Nucleic acid Chemistry

and Metabolism

Dr. Meghana.K.Padwal
Vice Principal (UG)
Professor and Head
Department of Biochemistry
BV(DU)MC, Pune-43
 BHARATI VIDYAPEETH
(DEEMED TO BE UNIVERSITY)
Medical College,
PUNE – SATARA ROAD, PUNE – 411 043
Department of Biochemistry

Paper II

I M.B.B.S: Sr No 4: Nucleic Acids: Chemistry & Metabolism

Long Answer Question(LAQs):

1) Describe structures & functions of DNA &RNA. (5+5)

2) Describe in brief the synthesis & catabolism of Purines in the body. Add a note
on Gout. (4+1+5)

3) Describe the synthesis & breakdown of Pyrimidines. Add a note on disorders of

its metabolism. Mention the differences in purine and pyrimidine metabolism.
(4+2+2+2)

4) Describe the chemistry, organization, functions and replication of DNA with its
inhibitor. Add a note on DNA repair mechanisms. (2+1+1+4+2)

5) Describe the different types of RNA’s. Add a note on Transcription with its
inhibitors. (6+4)
Short Essay Question(SEQs):

a) Purine salvage pathway

b) Synthetic and biologically important free nucleotides

LAo(
c) Types of RNA

d) Replication

e) Transcription

f) Cell cycle DNA damage and repair.

5
 Nucleic acid Chemistry
Nucleic acids: are biopolymer of nucleotides linked together by
phospho-diester bonds e.g DNA &RNA
components?
Nucleotide Nucleoside

Nucleoside + PO4 Sugar + Base

Base
PO4 N- Base

Sugar Sugar

Sugar: Pentose: Ribose (RNA) or Deoxyribose (DNA)

 Nitrogenous Bases

Purines & Pyrimidines

Purines

e.g. 1) Adenine (DNA & RNA)

2) Guanine (DNA & RNA)
 Pyrimidines

e.g in DNA e.g in RNA

1)Thymine 1)Uracil
2)Cytosine 2)Cytosine
Base Nucleoside Nucleotide
Base +Sugar Nucleoside + PO4

Adenine Adenosine AMP(Adenosine

Mono phosphate)

Guanine Guanosine GMP

Uracil Uridine UMP

Cytosine Cytidine
CMP
Thymine Thymidine TMP
Polynucleotides:
• Precursors of nucleic acid
• Nucleotide joined together by 3’-5’
phosphodiester linkages to form polynucleotide
Cyclic nucleotides:
• Hydroxyl group of ribose forms bond with
phosphate group and results cyclic structure
• Ex. Cyclic AMP, cyclic GMP
 Biologically Important Free Nucleotides
(not involved in formation of DNA & RNA)
A) Adenosine Derivatives:
1. ADP and ATP:
▪ High Energy Compounds.
▪ ATP “Energy Currency” of the cell.
2) AMP:Constituent of RNA and DNA
▪ cAMP: Second Messenger in Hormone action.
3)PAPS: Phospho adenosine phospho sulphate
▪ “Active Sulphate” Sulphate Donar for detoxification
4)SAM: S -Adenosyl Methionine
▪ -active form of methionine
▪ - Participates in transmethylation reactions.
5)Coenzymes: NAD,NADP,FMN,FAD, COASH
-Participating in oxidation reduction reactions.
B) Gunosine Derivatives:
1)GDP & GTP: High Energy Compounds
▪ Energy sources in “Protein Biosynthesis”.
▪ 7 methyl GTP cap at 5’end of mRNA
2) cGMP: Second Messenger in Hormone action.
c) Uracil Derivatives:
1)UDP Glucose: Glucosyl Donor for Biosynthesis of glycogen,
glycoproteins, galactose, proteoglycans.
2) UDP Glucuronic acid: Glycosidic acid donor in conjugation
reactions of detoxification.
E.g.: Bilirubin, Drugs
• D) Cytosine Derivatives:
1) CTP
2) CDP- Choline Phospholipid Synthesis
Synthesis of sphingomyelins
 Synthetic nucleotides
• Structural and synthetic analogues of purine and pyrimidine
nucleotides and nucleosides.

• They act as competitive inhibitors of the enzymes essential for

synthesis of naturally occurring nucleotides, therefore arrest
DNA replication and cell growth.

• They have tremendous pharmacological applications as:

• A)Anticancer agents:
▪ Azaserine,
▪ Mercaptopurine : analogue of inosine
▪ Fluorouracil: analogue of thymine
▪ Hydroxyurea : inhibits ribonucleotide reductase
B) Antiviral agents:
Inhibit viral DNA replication, so used as antiviral
agents.
Zidovudine/AZT- anti HIV drug (Human Immuno
Deficiency Virus)
Remdesivir: antiviral drug, replication and
proliferation of RNA virus by blocking RNA
polymerase (SARS –cov 2:Covid 19)
Arabinosyladenine: used in viral encephalitis
 Sources of Nucleoproteins
• Fish, Eggs, Meat and seafood.
• Dairy food such as milk and yoghurt
• Beans and pulses, Nuts
• Soy and tofu products.
 Digestion of dietary nucleic acids

Mouth Nucleoproteins

Nucleoproteins Proteins Amino acids

HCl
Stomach
Denatured Nucleic
acids
Nucleases, Phosphodiesterases
Pancreas
Nucleotides
Nucleotidases Pi
Circulation
Uric acid
Nucleosides
Intestinal
mucosal cell
Nucleosidases
Sugars
Bases
(Purines / Pyrimidines)
Urine
Synthesis of Purine Nucleotides
1) Denovo Synthesis(a new)
• Purine ring is synthesized on pre existing
ribose -5 -phosphate
• In all tissues except in brain, RBCs and WBCs
2) Purine Salvage Pathway:
• Utilizes Pre existing nucleosides
• Only in brain, RBCs and WBCs
How Purine
synthesis?
 PURINE NUCLEOTIDE SYNTHESIS: Denovo Pathway

Purine Ring : Sources

Carbon Dioxide
Glycine
Aspartate

N5N10 –Methenyl-THF
N10 – Formyl-THF

Glutamine

Synthesized by all tissues except Brain & RBC

Location:- Cytosol , Major site- Liver
 DENOVO PATHWAY
Ribose 5 Phosphate (HMP Shunt)
PRPP Synthase ATP
Mg+
Regulatory enzyme AMP
PRPP (Phospho ribosyl pyrophosphate)
Amidotransferase
(Rate limitting step,
AMP & GMP inhibit Glutamine
the enzyme)
Glycine
N5N10 Methenyl FH4
ATP, Mg2+
Key
Enzymes? CO2
Aspartate
N10 Formyl FH4
IMP (Inosine monophosphate)
(Product feedback inhibition)
 IMP IMP
ATP
GTP Glutamine
Aspartate NAD+
Synthase Dehydrogenase
Adenylosuccinase Amidotransferase
GDP+Pi Fumarate Glutamate NADH + H+
ADP + Pi

AMP GMP

Kinases Kinases

ADP and ATP GDP and GTP

Ribonucleoside diphosphate reductase
dADP and dGDP
 Inhibitors of purine nucleotides

1) Mercaptopurine:
inhibits formation of AMP &
GMP from IMP.
2) Azaserine:
Anticancer
acts as glutamine
agents
antagonists and affects
AMP & GMP formation.
3) Methotrexate:
Inhibit formation of THF
 Purine Salvage Pathway
• Salvage: Saved from being lost or waste.
• Free Purine Bases and nucleosides that are
formed during the course of turnover of cellular
nucleic acids, are recycled to form Purine
nucleotides.
• Thus pathway is used to recover & conserve
bases and nucleoside.
• Occurs in Brain, RBCs & WBCs where De novo
Pathway is not operative.
• Requires far less energy compared to denovo
synthesis.
Phosphoribosylation of Free Bases (major)
1) Adenine Phosphoribosyl Transferase
APRTase
PRPP PPi

Adenine AMP

Hypoxanthine Guanine Phosphoribosyl

Transferase
2) PRPP HGPRTase PPi

Guanine GMP
OR OR

Hypoxanthine IMP
 Phosphorylation of Nucleosides (minor)
1)

Adenosine ATP ADP

AMP
Kinase

2)
ATP ADP
Guanosine Kinase
GMP
 Catabolism of Purine nucleotide
• AMP and GMP are mainly catabolised
• End product is Uric acid
• Takes place mainly in Liver
 AMP GMP
Nucleotidase
Adenosine
H2O
Adenosine deaminase
NH3
Inosine Guanosine
Pi Purine nucleoside Pi
R-1-P phosphorylase
R-1-P
Hypoxanthine Guanine
O2 H2O
Xanthine Guanase
oxidase H2O2 NH3

H2O Xanthine
O2 H2O
H2 O 2 Xanthine oxidase
Uric Acid
• End product of purine catabolism is uric acid
• Normal Range of Uric acid : 2-7 mg/100 ml of serum

a) Hypouricemia: Uncommon

b) Hyperuricemia:
• Hyperuricemia is a condition in which serum urate
level is increased above normal level and exceeds its
solubility limit.

• Uric acid & urates are relatively insoluble molecules,

which can readily precipitate out of aqueous
solutions like urine or synovial fluid.
 Disorders of Purine metabolism:
1. Adenosine deaminase deficiency/SCID:
Severe combined immunodeficiency(SCID)-damage of
immune system

• Autosomal recessive.
• ADA deficiency leads to defective breakdown of purine
nucleotides which accounts to Hypouricemia

• In SCID, both T and B lymphocytes are deficient &

along with ADA deficiency , Purine nucleoside
phosphorylase deficiency (involved in uric acid synth)

• ADA therapy & for SCID-first disease successfully

treated with gene replacement therapy.
2. GOUT:
Clinical Syndrome characterized by acute or prolonged
Hyperuricemia (Normal Range 2-7mg/100ml)

Deposition of Sodium urate Crystals

in & around joints & renal tubules

Inflammation of Joints ,Urolithiasis

Acute arthritis & Severe pain

• Uric acid crystals are
deposited in the
extremities with a
surrounding area of
inflammation.

•This is called a tophus

and is often described
as an arthritic “great
toe”.
 Types and causes of Gout
Type A-Primary Gout:
Causes:
• HGPRTase deficiency (Lesch Nyhan Syndrome)
• Increased activity of PRPP synthase & Amidotransferase
• Glucose 6 Phosphatase deficiency (Von Gierke’s Disease)
Type B- Secondary Gout:
Causes:
• Increased cell destruction
• Alcoholism
• Impaired Renal function
• Lactic acidosis
 Types & Causes of Gout
A) Primary (Genetic):-
i) HGPRTase deficiency (Lesch Nyhan Syndrome)
HGPRTase Purine Salvage Pathway
PRPP
More Catabolism of Purines

Uric Acid Gout

ii) Increased activity of PRPP synthase & Amidotransferase

PRPP synthase & Amidotransferase PRPP AMP & GMP

Uric acid Gout

iii)Glucose 6 Phosphatase deficiency (Von Gierke’s Disease)
Glycogen / Glu-6- Phosphatase (Von Gierke’s Disease)
Glucose G6P Glucose
HMP shunt

Ribose 5-P

PRPP

GMP AMP

Uric acid
 Types & Causes of Gout
B) Secondary:-
i) Increased cell destruction: More formation of uric acid
e.g leukemia, polycythemia, Pregnancy Induced hypertension (PIH) :
etc.

ii) Alcoholism: Metabolism of alcohol in our body leads to

production of lactic acid. There is competition between lactic acid
and uric acid for excretion. Lactic acid being more soluble is
excreted and uric acid is retained in blood leading to acute gout.

iii) Impaired Renal function:- Eg. Chronic Renal Failure

Decreased Uric acid excretion through urine leading to accumulation
in blood ----Hyperuricemia.
Clinical features:
• Joint pain
• Tophus formation
• Stone formation
Diagnosis:
• Estimation of serum uric acid
• X-ray of joints
• Light polarising microscopy
• Urinary Uric acid (spot /24 hrs)
 Treatment of Gout
1) Diet: Low Purines, Avoid Non vegetarian Diet &
Alcohol
2)Anti-inflammatory Drugs: Colchicine

3)Allopurinol (Primary Gout): Competitive Inhibitor (Suicidal

Inhibitor) of Xanthine oxidase, forms alloxantine, more
potent Inhibitor

4)Probenecid (Secondary Gout) Uricosuric drug which

decreases reabsorption of uric acid via kidney tubules
and promotes excretion in urine.
 Catabolism of Purine Nucleotides
AMP Nucleotidase
GMP
Adenosine
H2O
Adenosine deaminase
NH3
Inosine Guanosine
Purine nucleoside Pi
Pi
R-1-P phosphorylase
R-1-P
Hypoxanthine Guanine
O2 H2O
Xanthine Guanase
oxidase H2O2 Allopurinol NH3

H2O Xanthine
O2 H2O Allopurinol
H2 O 2 Xanthine oxidase
Uric Acid
 Lesch Nyhan Syndrome
• Inherited X- linked, Affects only males
• Complete deficiency of HGPRTase (Purine Salvage
Pathway: Brain and RBCs)
• Symptoms: Hyperuricemia, Gout, Urinary stone,
neurological symptoms, Self mutilation (Self destructing
behaviour like biting own fingers,nails,tounge)
• Biochemical Basis:
• Brain is totally dependant on salvage pathway for
production of purine nucleotides,
• Therefore absence of the enzyme leads to deprivation
of purine nuclotides to brain leading to poor motor
activities and above symptoms.
• Treatment:- Allopurinol - reduces uric acid formation.
• Death occurs due to uric acid nephropathy
Biosynthesis of Pyrimidine
Nucleotide
Pyrimidine Ring:- Sources

Glutamine
Aspartate

Carbon
Dioxide
 CO2 + Glutamine + ATP
Carbamoyl phosphate
synthetase II Aspartate
transcarbamoylase
Carbamoyl phosphate Carbamoyl
aspartic acid

dehydrogenase
NAD

CO2 NADH +H+

UMP OMP
PRPP
Orotic acid
Decarboxylase phosphoribosyl
Transferase
 UMP
ATP d-CDP &
Kinase
Ribonucleotide
ADP d-UDP
+Pi reductase H2 O
UDP
NADPH + H+ NADP Pi
ATP
Kinase d- UMP
ADP + Pi N5N10 Methylene FH4 Thymidylate
UTP
Synthase
Synthetase Glutamine FH2
dTMP
Glutamate Thymidylate Kinase
CTP
dTDP
CATABOLISM OF PYRIMIDINES
 Cytosine
½ O2
NH3
Thymine
Uracil NADPH+ H+
NADPH+ H+
NADP
NADP
Dihydrouracil Dihydrothymine
H2O
H2 O
β Aminoisobutyrate
β-alanine

CO2 + NH3
What is difference between purine and
pyrimidine metabolism?
 Difference between Purine &
Pyrimidine Metabolism
Purine nucleotide Pyrimidine nucleotide
Metabolism Metabolism

Builds on the back bone PRPP is added much

of PRPP later

Product of Catabolism Products of catabolism

Uric acid is relatively CO2,NH3, Beta alanine
insoluble, thus results soluble, so no toxicity
hyperurecemia leads to symptoms
Gout
 Disorders of pyrimidine metabolism
Orotic aciduria:
• Autosomal recessive due to deficiency of
enzymes 1. orotate phosphoribosyl transferase &
2. decarboxylase

Reye syndrome: due to deficiency of ornithine

transcarbamoylase of urea cycle----accumulated
carbamoyl-P is diverted for orotic acid synthesis.
Structure and functions of DNA
&RNA
DNA (Deoxy -ribonucleic acid)
Hydrogen Bond

Major
groove

Minor
groove

Watson & Crick Model (B- DNA)

 Features
1. Right handed double helix:
DNA is composed of 2 strands twisted around each
other in right handed helical structure.
2. Antiparallel:
Two strands of DNA are antiparallel i.e one strand
runs in 5’-3’ direction and other in 3’-5’ direction.
3. Base Pairing(Chargaff’s rule):
Nucleotide bases on one strand interact with
nucleotide bases on other strand to form base pairs by
hydrogen bonding. e.g Adenine pairs with Thymine
by 2 hydrogen bonds & cytosine with Guanine by 3
hydrogen bonds.
 Erwin Chargaff – (1905-2002)

5’ 3’

T A G C A C
A T C G T G

3’ 5’
4. Backbone of DNA: Sugar & phosphate groups
(hydrophilic) oriented outside and bases
(hydrophobic) are stacked inside.
5. Winding of 2 chains creates minor (shallow)
and major (deep) groove.
6) One strand act as sense or coding or non
template strand and another one is non sense or
non coding or template strand.
 Organization of DNA molecule
• Just like structure of protein, primary structure
of DNA is its nucleotide sequence.
• It is coiled in the form of double helix which is
then further super coiled.
• Super coiling is the intrinsic property of DNA,
which is evident by packing of 2 meter of DNA
in a tiny nucleus along with protein histone.
 Chromosomes, Gene and DNA
• Chromosomes is the storage form of genetic
information visible under light microscope.
• In humans there are 23 pairs of
chromosomes(Diploid)
• Gene is a part on DNA that encodes (transcribes)
for protein.
• DNA is organized into chromosomes along with
protein histone (Forming Nucleoprotein) to form
a compact structure called chromatin.
Functions
1. DNA is the chemical basis of heredity &
regarded as reserve bank of genetic
information.
2. Serves as template for process of replication
& transcription.
3. DNA’s major function is to code for proteins.
Information is encoded in the sequence of the
nitrogenous bases.
Junk DNA:
•Almost 90% of DNA is transcriptionally inactive
(protein bound) ,this is known as Junk DNA.
•To discover the function of Junk DNA is the major
challenge in the field of molecular biology.
Active DNA:
• DNA that is transcriptionally active, expressed and
forms protein is called active DNA.
 Mitochondrial DNA (mtDNA)
• It has maternal inheritance.
• It is passed from mother to daughters and sons
but sons can not transfer to their children.
• It has information for synthesis of components of
ETC.
• It helps to track the ancestry.
• Defect in mitochondrial DNA will lead to OXPHOS
diseases. (Refer Biological Oxidation)
 RNA (Ribonucleic acid)
• Polymer of purine & pyrimidine nucleotides
linked by phosphodiester bridges.
• Sugar is ribose sugar.
• 3 main classes of RNA molecule in prokaryotes
& eukaryotes.
1. mRNA
2. tRNA
3. rRNA
 mRNA(Messenger RNA)
• Carries message from DNA to ribosomes.
• mRNA is nearly 5% of cellular RNA.
• This RNA is variable in size & relatively stable .
• Template strand of DNA forms hnRNA
(heterogenous nuclear RNA) which are processed to
form active mRNA.
• Active mRNA in eukaryotes have unique characteristics i.e
- 5’ end is capped by 7-methylguanosine triphosphate
- 3’ end has attached poly A tail (polymer of adenylate
residue).

• The cap is involved in recognition & stabilization of mRNA

• Poly A tail maintains intracellular stability of mRNA

• Message lies in linear sequence of nucleotide for

translating it into polypeptide chain.
tRNA (transfer RNA)
• Cellular composition is 10-20%
• tRNA vary in length from 74-95 nucleotides &
contain unusual nucleotides also.
• 20 species of tRNA in every cells, each differ from
other.
• Structure appears like clover leaf, contains 4 arms.
1. Acceptor arm
2. D arm
3. Anticodon arm
4. TψC arm
tRNA (transfer RNA)

Hydrogen bond

Extra arm
Acceptor arm:- consist of base paired stem ,
terminates in sequence CCA at 3’ end ,is
attachment site for amino acid .
D arm:- named D because of presence of base
dihydrouridine.
Anticodon arm:- Contain anticodon that base pairs
with codon on mRNA.It has nucleotide sequence
complementary to codon of mRNA& is responsible
for specificity of tRNA.
T ψ C arm:-for sequence T, pseudouridine, C
Extra arm:- known as variable arm, found between
anticodon & T ψ C arm.
Functions:
1. Transfer amino acids from cytoplasm to
ribosome.
2. tRNA serves as an adapter for translation of
information in the sequence of nucleotide of
mRNA into specific amino acids.
 rRNA(ribosomal RNA)
• Cellular composition is 50-80% of RNA.
• Ribosome act as machinery for synthesis of protein.
• In active protein synthesis ribosomes associated with
mRNA in assembly called polysome.
• Each ribosome dissociated into 2 subunits e.g

80S ribosome
of eukaryotes

60S

40S
Functions:-
1. Key role in binding of mRNA to ribosome & help
in translation.
2. May be acting as “peptidyl transferase” enzyme
in protein synthesis.

SnRNA(small nuclear RNA):-

• Subgroup of RNA
• Involved in RNA processing.
 RNA DNA
Sugar Ribose Deoxyribose
Purines Adenine, Guanine Adenine, Guanine
Pyrimidine Cytosine, Uracil Cytosine, Thymine
Chargaff’s Not Obeyed: Total purine Obeyed: Equal
Rule &pyrimidine Not equal
Structure Single stranded Double stranded
Location Present in cytoplasm & nucleus Present in nucleus &
mitochondria
Size Comparatively small Very large
Linkage Nucleotides linked with phosphodiester linkage
Function Protein synthesis • Chemical basis of heredity
• Protein synthesis
Interaction Interact with proteins but do not Interact with proteins
with to form chromatin and to form chromatin
 Human Genome Project
• Human Genome is the complete genetic
information of an organism occurring in the
DNA in each cell.
• Human Genome Project:( HGP)
• It is international scientific project with the
goal of determining the sequence of base
pairs which make up human DNA and
identifying mapping of all genes of human
genome.
 Central Dogma
Replication

DNA mRNA Protein

Transcription Translation

Reverse Transcriptase
•Retroviruses like HIV(AIDS) ,Rous Sarcoma (Cancer)contains the enzyme
reverse transcriptase. (Discovered by Baltimore and Tenin 1975:
awarded Noble Prize)
•These viruses invade the host cells, converts viral RNA to DNA by
reverse transcriptase and then introducing this DNA in host cell.
•This disturbs the whole process of Transcription and Translation in host
cell resulting in respective diseases.
 Replication(DNA synthesis)
Definition:
Replication is a process in which DNA copies itself to produce identical
daughter molecule of DNA during cell division.
Principles of Replication:
1) Copying is accurate:
As the genetic information is carried from generation to generation,
integrity of the DNA must be preserved at any cost. This needs
accurate replication. To overcome this task and to achieve fidelity ,
the process of replication is endowed with proof reading ability.
2) Semi-conservative replication:
Half of the original parent DNA molecule is conserved in each of the
daughter molecules. (replicated DNA contains 1 parent strand & 1
newly synthesized strand.)
3) Supercoils and Topoisomerase:
Topoisomerase enzymes helps in unwinding of supercoiled DNA which
needs access for unwinding purpose.
Requirements:
• Substrate:-d-ATP, d-GTP, d-CTP, d-TTP
• Template strand
• Enzymes: - DNA polymerases
- DNA topoisomerases
• Primer: RNA primer
Process of replication involves
1. Initiation
2. Elongation
3. Termination
• Initiation of DNA synthesis occurs at the site called origin
of replication(Ori)
• Site consist of short sequence of AT base pairs.
• DNA binding protein (A,B,C) binds to site & separates the
strand.
• Two strands of DNA separate at site of replication to form
bubble for rapid replication.
• Active synthesis starts at the region, that region is called
replication fork.
• Replication is bidirectional because fork moves in both
direction from origin (5’-3’ & 3’-5’).
• One strand is leading strand-continuous synthesis of DNA
& other strand is lagging strand- discontinuous synthesis.
• SSB(single stranded DNA binding protein): stabilizes the
separated strands & prevents their re-association.
• Okazaki fragments: small fragments of discontinuously
synthesized DNA (lagging strand)
• Proof reading: It checks incoming nucleotide by DNA
polymerase
Enzymes involved in replication
• Helicase: binds to DNA at the replication fork , causes
unwinding of the 2 strands, ATP dependant. (unzip effect)
• Polymerase: multiple types, responsible for the actual
synthesis of DNA
• Ligase: brings about joining of Okazaki fragments catalyzed
by DNA Ligase
• Primase: Adds an RNA primer so that DNA synthesis can
begin.
Replication

DNA helicase

SSB

Primer
The unwinding Primase
DNA
causes torsional HELICASE
Polymerase
strain in the helix RNA primer

SSB
REPLICATION
FORK

Topoisomerase
 3’

5’

3’
5’
3’

5’
Ligase
 Inhibitors of DNA replication
Drug Action(inhibition of) Enzymes
Antibacterial agents:
Ciprofloxacin Bacterial DNA gyrase
Nalidixic acid Bacterial DNA gyrase

Anticancer agents:
Doxorubicin, Human topoisomerase
Actinomycin D
 DNA Damage and Repair
➢ DNA stores, maintains and passes genetic information to
future generation by Replication.
➢ High degree of accuracy is maintained in copying the
information.
➢ Errors may occur during replication despite the proof
reading result in mutation that is, permanent change in
nucleotide sequence of DNA.
➢ Altered DNA may cause structural damage to DNA
molecule, and can eliminate cell’s ability for transcription.
➢ To avoid this the DNA repair process is constantly active
and cell immediately responds to damage in DNA structure.
• Causes of DNA damage
• Errors in replication
• Free radicals
• Environmental factors : Chemical agents ,UV
Radiations
• Chemotherapy

• Types of DNA damage:

▪ Single base alteration
▪ Two base alteration
▪ Chain breaks
▪ Cross linkage
 DNA Repair mechanisms
• DNA repair is a collection of processes by which a cell
identifies and corrects damage to the DNA molecules
that encode is genome.
• Repair of damaged DNA is essential for maintaining
genomic integrity, prevention of propagation of
mutation in somatic cells as well as germ cells.
• Failure of DNA repair leads to faulty replication,
transcription ,apoptosis or mutation and cancer.
• Mechanism of DNA repair:
• Base-excision
Special enzymes replace just the defective base
• Nucleotide-excision
Removal of larger segments of DNA (10 -100s of bases)
• Mismatch repair:
Special enzymes scan the DNA for bulky alterations.
Diseases associated with defective DNA
repair mechanism:
• Xeroderma Pigmentosum
• Cockyane Syndrome
• Ataxia Telangiectasia
• Fanconi’s anemia
• Bloom’s syndrome
 DNA ISOLATION FROM BLOOD/TISSUE

• Introduction:
• Isolation of DNA is a process of purification using a
combination of physical and chemical methods and
it requires careful handling to avoid sample
contamination.
• DNA extraction procedures have common elements
due to the presence of lipid layer on the outer
membrane, proteases, magnesium and coiling of DNA
around histones.
 The isolation method of choice is
dependent upon
a. The source of the DNA: blood, tissue, bacterial,
virus etc.;
b. The final application: PCR, sequencing,
fingerprinting, library construction, etc.
c. The type of DNA: genomic vs plasmid.
 Steps of DNA isolation
• Cells are lysed during a short incubation with
chemotropic salt, which immediately inactivates all
nucleases.
• Cellular nucleic acids bind selectively to special glass
fibers pre-packed in the purification filter tube.
• Bound nucleic acids are purified in a series of rapid
“wash and spin” steps to remove contaminating
cellular components.
• Finally, low salt elution releases the nucleic acids
from glass fiber.
The extraction of DNA
generally follows common
steps:
1.Lyse (break open) the cell
2.Separate the DNA from the
other cell components
3.Isolate the DNA
 Applications of isolated DNA:
• The isolated DNAs are principally used in the polymerase
chain reaction to:

a. Identify point source of community and hospital based

outbreaks. E.g. COVID 19, Tuberculosis etc.

b. Predict virulence of microorganisms.

c. Gene cloning.

d. In forensic science identification of rapists, accident, war

victims and paternity determination.
Q1. Identify the procedure. 1 Mark

DNA isolation

Q2. Mention any two indications. 1 Mark

a. Polymerase chain Reaction: Tuberculosis, COVID 19
b. To predict virulence of microorganisms
c. Gene cloning
d. In forensic science
 Assignment
Assignment correction will be done batchwise from 29/5/2024 to 03/06/2025 during
Revision cycle

Batch Date Question to be written in journal on page no.166

D 29/05/2025
Draw the diagram showing the steps of DNA isolation

C 30/05/2025
Write the principle of DNA isolation

B 02/06/2025
Draw the diagram showing the steps of DNA isolation

A 03/06/2025
Write the applications of DNA isolation
 Cell Cycle
• Cell cycle is a life cycle of a cell.
• The term cycle refers to the events occurring during the
period between two mitotic divisions
• Total duration is 12 to 24 hrs., depending on type of cells.
• Cell cycle represents the formation of two New daughter
cells by copying its genetic material (DNA) and physically
split into two daughter cells by the division of mother cell.
• Phases of cell cycle:
1) Interphase: Cell grows and makes a copy of its DNA.
2) Mitotic Phase : (M Phase): Cell separates its DNA into
two sets and divides its cytoplasm (cytokinesis ) forming
two new cells.
• It is divided into G1(gap-1), S (synthesis),G2 (gap-2), and
M(mitosis) phases.
 Cell cycle control/Regulation or checkpoints
• Checkpoint is a stage in eukaryotic cell cycle where the cell
examines internal and external cues and decides whether
to move forward or not with cell division.

• Four types of cyclins(A,B,D,and E) and five different types of

cyclin dependent kinases(CDK1,2,4,5 and 6) control the
cycle.

• Cancer and Cell cycle:

• The genes that code for positive cell cycle regulators are
called as proto oncogenes , which when mutated gets
converted to oncogenes which can cause cancer.

• The genes that code for negative cell cycle regulators are
called as Tumor Suppressor genes, which can halt cell cycle
and stop its multiplication.
 Cancer and Cell cycle:
• Retinoblastoma protein(Rb): inhibits cell cycle at
G1phase.
• Certain tumor antigens derived from viruses such
as SV40,HSV,HPV may combine with Rb.
• Rb cannot inhibit cell cycle, leading to continous
cell division and cancer.

• p53 oncosuppressor protein:

Inhibits cell division, allowing them to repair.
If damage is extensive and repair not possible, p53
then directs the cell to apoptosis.
 Transcription
Definition: Transcription is defined as the synthesis of
RNA molecule from DNA. It results in transfer of
information stored in DNA to RNA, which is used for
protein synthesis.

• One strand serves as template i.e coding strand or

sense strand, which produces working copies of RNA

• Other strand does not participate in transcription is

referred as non-coding or antisense strand.
 Selective transcription

• Entire molecule of DNA not expressed in

transcription.

• RNAs are synthesized from some selected

regions of DNA (this may be due to some
inbuilt signals in DNA).
• Eukaryotes possess 3 RNA polymerases , synthesize all
the RNAs.
Initiation: RNA polymerase bind to DNA to start
transcription
• The enzyme binding site is called promoter region.
Enzyme recognizes a specific sequence on DNA for
transcription
Elongation: Addition of nucleotide to growing chain .
Mistakes in RNA are less dangerous & not transmitted to
daughter cell.
Termination: by termination signals like Rho dependant
& Rho independent termination
Transcription mRNA transcript (hnRNA)
Polymerase

mRNA
Post transcriptional
modifications
(splicing, base addition
cleavage)
Active mRNA
 Inhibitors of Transcription
Inhibitor Mode of action

Alpha Amantinin Inactives RNA polymerase II.

(Toxin of Mushroom) Leading to cytolysis of hepatic
cells, diarrhea, cramps,
leading to hepato- reanal
failure. etc
Rifampicin Inactivates RNA polymerase
(Ant tubercular Drug)
 Genetic Code
• Definition:
• The three nucleotide (triplet) base sequences in mRNA
that code for amino acids in protein synthesis are
called codons or genetic code.
• Codons are composed of four Bases :
• A, U, G, C
• (4)3 = A set of 64 Codons
• Chain Initiation codon: AUG (methionine)
• Chain termination (Nonsense/Stop) codons:
3 codons (UAA,UAG,UGA).
• 61 code for 20 amino acids.
Importance of Genetic Code
1) To understand protein biosynthesis.
2) To understand mutation.
3) Diagnosis and probable treatment of genetic
disorders.
4) To understand pathophsiology of viral
infection that can invade host cell and disturb
protein biosynthesis.
114
 Characteristics /Properties of
Genetic Code
1. Universal:
• Codons code for same amino acid through out all
the species. It has been highly preserved during
evolution
• e.g. AUG code for methionine.
2. Non-overlapping :
• Linear sequence, read one after another, Starting
point is extremely important, does not overlap.
• The addition or deletion of 1 or more alters the
message sequence in mRNA & results in synthesis
of different protein.
3. No punctuations: Read Continuously
4. Reads in 5’ → 3’ direction
5. Degeneracy :
• 61 codons: 20 amino acid
• So 1 amino acid may have more than one codon.
• UUU & UUC → Phenylalanine
• Amino acids No. of codons
Histidine 2
Serine 6
6. Unambiguous:
• There is no ambiguity (confusion)
• One codon will code for one amino acid only.
• e.g UUU code for Phenylalanine, not for other amino acid.
7. Wobble Hypothesis
• The first two bases of the codon strictly follow the
Watson-Crick base pairing rule with anticodon while
the third base is less stringent.
• Pairing of codon and anticodon can wobble at 3rd
base
• The first two bases of the codon are important in
determining the amino acid in polypeptide chain.
• One tRNA will recognize multiple codons of that
amino acid
• AGA/AGG: Arginine
• UCU, UCC, UCA & UCG: Serine
 Mitochondrial Codons
• Mitochondrial DNA(mtDNA) is not transmitted
through nuclear DNA.
• Inherited only from the mother’s ovum.
Codon Cytoplasm Mitochondria
AUA Isoleucine Methionine
UGA Stop codon Tryptophan
AGA/AGG Arginine Stop codons
119
 Mutation
•Definition: Sudden permanent change in the
nucleotide sequence of DNA.
•Mutagens: Substances that induce mutation.
•Causes of mutation: faulty replication, use of anti-
malignant drugs, use of nitrous acid which
deaminates adenine , adenine pairs with thymine
while hypoxanthine with cytosine,viruses.
•Carcinogenic mutation leads to change of somatic
cell to carcinogenic cell./ Mutation in somatic cell
may result in uncontrolled cell division leading to
cancer.
 Point Transition: Purine by Purine,
Mutation Pyrimidine by Pyrimidine
T ↔C OR A ↔ G

Mutation
Transversion: Purine by Pyrimidine
Pyrimidine by Purine
T ↔ G OR C ↔ A
Frameshift
mutation
 Effects of point mutation

Point
mutation

Silent Nonsense
Missense

Partially
Acceptable acceptable
Unacceptable

Functional Abnormal Non-functional

protein function protein protein
e.g. Hb Hikari e.g. Hb S e.g. Hb M
Silent Mutation:
• The changed base could be the third base of the codon
• Due to degeneracy of the genetic code it may code for the same
amino acid or it may be in the nonfunctional region of the
protein
• (GAA and GAG both code for glutamic acid)
• This does not reflect any change in the protein, thus silent
mutation
Missense Mutation:
• The changed base codes for a different amino acid GAG codes
for glutamic acid, while GUG codes for valine
Nonsense Mutation:
• The changed base in the codon may convert the codon to a
termination codon .
• E.g. : UCA for serine to UAA termination codon. The altered
codon terminates protein synthesis. (Thalassemia)
Missense Exampl Base
Effect on function
Mutation e change

β chain 61 -
Acceptable
Hb Hikari Lysine to No change in Hb function
Missense
Asparagine

β chain
Partially 6– Hb S can bind to oxygen but
acceptable Hb S Glutamic deoxygenated form causes
Missense acid to polymerisation
Valine
Alpha
chain, oxidation of ferrous iron in heme to
Unacceptabl
Hb M 58- ferric state, does not bind to
e Missense
Histidine to oxygen at all
Tyrosine
 Frame Shift Mutation
Entirely different sequence

Protein -- Met----------Ala--------Leu----------Ala----------Lys--------n

mRNA -- AUG--------GCC--------CUU--------GCA--------AAG--------n

- 1 U (Deletion)
mRNA -- AUG--------GCC--------UCU--------UGC--------AAA
Normal
Protein -- Met--------Ala---------Ser----------Cys----------Lys

+ 1 C (Insertion)
mRNA -- AUG--------GCC-------CUC--------UUG--------CAA-----------n

Protein -- Met----------Ala------Leu----------Leu----------Gly-----------n

Entirely different sequence

 Frame shift Mutations

• Insertion or deletion of a base leads to an altered

reading frame on the mRNA.

• As there are no punctuation marks on Genetic code,

this change won’t be understood.

• This will lead to formation of a protein with several

altered amino acids and/or premature chain
termination.
 Mitochondrial DNA mutations

• Mt DNA is highly susceptible to somatic or non-inherited

mutations as they lack histone proteins.
• Eg. OXPHOS diseases:. A group of clinically heterogeneous
diseases, lack of cellular energy due to defects of oxidative
phosphorylation (OXPHOS), resulting from pathogenic
mutations in the nuclear DNA.
-Leber’s hereditary optic neuropathy(LHON)
- Mitochondrial encephalopathy, lactic acidosis,
stroke like episodes(MELAS)