Replication

Definition: It is the process in which two

identical copies of daughter DNA molecules
are formed from parent DNA during cell
division.
Replication is semiconservative:Each daughter
DNA gets one strand from parent DNA. The
other strand is newly synthesized.
Requirements for DNA replication
➢ Template
➢ Four types of deoxy nucleoside triphosphates

1. dATP

2. dGTP

3. dCTP

4. dTTP.

➢Primer
• Enzymes :
• DNA helicases: Unwind the double helix using
energy ( ATP )
• DNA topoisomeraseI & II: Relieves the
supercoiling
• DNA polymerases :
DNA polymerase I: It removes the RNA
primer and catalyses the synthesis of a
fragment of DNA that replaces RNA primer
DNA polymerase II : participates in DNA repair.
DNA polymerase III : It is the main replication
enzyme, also required for proofreading (5’-3’
polymerase activity and 3’-5’ exonuclease
activity).
DNA ligase : Seals thenick in single strand as
well as in Okazaki fragments.
Primase: Initiates synthesis of short segment of
RNA (RNA primer)
Proteins :
• dnaA: Binds to AT-rich region → unwinding of
DNA

• Single-stranded DNA- binding proteins (SSB)

proteins): Prevent annealing of separated DNA
strands.
 Steps in DNA Replication
1. Identification of site of origin of replication
(ori) by specific proteins
2. Unwinding of double-stranded DNA to
form 2 single stranded DNA (ssDNA)
3. Formation of replication fork, synthesis of
primer and initiation of DNA synthesis
4. Elongation
 Steps in DNA Replication
1. Identification of site of origin of replication
(ori) by specific proteins
• Origin of replication is a unique nucleotide
sequence from where DNA replication begins.
/*
2.Unwinding of double-stranded DNA to
form 2 single stranded DNA (ssDNA).
• dnaA protein binds to AT-rich region in the
origin of replication → local
melting/unwinding of dsDNA → short
segment of ssDNA formed
• Single strand binding (SSB) proteins attach to
each strand and prevent their annealing (
maintain DNA in single strand form).
Separation of strands of DNA is required as
DNA polymerase binds t o single strand of
DNA
3.Formation of replication fork, synthesis of primer and
initiation of DNA synthesis :
• Unwinding of DNA causes formation of replication fork
→ DNA helicase binds to the fork → unwinding of
adjacent double-stranded region → primase binds to
DNA at 3’ end of each strand → synthesis of short
segment of RNA (RNA primer) → DNA polymerase
using RNA primer, initiates synthesis of daughter strand
(complementary to template) in 5’ → 3’ direction.
• Both parent strands are simultaneously replicated
in the 5' → 3' direction. The replication forks, thus,
advance in opposite direction from their origin.
This process results in the formation of 'replication
bubbles'.
• Direction of DNA synthesis
Leading strand :DN A is synthesized continuously in
5 ’ → 3 ’ direction towards the replication fork
and it needs only one RNA primer.
Lagging strand : It is synthesized in short stretches
in 5 ’ → 3 ’ direction , but away from replication
fork . These short stretches of discontinuous DNA
are called Okazaki fragments. It requires
many RNA primers.
4. Elongation
a. It is the process where there is sequential
addition of deoxynucleotides via phosphodiester
bonds.
b. First phosphodiester bond is formed between
3’– OH groupof RNA primer and 5’ phosphate
group offirst entering deoxynucleotide.DNA
polymerase(DNA pol-III) elongates new DNA
strand by adding deoxynucleotides (dATP, dGTP,
dT TP, dCT P) one at a time, to the 3’ end of
growing chain , complementary to bases in the
template strand.
C. Proofreading of newly formed DNA: Any
errors due to mismatched nucleotides
during replication are immediately repaired
to prevent lethal mutations. This is mainly
done by DNA polymerase III and DNA
polymerase I.
d.Excision of RNA primer: DNA polymerase I,
which removes the RNA primer (5’- 3’
exonuclease activity) and synthesizes DNA
that replaces RNA using 5’-3’ polymerase
activity; it also proofreads the new chain
using 3’-5’ exonuclease activity.
• DNA ligase: Catalyzes the formation of
phosphodiester bond between DNA
synthesized by DNA polymerase III and that
formed by DNA polymerase-I using energy.
Inhibitors of replication:
Drug Inhibited enzyme action

Nalidixic acid DNA helicase Antibacterial

Ciprofloxacin DNA helicase Antibacterial

5- fluorouracil DNA polymerase Anticancer

6-mercaptopurine DNA polymerase Anticancer

 MUTATIONS
• A mutation is defined as a change in
nucleotide sequence of DNA
• Mutagens and Mutagenesis :Any agent which
will increase DNA damage or cell proliferation
can cause increased rate of mutation, Such
substances are called mutagens.
• X-ray, gamma-ray, UV ray, acridine orange,
etc. are well known mutagens.
• Types of mutations :
1. Point mutations
2. Frameshift mutations
• 1. Point mutations : The replacement of one
base pair by another results in point
mutation. They are of two sub-types.
(a) Transitions : In this case, a purine (or a
pyrimidine) is replaced by another.
(b) Transversions : These are characterized by
replacement of a purine by a pyrimidine or
vice versa.
2. Frameshift mutations : The insertion or
deletion of a base in a gene results in an
altered reading frame of the mRNA (hence
the name frameshift).
• These occur when one or more base pairs are
inserted in or deleted from the DNA,
respectively, causing insertion or deletion
mutations.
Consequences of mutations
1. Silent mutation : The codon (of mRNA)
containing the changed base may code for
the same amino acid.
• For example, CUA is mutated to CUC; both
code for leucine, and so this mutation has no
effect.
2. Missense mutation : In this case, the changed
base may code for a different amino acid.
• For example, UCA codes for serine while ACA
codes for threonine.
• The mistaken (or missense) amino acid may
be acceptable, partially acceptable or
unacceptable with regard to the function of
protein molecule. Sickle-cell anemia is a
classical example of missense mutation.
3. Nonsense mutation : Sometimes, the
codon with the altered base may become
a termination (or nonsense) codon.
• For instance, change in the second base of
serine codon (UCA) may result in UAA. The
altered codon acts as a stop signal and
causes termination of protein synthesis, at
that point.