Unit-4

Nature and function of genetic materials

Genetic material is that substance which not only controls the inheritance of traits from one
generation to the next but is also able to express its effect through the formation and
functioning of the traits.

Components of genetic material which control characters are called genes. Genes are located
over chromosomes. In sexually reproducing organisms, an individual receives one genome or
one set of chromosomes (and hence genes) from male parent and a second genome or set of
chromosomes from female parent.

There are three fundamental characteristics of genes:

(i) Storage and expression of hereditary information’s,

(ii) Replication and transmission to progeny,

(iii) Mutability.

Properties of Genetic Material:

A molecule that can act as a genetic material must fulfill the following criteria :

1. The hereditary information must be present in the coded form in the structure of genetic
material and its genes.

2. The structural elements of the genetic material must be ubiquitous in their distribution.

3. It should have vast diversity as is found in the innumerable forms of life.

4. It should be able to replicate or form its carbon copies.

5. It should be present in all the cells.

6. It should be same both in quantity and quality in all the somatic cells of an individual.

7. The replicated genetic material must be transferred faithfully from a cell to its daughters
and from one generation to the next.
8. Genetic material should be able to express itself through formation of specific bio-
chemicals.

9. There should be some intrinsic control system for differential functioning of genetic
material or its genes so that different parts of an organism are able to have specific size,
structure and functions.

10. There should be a sort of biological clock in the expression of genetic material that
governs development of embryo, juvenile state, mature state, sexual maturation and ageing.

11. There are occasional changes or mutations in the structure and functioning of its genes
which are of permanent nature and inheritable. Mutations are essential for evolution and
adaptability.

12. It should be stable both chemically and physically.

13. Genetic material must be able to express its effect in the form of Mendelian characters.

Nucleic Acid

UNIT-4

Nucleic Acid

 Nucleic acid, naturally occurring chemical compound that is capable of being broken
down to yield phosphoric acid, sugars, and a mixture of organic bases (purines and
pyrimidines).
 Nucleic acids are the main information-carrying molecules of the cell, and, by
directing the process of protein synthesis, they determine the inherited characteristics
of every living thing.
 The two main classes of nucleic acids are deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA).
 DNA is the master blueprint for life and constitutes the genetic material in all free-
living organisms and most viruses.
 RNA is the genetic material of certain viruses, but it is also found in all living cells,
where it plays an important role in certain processes such as the making of proteins.

Base Pairing
  Two nitrogen-containing bases (or nucleotides) that pair together to form the structure
of DNA. The four bases in DNA are adenine (A), cytosine (C), guanine (G), and
thymine (T). These bases form specific pairs (A with T, and G with C). Base pair may
also refer to the actual number of base pairs, such as 8 base pairs, in a sequence of
nucleotides.
 DNA base pair. Under normal circumstances, the nitrogen-containing bases adenine
(A) and thymine (T) pair together, and cytosine (C) and guanine (G) pair together.
The binding of these base pairs forms the structure of DNA.

Chargaff’s Rule

 Chargaff’s rule is something that relates to the DNA of a species. Also, it is named after
its founder Erwin Chargaff. Furthermore, in this, we will discuss what is Chargaff’s rule.
 The accepted tetranucleotide hypothesis, most works assumed the derivations from the
equimolar base ratios were because of experimental error. Also, Chargaff through
experiment shows that the base composition of species varies among species.

 However, in all the species the molar ratios [A] = [T] and [C] = [G], and that ratio [C +
G] / [A + T] was typically less than the unity with [C + G] is less abundant. And these
ratios are referred as Chargaff’s rule.

Chargaff’s Rule of Base Pairing

In the Chargaff’s rules of base pairing are:

 Relation of A with T: The Pyrimidine Thymine (T) always pairs with the Purine Adenine
(A)

 Relation of C with G: The Purine Guanine (G) always pair with the Pyrimidine Cytosine
(C)
It is steady with there not being enough space (20 Å) for two purines to fit within the spiral and
too much space for two pyrimidines to get near enough to each other to form hydrogen bonds
among them.

DNA Structure

1. In eukaryotes, such as plants and animals, DNA is found in the nucleus, a specialized,
membrane-bound vault in the cell, as well as in certain other types of organelles (such
as mitochondria and the chloroplasts of plants).
2. In prokaryotes, such as bacteria, the DNA is not enclosed in a membranous envelope,
although it's located in a specialized cell region called the nucleoid.
 3. In eukaryotes, DNA is typically broken up into a number of very long, linear pieces
called chromosomes, while in prokaryotes such as bacteria, chromosomes are much
smaller and often circular (ring-shaped).
4. A chromosome may contain tens of thousands of genes, each providing instructions
on how to make a particular product needed by the cell.

Nucleotides

 DNA and RNA are polymers (in the case of DNA, often very long polymers), and are
made up of monomers known as nucleotides.
 Each nucleotide is made up of three parts: a nitrogen-containing ring structure called a
nitrogenous base, a five-carbon sugar, and at least one phosphate group.
 The sugar molecule has a central position in the nucleotide, with the base attached to
one of its carbons and the phosphate group (or groups) attached to another.

Nitrogenous bases

 The nitrogenous bases of nucleotides are organic (carbon-based) molecules made up

of nitrogen-containing ring structures.
 Each nucleotide in DNA contains one of four possible nitrogenous bases: adenine (A),
guanine (G) cytosine (C), and thymine (T).
 Adenine and guanine are purines, meaning that their structures contain two fused
carbon-nitrogen rings.
 Cytosine and thymine, in contrast, are pyrimidines and have a single carbon-nitrogen
ring. RNA nucleotides may also bear adenine, guanine and cytosine bases, but instead
of thymine they have another pyrimidine base called uracil (U).

Sugars

In addition to having slightly different sets of bases, DNA and RNA nucleotides also have
slightly different sugars. The five-carbon sugar in DNA is called deoxyribose, while in RNA,
the sugar is ribose.

Phosphate

Nucleotides may have a single phosphate group, or a chain of up to three phosphate groups,
attached to the 5’ carbon of the sugar.
Polynucleotide chains

A consequence of the structure of nucleotides is that a polynucleotide chain

has directionality – that is, it has two ends that are different from each other. At the 5’ end,
or beginning, of the chain, the 5’ phosphate group of the first nucleotide in the chain sticks
out. At the other end, called the 3’ end, the 3’ hydroxyl of the last nucleotide added to the
chain is exposed. DNA sequences are usually written in the 5' to 3' direction, meaning that
the nucleotide at the 5' end comes first and the nucleotide at the 3' end comes last.

Properties of DNA

Deoxyribonucleic acid, or DNA, chains are typically found in a double helix, a structure in
which two matching (complementary) chains are stuck together, as shown in the diagram at
left. The sugars and phosphates lie on the outside of the helix, forming the backbone of the
DNA; this portion of the molecule is sometimes called the sugar-phosphate backbone. The
nitrogenous bases extend into the interior, like the steps of a staircase, in pairs; the bases of a
pair are bound to each other by hydrogen bonds.

The two strands of the helix run in opposite directions, meaning that the 5′ end of one strand
is paired up with the 3′ end of its matching strand. (This is referred to
as antiparallel orientation and is important for the copying of DNA.) . Because of the sizes
and functional groups of the bases, base pairing is highly specific: A can only pair with T,
and G can only pair with C, as shown below. This means that the two strands of a DNA
double helix have a very predictable relationship to each other.
 Types of DNA
Eukaryotic organisms such as animals, plants and fungi, store the majority of their DNA
inside the cell nucleus and some of their DNA in organelles such as mitochondria.
Based on the location DNA may be:
Nuclear DNA
 Located within the nucleus of eukaryote cells.
 Usually has two copies per cell.
 The structure of nuclear DNA chromosomes is linear with open ends and includes
46 chromosomes containing 3 billion nucleotides.
 Nuclear DNA is diploid, ordinarily inheriting the DNA from two parents. The mutation
rate for nuclear DNA is less than 0.3%.
Mitochondrial DNA
 Mitochondrial DNA is located in the mitochondria.
 Contains 100-1,000 copies per cell.
 Mitochondrial DNA chromosomes usually have closed, circular structures, and contain
for example 16,569 nucleotides in human.
 Mitochondrial DNA is haploid, coming only from the mother.
 The mutation rate for mitochondrial DNA is generally higher than nuclear DNA.
Forms of DNA
 Most of the DNA is in the classic Watson-Crick model simply called as B-DNA or B-
form DNA.
 In certain condition, different forms of DNAs are found to be appeared like A-DNA,Z-
DNA,C- DNA,D-DNA,E-DNA.
 This deviation in forms are based on their structural diversity.
1. B-DNA
Most common, originally deduced from X-ray diffraction of sodium salt of DNA fibres at
92% relative humidity.
2. A-DNA
Originally identified by X-ray diffraction of analysis of DNA fibres at 75% relative humidity.
3. Z-DNA
Left handed double helical structure winds to the left in a zig- zag pattern.
4. C-DNA
Formed at 66% relative humidity and in presence of Li+ and Mg2+ ions.
5. D-DNA
Rare variant with 8 base pairs per helical turn, form in structure devoid of guanine .
6. E- DNA
Extended or eccentric DNA.
 Functions of DNA
DNA has a crucial role as genetic material in most living organisms. It carries genetic
information from cell to cell and from generation to generation.
Thus its major functions include:
 Storing genetic information
 Directing protein synthesis
 Determining genetic coding
 Directly responsible for metabolic activities, evolution, heredity, and differentiation.
It is a stable molecule and holds more complex information for longer periods of time.

DNA Denaturation and Renaturation

Denaturation of DNA Renaturation of DNA

Double-stranded DNAs are converted to single Denatured single strands of DNA, which are
strands complementary, form double strands

Denaturation occurs on heating Renaturation occurs on cooling

Unwinding of DNAs take place Rewinding of DNAs take place

In this process, hydrogen bonds between There is a formation of hydrogen bonds between
complementary base pairs of two DNA strands complementary base pairs of two strands to form
are broken double strands

The rate of UV absorbance (260nm) increases The rate of UV absorbance decreases

Viscosity decreases Viscosity increases

DNA Replication in prokaryotes

DNA Replication
In the process of DNA replication, the DNA makes multiple copies of itself. It is a biological
polymerisation, which proceeds in the sequence of initiation, elongation, and termination. It
is an enzyme-catalysed reaction. DNA Polymerase is the main enzyme in the replication
process.
DNA Replication Steps
Following are the important steps involved in DNA replication:

Initiation
 DNA replication demands a high degree of accuracy because even a minute mistake
would result in mutations. Thus, replication cannot initiate randomly at any point in
DNA.
 For the replication to begin there is a particular region called the origin of replication.
This is the point where the replication originates. Replication begins with the spotting
of this origin followed by the unwinding of the two DNA strands.
 Unzipping of DNA strands in their entire length is not feasible due to high energy
input. Hence, first, a replication fork is created catalysed by the helicase enzyme,
which unzips the DNA strand.

Elongation
 As the strands are separated, the polymerase enzymes start synthesising the
complementary sequence in each of the strands. The parental strands will act as a
template for newly synthesising daughter strands.
 It is to be noted that elongation is unidirectional i.e. DNA is always polymerised only
in the 5′ to 3′ direction.
 Therefore, in one strand (the template 3‘→5‘) it is continuous, hence called
continuous replication while on the other strand (the template 5‘→3‘) it is
discontinuous replication.
  They occur as fragments called Okazaki fragments. The enzyme called DNA ligase
joins them later.

Termination
Termination of replication occurs in different ways in different organisms. In E.coli like
organisms, chromosomes are circular. And this happens when the two replication forks
between the two terminals meet each other.

Role of Enzymes in DNA Replication

DNA replication is a highly enzyme-dependent process. There are many enzymes involved in
DNA replication, which includes the enzymes, DNA-dependent DNA polymerase, helicase,
ligase, etc. Among them, DNA-dependent DNA polymerase is the main enzyme.

DNA-dependent DNA polymerase

It helps in the polymerisation, catalyses and regularises the whole process of DNA replication
with the support of other enzymes. Deoxyribonucleoside triphosphates are the substrate as
well as the energy provider for the replication process. DNA polymerase is of three types:
DNA Polymerase I
It is a DNA repair enzyme. It is involved in three activities:

 5′-3′ polymerase activity

 5′-3′ exonuclease activity
 3′-5′ exonuclease activity
DNA Polymerase II
It is responsible for primer extension and proofreading.
DNA Polymerase III
It is responsible for in vivo DNA replication.

Helicase
Helicase is the enzyme, which unzips the DNA strands by breaking the hydrogen bonds
between them. Thus, it helps in the formation of the replication fork.

Ligase
Ligase is the enzyme which joins together the Okazaki fragments of the discontinuous DNA
strands.

Primase
This enzyme helps in the synthesis of RNA primer complementary to the DNA template
strand.

Endonucleases
These produce a single-stranded or a double-stranded cut in a DNA molecule.

Single-stranded Binding Proteins

It binds to single-stranded DNA and protects it from forming secondary structures.

DNA Replication Process in Prokaryotes

The DNA replication in prokaryotes takes place in the following place:

1. The two strands of DNA unwind at the origin of replication.

2. Helicase opens the DNA and replication forks are formed.
3. The DNA is coated by the single-strand binding proteins around the replication fork
to prevent rewinding of DNA.
4. Topoisomerase prevents the supercoiling of DNA.
5. RNA primers are synthesised by primase. These primers are complementary to the
DNA strand.
6. DNA polymerase III starts adding nucleotides at the end of the primers.
7. The leading and lagging strands continue to elongate.
8. The primers are removed and the gaps are filled with DNA Polymerase I and sealed
by ligase.

RNA STRUCTURE AND TYPES

 RNA, abbreviation of ribonucleic acid, complex compound of high molecular

weight that functions in cellular protein synthesis and replaces DNA (deoxyribonucleic
acid) as a carrier of genetic codes in some viruses.
 RNA consists of ribose nucleotides (nitrogenous bases appended to a ribose sugar)
attached by phosphodiester bonds, forming strands of varying lengths.
 The nitrogenous bases in RNA are adenine, guanine, cytosine, and uracil, which
replaces thymine in DNA.
 The ribose sugar of RNA is a cyclical structure consisting of five carbons and
one oxygen.
 The presence of a chemically reactive hydroxyl (−OH) group attached to the second
carbon group in the ribose sugar molecule makes RNA prone to hydrolysis.

Types and functions of RNA

 RNAs play important roles in both normal cellular processes and diseases.
  Of the many types of RNA, the three most well-known and most commonly studied
are messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA),
which are present in all organisms.

Types of RNA
In both prokaryotes and eukaryotes, there are three main types of RNA – messenger RNA
(mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA). These 3 types of RNA are
discussed below.

Messenger RNA (mRNA)

 mRNA accounts for just 5% of the total RNA in the cell.

 It carries complementary genetic code copied, from DNA during transcription, in the
form of triplets of nucleotides called codons.

Ribosomal RNA (rRNA)

 rRNAs are found in the ribosomes and account for 80% of the total RNA present in
the cell.

 Ribosomes are composed of a large subunit called the 50S and a small subunit called
the 30S, each of which is made up of its own specific rRNA molecules.

 Different rRNAs present in the ribosomes include small rRNAs and large rRNAs,
which belong to the small and large subunits of the ribosome, respectively.

 rRNAs combine with proteins and enzymes in the cytoplasm to form ribosomes,
which act as the site of protein synthesis.

Transfer RNA (tRNA)

 tRNA is the smallest of the 3 types of RNA, possessing around 75-95 nucleotides.

 tRNAs are an essential component of translation, where their main function is the
transfer of amino acids during protein synthesis. Therefore, they are called transfer
RNAs.

 Each of the 20 amino acids has a specific tRNA that binds with it and transfers it to
the growing polypeptide chain.
The secondary folded structure of tRNA has three hairpin loops, which give it an appearance
of three-leafed clover. The main constituents of tRNA are:
Acceptor arm
 It is formed by the base pairing of 7-9 nucleotides of 5’ terminal and 3’ terminal. The
5’ terminal has a phosphate group and the 3’ ends with a specific sequence of CCA or
CCA tail. The amino acid attaches to the 3’ hydroxyl group of the acceptor arm.
 The aminoacylation of tRNA or charging of tRNA is the first step of the translation
process. The enzyme aminoacyl tRNA synthetases catalyse the reaction.
DHU Loop
D arm has a stem of 3-4 base pairs and it ends in a loop called D loop as it generally contains
dihydrouridine, a modified nucleotide.
Anticodon Loop
It has a 5 base pair long stem. It has an anticodon loop, which contains the complementary
codon (3 nucleotides sequence) present on mRNA for the amino acid it carries. These
unpaired bases of anticodon loop pair with the mRNA codon. Each codon is identified by a
specific tRNA.
TΨC Loop
The T arm consists of a stem of 4-5 bp and a loop containing pseudouridine, modified
uridine.
Variable Loop
It is present between the TΨC loop and the anticodon loop. Its size varies from 3-21 bases. It
helps in the recognition of the tRNA molecule.
Other types of RNA
Beyond the primary role of RNA in protein synthesis, several varieties of RNA exist that are
involved in post-transcriptional modification, DNA replication, and gene regulation. Some
forms of RNA are only found in particular forms of life, such as in eukaryotes or bacteria.

DNA Repair Mechanism

DNA repair can be divided into a set of mechanisms that identify and correct damage in
DNA molecules. There are two general classes of DNA repair; the direct reversal of the
chemical process generating the damage and the replacement of damaged nucleotide
bases.

Excision is the general mechanism by which repairs are made when one of the double helix
strands is damaged. The non-defective strand is used as a template with the damaged DNA on
the other strand removed and replaced by the synthesis of new nucleotides. There are three
types of excision repair:

1. Base-excision repair.
2. Nucleotide excision repair.
3. Mismatch repair.

Base-excision repair involves the recognition and removal of a single damaged base. The
mechanism requires a family of enzymes called glycosylases. The enzymes remove the
damaged base forming an AP site which is repaired by AP endonuclease before the
nucleotide gap in the DNA strand is filled by DNA polymerase.

Nucleotide excision repair is a widespread mechanism for repairing damage to DNA and
recognizes multiple damaged bases. This mechanism is used to repair the formation of
pyrimidine dimers from UV light within humans. The process involves the recognition of
damage which is then cleaved on both sides by endonucleases before resynthesis by DNA
polymerase.

The third excision mechanism is called mismatch repair and occurs when mismatched bases
are incorporated into the DNA strand during replication and are not removed by proofreading
DNA polymerase. In mismatch repair, the missed errors are later corrected by enzymes which
recognize and excise the mismatched base to restore the original sequence.

DNA double strand break repair

The repair of damage to both DNA strands is particularly important in maintaining genomic
integrity. There are two main mechanisms for repairing double strand breaks: homologous
recombination and classical nonhomologous end joining.
Homologous recombination involves the exchange of nucleotide sequences to repair damaged
bases on both strands of DNA through the utilization of a sister chromatid. Classical
nonhomologous end joining connects the break ends without a homologous template through
the use of short DNA sequences called microhomologies. The mechanism is prone to error
but protects genome integrity from possible chromosomal translocations that can occur
through homologous recombination.