

- - DNA polymerases are responsible for synthesizing DNA: they add

nucleotides one by one to the growing DNA chain, incorporating only
those that are complementary to the template.
 They always need a template
 They can only add nucleotides to the 3' end of a DNA strand
o (They use the free -OH group found at the 3' end as a
"hook," adding a nucleotide to this group in the
polymerization reaction.)
 They can't start making a DNA chain from scratch, but require a pre-existing chain or short
stretch of nucleotides called a primer
 They proofread, or check their work, removing the vast majority of "wrong" nucleotides that are
accidentally added to the chain
3. Primase
- Primase 

- makes an RNA primer, or short stretch of nucleic acid

complementary to the template, that provides a 3' end for DNA
polymerase to work on. 
- A typical primer is about five to ten nucleotides long. The
primer primes DNA synthesis, i.e., gets it started.
- Once the RNA primer is in place, DNA polymerase "extends" it,
adding nucleotides one by one to make a new DNA strand that's
complementary to the template strand.

4. Topoisomerase
- prevents the DNA double helix ahead of the replication fork from
getting too tightly wound as the DNA is opened up.
- It acts by making temporary nicks in the helix to release the tension,
then sealing the nicks to avoid permanent damage.
5. DNA ligase
- Finally, there is a little cleanup work to do if we want DNA that
doesn't contain any RNA or gaps. The RNA primers are removed and
replaced by DNA through the activity of DNA polymerase I, the other
polymerase involved in replication. The nicks that remain after the
primers are replaced get sealed by the enzyme DNA ligase.
Proteins
1. single-strand binding proteins
- coat the separated strands of DNA near the replication fork, keeping
them from coming back together into a double helix.

2. sliding clamp
- holds DNA polymerase III molecules in place as they synthesize
DNA. The sliding clamp is a ring-shaped protein and keeps the DNA
polymerase of the lagging strand from floating off when it re-starts at a
new Okazaki fragment^44start superscript, 4, end superscript.