DNA Replication

Introduction

During cell division, each daughter cell gets an exact copy of the genetic information of the
mother cell. This process of copying the DNA is known as DNA Replication,

Semi-conservative type:

In daughter cell, one strand is derived from the mother cell, while the other strand is newly
synthesized. This is called Semi-conservative type of DNA Replication.

Basic requirement:

a) Substrates - dATP, dGTP, dCTP,dTTP.

b) Template - Each strand serves as a template strand for which new
complementary strand is synthesized.
c) Enzymes -
1. DNA Polymerase:

Prokaryotic Eukaryotic Function

DNA polymerase I α Gap filling

Synthesis between okazaki

fragments of lagging strand

DNA polymerase II β DNA Repair

ε DNA proof reading & DNA repair

γ Mitochondrial DNA synthesis

DNA polymerase III δ Leading &lagging strand synthesis

2. DNA Topoisomerase
Type I: Relaxation of supercoiled DNA by breaking one strand of DNA.
Type II: or DNA gyrase: cleaves both strand.
3. Primase : synthesis of RNA primer
A short piece of RNA, about 100 – 200 nucleotides length.

Stages of Replication

A) Initiation
1. DNA Replication starts with recognition of the site of origin of replication (ORI).
2. Origin recognition complex: DNA - A protein recognizes & binds to the ‘ORI’ &
denatures the DNA.
3. DNA helicase unwinds the DNA in both directions& form a V where active
synthesis occurs. This region is called Replication fork.
 4. Topoisomerases relieves the stress produced by supercoiling. Makes a transient
break in one strand of DNA and is immediately resealed after the coiling.
5. Single stranded DNA binding protein SSB stabilizes the complex, prevents the
rewinding of DNA. This forms the ‘Replication bubble’.
6. Requires RNA primer. RNA primers are short pieces of RNA formed by the
enzyme primase using as DNA as template.

B) Elongation of DNA strand

1. Polymerization of new strand of DNA is taking placing from 5’ to 3’ direction.( the

template strand is read in 3’ to 5’ direction.

2. DNA chain which runs in 3’ 🡪 5’ direction is copied by DNA polymerase III in 5’ 🡪 3’

direction as a continuous strand. Adds nucleotides complementary to the template strand.
This new strand is known as ‘Leading strand’.

3. DNA chain which runs in 5’ 🡪 3’ direction is copied by DNA polymerase III but in
discontinuous manner because synthesis can proceed only in 5’ 🡪 3’ direction. This new
strand is known as ‘Lagging strand’.

4. Requires many RNA primers, synthesized by RNA primase followed by synthesis of

segments of DNA by DNA polymerase I.

5. These short segments of DNA of 1000 – 2000 nucleotides are referred as “Okazaki
fragments”.

6. As lagging strand is synthesized, RNA primers are removed. The gaps are filled by
DNA polymerase I. Two polynucleotide chains are joined together by DNA ligase.
 B) Termination
- ‘Ter’ Termination sequences.
- Ter binding proteins binds these sequences & prevents Helicase from unwinding
of DNA.
- Facilitates termination of Replication.
C) Proof reading
- DNA polymerase I & II removes erroneous nucleotides before the introduction of
the next nucleotide.

Inhibitors of DNA Replication - Mechanism: Blocks replication of DNA & Multiplication

cells.

I Anti-bacterial agents – widely used antibiotics.

Ciprofloxacin, Nalidixic acid, Novobiocin.

Action: Inhibits DNA gyrase.

II Anti-Cancer agents –

Etoposide, Adriamycin, Doxorubicin – [Mechanism: Inhibits Human Topoisomerase]

6-Mercaptopurine - [DNA polymerase]

5 – Fluoro uracil - [Thymidylate synthase]

Transcription
Intro

Transcription is defined as the synthesis of RNA molecules using DNA as a template, that
results in the transfer of the information stored in double stranded DNA into single stranded
RNA.

5’ 3’

3’ 5’ Template strand

5’ 3’ mRNA [ TRANSCRIPTION]

Primary Transcript 🡪 post Transcriptional modification 🡪 active RNA molecule

Basic requirement
 1. Template: single strand of DNA acts as Template strand.
2. Substrate: ATP, GTP, CTP, UTP
3. Enzyme: DNA dependent RNA polymerase called as RNA polymerase
a) Prokaryotic RNA polymerase
It is a holoenzyme with 5 subunits. 2α, 1β, 1β’, Ꙍ & 1 sigma factor.
Sigma factor is required for binding of the polymerase to specific promotor
regions of DNA template.
b) Eukaryotic RNA polymerase
RNA polymerase I
RNA polymerase II
RNA polymerase III

Stages of Transcription

I Initiation

Initiation of transcription involves binding of RNA polymerase (+ sigma factor) to the

template at the promoter site.

Promoter sequences are responsible for directing RNA polymerase to initiate transcription at
a particular point known as Initiation site.

Prokaryotic promoters

a) Pribnow box (TATA)

Consist of –TATAAT- consensus sequences, with 10 base pairs upstream the
starting point of transcription.

b) -35 sequence:
Consist of –TTGACA- sequences, with 35 base pairs upstream the starting point
of transcription.

Eukaryotic promoters

a) Hogness box
Consist of –TATAAT- sequence.
-25 base pairs upstream to start point.
b) CAAT box
Consist of – GGCAATCT- sequences.
-70 base pairs upstream to start point.
c) Enhancers increase the rate of transcription.
Silencers decrease the rate of transcription.

Steps in Initiation:

1. RNA polymerase 🡪 sigma factor recognizes the promoter region and binds with
promoter site of DNA forming ‘Pre-initiation complex’.
2. Unwinding of the DNA helix.
 3. First nucleotide complementary to the base present in DNA, attaches to the initiation
site on beta subunit of RNA polymerase.
4. RNA polymerase catalyses formation of phosphodiester bond between first 2 bases.
5. Enzyme moves to the next base on DNA template.

Elongation

1. RNA polymerase utilises ATP, GTP, CTP, UTP for the formation of RNA.
2. As 10 nucleotides are added, sigma factor dissociates (Promoter clearance).
3. The core enzyme continues the elongation of the transcript, until a termination signal
is reached.
4. RNA polymerase
- Doesn’t require primer
- Moves synthesizing 5’ to 3’ direction
- Obeys Base pairing rule.
- No nuclease activity & no proof reading activity.

Termination

a) Rho- dependent termination

- Rho factor, a specific protein factor binds rut (Rho utilisation) sites on the
growing RNA.
- Recognises termination signal
- Its ATP- dependent Helicase activity displaces RNA polymerase 🡪 Termination of
RNA synthesis.
b) Rho-independent termination
- Involves formation of Hairpin loop in newly synthesized RNA.
- Hair loop is followed by sequence of uracil residues.
- RNA transcription ends.

Post-transcriptional modification

▪ Def: Primary transcript made by RNA Polymerase undergoes further enzymatic

alteration called post-transcriptional processing.
▪ mRNA formed and released from the DNA template in known as ‘Primary
transcript’[Hetero nuclear hn RNA].
▪ Prokaryotic post-transcriptional processing.
1. Cleavage of large precursor of RNA – removal of excess sequences from the
primary transcript by endonucleases or exonucleases.
2. Terminal addition of nucleotides eg. CCA sequence at 3’ end of tRNA.
3. Nucleotide modification eg. Uridylate residues modified to form ribothymidylate
& pseudouridylate.
▪ Eukaryotic post-transcriptional processing
▪ Undergoes extensive processing to become mature RNA.
1. Endonuclease cleavage:
Large precursor RNA 🡪 excess sequences are cleaved from it.
 Eg. Processing of ribosomal RNA
2. Poly A tail
- 3’ end is polyadenylated.
- Possess (20 – 250) adenine nucleotides.
- Importance: Protects mRNA, stabilizes and determines half-life of mRNA.
3. Capping at 5’ end
- mRNA is capped at 5’ end by 7-methyl guanosine triphosphate.
- S-Adenosyl Methionine is the methyl donor.
- Importance: Stabilises mRNA and recognition of mRNA by translating
machinery.
4. Methylation
- Methylation of N6 of adenine and 2’-hydroxy group of ribose.
5. Splicing
- Exons are the coding sequence of the gene.
- Introns are the non-coding sequence or intervening sequences between exons.
- Def: The process by which introns are excised and exons are linked to form the
functional mRNA is called as Splicing.
- Formation of Spliceosome:
a) Small nuclear RNA eg. U1, U2, U4, U5, U6, U7.
b) It recognises 5’ & 3’ splice site of introns.
c) Formation of looped structure.
d) Cleavage & removal of introns.
e) The exons are joined by 3’ 5’ phosphodiester linkage.

Inhibitors of Transcription
- Actinomycin D &Mitomycin: Intercalate with DNA strand, thus blocking
transcription.
- Rifampicin: (Anti-Tuberculosis) Binds to β subunit of RNAP.
- α-amanitin: (Mushroom poison) Inactivates Eukaryotic RNAP II.
 Recombinant DNA Technology
Def: Recombinant DNA Technology is the artificial means when a gene from one species is
transferred to another living organism.

Basic requirement:

1. Restriction endonuclease
2. DNA polymerase
3. DNA ligase
4. Vectors – are required to carry a fragment of DNA into host cell. Eg. Plasmid,
cosmids, bacteriophage, artificial chromosome.

Principle:

1. Isolation of desired piece of DNA

2. Insertion of isolated DNA into vector to form Chimeric DNA or Hybrid DNA.
3. Introduction of recombinant vectors into host cells.
4. Multiplication & selection of clones
5. Expression of gene.

Construction of Recombinant DNA

1. Identification and isolation of specific mRNA.

2. DNA synthesis from mRNA by reverse transcriptase.
3. RNA is hydrolysed.
4. From DNA Complementary DNA is produced.
5. Fragmentation of DNA and vector DNA with the same Restriction endonuclease
which cuts at restriction site. This results in sticky ends of the cut piece.
6. Insertion of isolated DNA into vector to form Recombinant / Hybrid DNA.
(Annealing)
7. Nick is sealed by DNA ligase.
8. Introduction of rDNA into host cell by transformation or transfection.
9. Host cells with insert DNA is incubated 🡪 Replication of DNA 🡪 Isolation of proteins.

Applications:

a) Molecular basis of disease – Recombinant DNA technology is used to understand the

molecular basis of diseases like Familial Hypercholesterolemia, Sickle cell disease,
Cystic Fibrosis, Muscular Dystrophy, Thalassemia.
b) Diagnosis of disease
c) Production of proteins –
 d) Gene therapy – It is the introduction of normal genes into individuals who have
defective genes. Adenosine deaminase, Cystic fibrosis, Sickle cell disease – at
experimental level.
e) Transgenic animals
f) Genetic counselling& screening tests to prevent passage of defective genes to
offspring.
g) Forensic medicine – DNA finger print is used in criminal cases.
h) Commercial application
i) Agricultural application – Using recombinant DNA technology, plants are made
resistant to insects.
j) Industrial application

RFLP – Restriction Fragment Length Polymorphism

Introduction
Human genome is heterogeneous.
Normal variation of DNA sequence.
Variation of DNA sequence is called Polymorphism.
Occurs in every 500 nucleotides
Def: An inherited difference in the pattern of restriction sites is known as Restriction
Fragment length polymorphism.

Polymorphism: It is variation in nucleotide sequence from one individual to another.

It is clinically harmless DNA variation. Does not affect phenotype.Occurs in intervening
sequences that do not code protein.

RFLP may be due to

1. Single base changes
2. Deletions or insertions of DNA into a restriction fragment

DNA

DNA cleaved into fragmentsof different length

Electrophoresis is done

DNA fragments are separated according to their molecular size

Separated DNA on agarose gel is transferred to nitrocellulose paper

Hybridized with labelled probe sequences

Genotypic changes can be recognized by altered restriction fragments

Applications

Used to detect human genetic defect:

1. Sickle cell anemia 2. Thalassemia

Forensic medicine: Finger printing with unique pattern of DNA fragments.

Lac Operon Concept

Intro
Jacob & Monod – Lac operon concept based on the lactose metabolism in Escherichia coli.
It is the co-ordinated unit of gene expression to synthesize the enzymes necessary to
metabolise lactose by E. coli.
Lac operon consist of:
1. Structural genes: Z - Beta galactosidase
Y -permease
A -Thiogalactosidetranscetylase
2. Operator gene
3. Regulator gene – produces repressor molecules, which has strong affinity to the
operator site. Transcription of the structural gene is under the control of regulator
gene.
4. Promoter site – RNA polymerase binds to the promoter site and transcribes the
structural genes.

Lac operon Regulation

a) Negative regulation by Lac repressor in absence of lactose & presence of glucose.

Repression of Lac Operon
❖ In absence of Lactose🡪 Regulator (i) gene – produces ‘Lac repressor 🡪Binds to
Operator gene.
❖ Prevents binding of RNA polymerase to Promoter site 🡪No transcription of Z, Y, A
genes
b) Positive regulation by Induction of expression in presence of lactose & absence of
glucose.
❖ In presence of lactose & absence of glucose.
❖ Metabolite of lactose is called ‘ Allolactose’🡪binds to Lac repressor protein &
inactivates it 🡪 no free lac repressor to bind to the Operative site.
❖ RNA Polymerase binds to promoter & transcribe the structural genes of the operon to
produce the enzymes required for lactose metabolism.

c) Positive control by Catabolite repression in presence of both glucose & lactose.

❖ Bacteria 🡪accumulates cAMP when starved 🡪 when glucose is exhausted 🡪c-AMP
levels ↑ 🡪 favours CAP-cAMP
❖ CAP-cAMP required for the attachment of RNA polymerase to promoter site.
❖ When no glucose but lactose is available🡪CAP-cAMP binds to promoter
site🡪Stimulates transcription of structural genes.