DNA REPLICATION

 DNA is made up of millions of nucleotides, which are composed of deoxyribose sugar,

with phosphate and a base.
 DNA replication is semi-conservative, meaning that each strand in the DNA acts as a
template for the synthesis of a new complementary strand. Semi conservative
because once DNA molecule is synthesized it has one strand from the parent and the
other strand is a newly formed strand.
 DNA replication starts by taking one DNA molecule and giving two daughter
molecules, with each newly synthesized molecule containing one new and one old
strand.
 DNA replication simply is the process by which a DNA makes a copy of itself.
 The complementary pairing of these bases keeps the double strands intact. So, to
make two copies of one DNA, these hydrogen bonds in between the bases should be
broken to begin replication.

Enzymes and Proteins Used in DNA Replication

Nucleases
 A nuclease is an enzyme that can cleave the phosphodiester bonds present in
between the nucleotides.
 On the basis where they cleave, they are characterized as Exo and endonucleases.
 Exonucleases cleave nucleotides from their respective ends.
 Endonucleases act on the region in the middle of the targeted nucleotide. sub-
divided as DNase for DNA for cleaving and RNase for RNA cleaving.
 Restrictive endonuclease or restriction enzymes are the ones that cleave DNA into
fragments at or near the specific recognition sites within the molecule known as
restriction sites.

DNA Polymerase

 DNA polymerases are the enzyme that is responsible for adding new nucleotides and
synthesizing a new strand of DNA by taking the old fragmented strand as a template.
 DNA Polymerases also possess exonuclease activity, that cuts incorrectly added
nucleotides, and allows the DNA replication to happen without errors.
 DNA Polymerase is of many types and functions based on the cell they are found in.
 In prokaryotic cells, there are three DNA polymerases: DNA Polymerase Ι, DNA
Polymerase ΙΙ and DNA Polymerase ΙΙΙ.
 In eukaryotic cells, there are five DNA polymerases: DNA Polymerase α, β, γ, δ and ε

DNA ligase
 DNA ligase is a specific type of enzyme that facilitates the joining of DNA strands
together by catalyzing the formation of a phosphodiester bond.

DNA helicase
  DNA helicase is a motor protein that moves directionally along a nucleic acid
phosphodiester backbone, separating two nucleotides of DNA molecule.
 They separate double-stranded DNA molecules into single strands allowing each
strand to be copied.
 During DNA replication, this DNA helicase unwinds DNA at the origin, a site where
the replication is to be initiated.
 DNA helicase continues to unwind the double helix of DNA and thus forms a
structure called replication fork, named after the forked appearance of two strands
of DNA when unzipped apart.
 It is an energy-driven process as it involves the breaking of Hydrogen bonds between
annealed nucleotide bases.

DNA primase
 Primase is an enzyme that is capable to synthesize short stretches of RNA sequences
known as a primer.
 Primers are an integral part of DNA replication. These primers serve as an initiating
site for the addition of nucleotides by DNA polymerase.
 DNA polymerase can only add nucleotide at pre-existing 3′ Hydroxyl group which is
thus provided by the primers.

DNA topoisomerase
 DNA topoisomerase is a class of enzymes that release helical tension during
transcription and replication by creating transient nicks within the phosphate
backbone on one or both strands of the DNA.
 This tension is aroused when the DNA molecule unwinds due to helicase activity and
forms a replication fork. The progress of the replication fork generates supercoils
 Class Ι DNA topoisomerase makes a single-stranded break to relax the helix, Class ΙΙ
DNA topoisomerase break both the strands of DNA helix

Single strand binding proteins

 The single-strand binding (SSB) protein are DNA binding proteins, that binds to
single-stranded DNA to facilitate DNA replication.
 SSB proteins prevent the hardening of strands during DNA replication. It also
protects strands from nuclease degradation and prevents the rewinding of DNA.
 Thus, SSB proteins stabilize the single-stranded DNA structure that is important for
genomic progression.

Steps in DNA Replication

Step 1: Formation of Replication Fork
 Before DNA can replicate, this double-stranded molecule must unwind into two
single strands to initiate the replication process.
 DNA unwinds when the complementary base pairing between the double-stranded
is broken, and the site to initiate this unwinding is denoted by specific regions
(Adenine and Thymine rich).
 These specific coding regions are referred to as Origin of Replication (Ori) and thus
the replication process begins.
  These origins are targeted by initiator proteins, which go on to recruit more proteins
that can help the replication process by forming a replication fork around the Ori.

 Within this replication protein complex is an enzyme DNA helicase, which starts to
unwind the DNA from its Ori and exposes two strands resembling a Y-like structure
referred to as replication fork.
 The activity of helicase causes topological stress to the un-winded strand forming
supercoiled DNA, this stress is relieved by Topoisomerase by negative supercoiling.
 The replication fork is bidirectional; one strand is oriented to 5′ to 3′ direction
(leading strand) and the other strand is oriented to 3′ to 5′ direction (lagging strand)
but the addition of nucleotide progress only in 5′ to 3′ direction.
 The formation of a replication fork exposing two single-stranded strands marks the
beginning of Initiation.
Step 2: Initiation
 One strand runs from 5′ to 3′ direction towards the replication fork and is referred to
as leading strand and the other strand runs from 3′ to 5′ away from the replication
fork and is referred to as lagging strands.
 To this exposed single-stranded DNA, SSB proteins are adhered to prevent recoiling
of DNA and to stabilize it.
 After which another enzyme DNA primase comes into action to synthesize a short
stretch of RNA primer, which provides a free 3′ hydroxyl group for DNA polymerase
can now add nucleotides and extend the new chain of nucleotides.

Step 3: Elongation
 Now that primer is added to unzipped two single-stranded DNA, these strands now
act as a template for synthesizing new DNAs.
 The enzyme DNA polymerase synthesizes new nucleotide to match the template and
add on to the free 3′ hydroxyl group provided by the primer in each single-stranded
DNA.
 The leading strand runs from 5′ to 3′ so the addition of nucleotides by DNA
polymerase happens from 5′ to 3′ direction. As the replication fork progresses the
addition of nucleotide is continuous thus only requiring the primer once.
 However, lagging strands is antiparallel and run from the 5′ to 3′ direction, the
continuous addition of nucleotides is not possible as the replication fork progresses,
DNA polymerase cannot add complementary nucleotides to the 5′ end. Therefore,
multiple primers are required. Due to this phenomenon, the DNA nucleotides
synthesis from lagging strands occurs in fragments. These fragments are termed
Okazaki fragments.
 Hence, the leading strand using only one primer synthesizes nucleotides
continuously, while the lagging strand uses multiple primers and thus synthesizes
nucleotides discontinuously.

Step 4: Termination
 RNA primers of both leading and lagging strands are cleaved out or degraded by
exonucleases activity of DNA polymerase, and the nicks or gaps so formed are filled
with DNA and sealed by the enzyme DNA ligase.
 DNA polymerase also shows proofreading activity and check, remove and replace
any errors.
 Eventually, the replication forks terminate at terminating
recognizing sequences (ter). This ter- TUS complex arrest replication
fork and terminate

RECOMBINANT DNA TECHNOLOGY

 Recombinant DNA technology refers to the joining together of DNA molecules from
two different species that are inserted into a host organism to produce new genetic
combinations that are of value to science, medicine, agriculture, and industry.
 Recombinant DNA (rDNA), on the other hand is the general name for a piece of DNA
that has been created by the combination of at least two strands.

1. Isolation of Genetic Material

 The first step in rDNA technology is to isolate the desired DNA in its pure form i.e.
free from other macromolecules.
 Since DNA exists within the cell membrane along with other macromolecules such as
RNA, polysaccharides, proteins, and lipids, it must be separated and purified which
involves enzymes such as lysozymes, cellulase, chitinase, ribonuclease, proteases etc.
 Other macromolecules are removable with other enzymes or treatments. Ultimately,
the addition of ethanol causes the DNA to precipitate out as fine threads. This is
then spooled out to give purified DNA.

2. Restriction Enzyme Digestion

 Restriction enzymes act as molecular scissors that cut DNA at specific locations.
These reactions are called ‘restriction enzyme digestions’.
 They involve the incubation of the purified DNA with the selected restriction
enzyme, at conditions optimal for that specific enzyme.
 The technique ‘Agarose Gel Electrophoresis’ reveals the progress of the restriction
enzyme digestion.
 This technique involves running out the DNA on an agarose gel. On the application of
current, the negatively charged DNA travels to the positive electrode and is
separated out based on size. This allows separating and cutting out the digested
DNA fragments.
 The vector DNA is also processed using the same procedure.

3. Amplification Using PCR

 Polymerase Chain Reaction or PCR is a method of making multiple copies of a DNA
sequence using the enzyme – DNA polymerase in vitro.
 It helps to amplify a single copy or a few copies of DNA into thousands to millions of
copies.
 PCR reactions are run on ‘thermal cyclers’ using the following components:
Template – DNA to be amplified
Primers – small, chemically synthesized oligonucleotides that are complementary to
a region of the DNA.
Enzyme – DNA polymerase
 Nucleotides – needed to extend the primers by the enzyme.
 The cut fragments of DNA can be amplified using PCR and then ligated with the cut
vector.
4. Ligation of DNA Molecules
 The purified DNA and the vector of interest are cut with the same restriction
enzyme. This gives us the cut fragment of DNA and the cut vector, that is now open.
 The process of joining these two pieces together using the enzyme ‘DNA ligase’ is
‘ligation’.
 The resulting DNA molecule is a hybrid of two DNA molecules – the interest
molecule and the vector. In the terminology of genetics this intermixing of different
DNA strands is called recombination. Hence, this new hybrid DNA molecule is also
called a recombinant DNA molecule and the technology is referred to as the recom-
binant DNA technology.
5. Insertion of Recombinant DNA Into Host
 In this step, the recombinant DNA is introduced into a recipient host cell mostly, a
bacterial cell. This process is ‘Transformation’.
 Bacterial cells do not accept foreign DNA easily. Therefore, they are treated to make
them ‘competent’ to accept new DNA. The processes used may be thermal shock,
Ca++ ion treatment, electroporation etc.
6. Isolation of Recombinant Cells
 The transformation process generates a mixed population of transformed and non-
trans- formed host cells.
 The selection process involves filtering the transformed host cells only.
 For isolation of recombinant cell from non-recombinant cell, marker gene of plasmid
vector is employed.

Application of Recombinant DNA technology

 Recombinant DNA is widely used in biotechnology, medicine and research.
 Recombinant DNA is used to identify, map and sequence genes, and to determine
their function.
 Recombinant proteins are widely used as reagents in laboratory experiments and to
generate antibody probes for examining protein synthesis within cells and
organisms.
 Many additional practical applications of recombinant DNA are found in industry,
food production, human and veterinary medicine, agriculture, and bioengineering.
1. DNA technology is also used to detect the presence of HIV in a person.
2. Application of recombinant DNA technology in Agriculture – For example,
manufacture of Bt-Cotton to protect the plant against ball worms.
3. Application of medicines – Insulin production by DNA recombinant technology is a
classic example.
4. Gene Therapy – It is used as an attempt to correct the gene defects which give rise
to heredity diseases.
5. Clinical diagnosis – ELISA is an example where the application of recombinant DNA is
possible.
 CHROMOSOME STRUCTURE
 In the nucleus of each cell, the DNA molecule is packaged into thread-like structures
called chromosomes.
 Each chromosome is made up of DNA tightly coiled many times around proteins
called histones that support its structure.
 Chromosomes were first described by Strasburger (1815), and the term
‘chromosome’ was first used by Waldeyer in 1888.
 Each chromosome typically has one centromere and one or two arms that project
from the centromere.
 Structurally, each chromosome is differentiated into three parts—
1. Pellicle
2. Matrix
3. Chromonemata
Pellicle
 It is the outer envelope around the substance of chromosome.
 It is very thin and is formed of achromatic substances.
Matrix
 It is the ground substance of chromosome which contains the chromonemata.
 It is also formed of non-genic materials.
Chromonemata
 Embedded in the matrix of each chromosome are two identical, spirally coiled
threads, the chromonemata.
 The two chromonemata are also tightly coiled together that they appear as single
thread of about 800A thickness.
 Each chromonemata consists of about 8 microfibrils, each of which is formed of a
double helix of DNA.

Chromatid:
 Each chromosome has two symmetrical structures called chromatids or sister
chromatids which is visible in mitotic metaphase.
 Each chromatid contains a single DNA molecule
 At the anaphase of mitotic cell division, sister chromatids separate and migrate to
opposite poles
Centromere and kinetochore:
 Sister chromatids are joined by the centromere. The centromere is called primary
constriction.
 Spindle fibres during cell division are attached at the centromere
 The number and position of the centromere differs in different chromosomes
 Centromere divides the chromosome into two parts, the shorter arm is known as ‘p’
arm and the longer arm is known as ‘q’ arm.
 The centromere contains a disc-shaped kinetochore, which has specific DNA
sequence with special proteins bound to them. The kinetochore provides the centre
for polymerisation of tubulin proteins and assembly of microtubules
Secondary constriction and nucleolar organisers:
  Other than centromere, chromosomes possess secondary constrictions.
 Secondary constrictions can be identified from centromere at anaphase because
there is bending only at the centromere (primary constriction)
 Secondary constrictions, which contain genes to form nucleoli are known as
the nucleolar organiser
Telomere:
 Terminal part of a chromosome is known as a telomere.
 Telomeres are polar, which prevents the fusion of chromosomal segments
Satellite:
 It is an elongated segment that is sometimes present on a chromosome at the
secondary constriction.
 The chromosomes with satellite are known as sat-chromosome
Chromatin:
 Chromosome is made up of chromatin. Chromatin is made up of DNA, RNA and
proteins. At interphase, chromosomes are visible as thin chromatin fibres present in
the nucleoplasm. During cell division, the chromatin fibres condense and
chromosomes are visible with distinct features.
 The darkly stained, condensed region of chromatin is known as heterochromatin. It
contains tightly packed DNA, which is genetically inactive
 The light stained, diffused region of chromatin is known as euchromatin. It contains
genetically active and loosely packed DNA
 At prophase, the chromosomal material is visible as thin filaments known
as chromonemata
 At interphase, bead-like structures are visible, which are an accumulation of
chromatin material called chromomere. Chromatin with chromomere looks like a
necklace with beads

EUKARYOTIC TRANSCRIPTION

Eukaryotic transcription is inferred as a process in which the eukaryotic cells

replicate the data in a strand of DNA and copy the genetic knowledge into a new
molecule of RNA.
Eukaryotic transcription is referred to as the first step towards gene expression in
which some segments of DNA are traced into RNA by a special enzyme called RNA
polymerase.
This results in forming an antiparallel RNA fibre known as a primary transcript.
Procedure of eukaryotic transcription
Transcription is the formation of RNA over the template of DNA. It creates single
stranded RNA which has coded information similar to the sense or coding strand of
DNA with the exception that T is replaced by U.
The DNA strand which functions as a template for RNA synthesis is called template
or antisense strand.
The segment of antisense DNA that takes part in transcription is called the
transcription unit. It may consist of one or more cistrons (¨ genes).
Each transcription unit has a promoter region, generally in the beginning and
terminator region where transcription ends.
Mode of transcription
 The enzyme taking part in transcription is called RNA polymerase.
There is single RNA polymerase (RNAP) in case of prokaryotes.
Eukaryotes have three types of RNA polymerase.
(i) RNA polymerase I – For synthesis of ribosomal RNAs except 5 S
(ii) RNA polymerase II – For mRNA and many snRNAs
(iii) RNA polymerase III – For tRNAs, 5S rRNA and some snRNAs
Prokaryotic RNA polymerase has a sigma factor for recognising the start signal of the
promoter region.
The remaining part of RNA polymerase is called core enzyme.
In eukaryotes separate protein factors take part in recognition and initiation.
They are called transcription factors, e.g., TFII, TFIII.
Promoter region of the transcription unit has a separate recognition site and
polymerase binding site.
Terminator region of the transcription unit has either palindromic sequences or poly
A sequences.
Termination of transcription requires a separate termination factor called rho (r)
factor.
The various steps in transcription are as follows –
1. Activation of Ribonucleotides
 Four types of ribonucleotides take part in the synthesis of RNA over DNA. They are
AMP, GMP, UMP and CMP.
 The nucleotides are available in the nucleoplasm. Before their incorporation, the
nucleotides are converted into activate through phosphorylation.
 It produces [Link], phosphate and enzyme phosphorylase are
required.
2. DNA Template
 Only one DNA strand functions as a template strand.
 It is also called an antisense strand.
 The unit of transcription begins with a promoter and ends in a terminator.
 The separation of template strand does not require specific chemicals as in case of
DNA replication.
 Instead, the core enzyme of RNA polymerase travels along the template strand with
the help of a protein factor called Nus A protein.
3. Initiation
 RNA polymerase reaches the promoter region.
 Sigma factor (s) recognises the promoter region.
 In eukaryotes there are separate transcription factors for recognition of promoter
regions.
 As soon as the enzyme RNA polymerase gets attached, the template DNA of the
transcription unit begins to unzipper.
4. Base Pairing
 The activated phosphorylated ribonucleotides come to lie opposite complementary
nitrogen bases of the template strand – A opposite T, U opposite A, G opposite C and
C opposite G.
 With the help of enzyme phosphatase the extra phosphate radicals of nucleotides
are hydrolysed.
 It releases energy which is helpful in establishing temporary bonds between
complementary base pairs.
5. Formation of RNA chain(Elongation)
 The core enzyme with the help of energy and Mg2+ builds phosphodiester bonds
between adjacent ribonucleotides forming the RNA chain.
 As the enzyme moves along the DNA template, the RNA chain elongates.
 Synthesis continues till the enzyme reaches the termination region.
 In the terminator region RNA polymerase is separated from the DNA template by
means of rho factor (r) and Nus G.
6. Chain Separation(Termination)
 Rho factor has ATPase activity.
 This separates RNA polymerase as well as the newly built RNA strand.
 As soon as the RNA strand separates, the sense and antisense strands of DNA re-
establish hydrogen bonds between their complementary base pairs.
 The duplex nature is restored.