DNA REPLICATION

Dr. A. D. NAVEEN KUMAR

Asst.Professor in Biochemistry
College of Medical and HEALTH Sciences
ADIGRAT University
Ethiopia
 DNA Replication
 Types of DNA replication
Semi-conservative model of DNA replication
Prokaryotic DNA replication
Eukaryotic DNA replication
Inhibitors of DNA replication
(Analogues, Intercalation, Polymerase Inhibitors)

 DNA damage
Types and agents of mutations
Spontaneous, Radiation, Chemicals.

 Repair mechanisms
Base Excision, Nucleotide Excision, Mismatch Repair.
 DNA-recombination
In meiosis
Transposition
 Central dogma

Replication Transcription Translation

DNA RNA PROTEIN

Reverse
transcription
 DNA replication is a biological process that
occurs in all living organisms and copies their
exact DNA. It is the basis for biological
inheritance.
 Replication is the process of synthesis of
daughter DNA from parental DNA by the
enzyme DNA Polymerase.

 ( dNMP )n + dNTP ( dNMP )n+1+ PPi

DNA Lengthened DNA
DNA Replication

Parental strand

Daughter stand
 DNA Replication
 A reaction in which daughter DNAs are
synthesized using the parental DNAs as
the template.
 Transferring the genetic information to
the descendant generation with a high
fidelity.
Replication

Parental DNA
Daughter DNA
6
Three possible replication patterns:

1. Semiconservative replication
2. Conservative replication
3. Dispersive replication
Semiconservative
replication

Conservative
replication

Dispersive
replication
 Each parent strand serves as a template
for a new strand and the two new DNA
Semiconservative replication
strands each have one old and one new
strand

Parent strands

New / Daughter
strand
Characteristics of Replication

 Semi-conservative replication
 Bidirectional replication
 Semi-continuous replication
 High fidelity

10
Meselson and Stahl experiment [1958]
demonstrated semiconservative
replication
 15N
1
medium E. coli grown in the presence
of 15N (a heavy isotope of
Nitrogen) for many generations

• Cells get heavy-labeled DNA

2
E. coli placed in
medium containing
only 14N (a light
Sampled 20 40 isotope of Nitrogen)
at: min min
0 min
3 Cells broken open
to extract DNA

4 Suspended DNA in Cesium

chloride (CsCl) solution.
 5
CsCl density gradient
centrifugation

0 min 20 min 40 min

DNA
14N 15N

F1 F2 generation
Both
generation DNA:
strands
DNA (one  Two light
heavy heavy/one strands
light strand)  One heavy/One
light strand
 Original 0 min
DNA
Three
1st Round:
rounds of 20 min

replication:
2nd 40 min
Round:

60
min? 3rd
Round:
 5’ 3’

Identical
5’ base sequences

3’

3’ 5’ 3’
5’
Semiconservative Replication
Half of the parental DNA molecule
is conserved in each new double helix,
paired with a newly synthesized
complementary strand. This is called
semiconservative replication.
Direction of the DNA Replication
 Bidirectional Replication

• Replication starts from unwinding the

dsDNA at a particular point (called
origin / ori site), followed by the
synthesis on each strand.
• The parental dsDNA and two newly
formed dsDNA form a Y-shape
structure called Replication fork.
Replication of Prokaryotes
The replication
process starts
from the origin,
and proceeds in
two opposite
directions.
It is named
- Replication.

21
 Replication Enzymes & Proteins
 DNA Polymerase - Matches the correct
nucleotides then joins / polymerizes
adjacent nucleotides to each other.
 Helicase - Unwinds the DNA and melts
it.
 Primase - Provides an RNA primer to
start polymerization.
 Single Strand Binding Proteins - Keep the
DNA single stranded after it has been melted
by helicase
 Gyrase - A topisomerase that Relieves
torsional strain in the DNA molecule.
• Ligase - Joins adjacent DNA strands
together (fixes “nicks”)
 Telomerase - Finishes off the ends of DNA
strands in Eukaryotes
 Enzymes and proteins of DNA
Replication
Protein MrW Sub Function
units
Dna A protein 50,000 1 Recognizes ori
sequences
Dna B protein 300,000 6 Unwinds/opens dsDNA
(DNA Helicase)
Dna C protein 29,000 1 Assists Dna B to bind at
ori-site
DNA polymerases Synthesizes the new
DNA strands
Dna G protein 60,000 1 Synthesize RNA primer
(DNA Primase)
Single Strand Binding 75,600 4 Binds single-stranded
Proteins (SSB) DNA
DNA Gyrase 400,000 4 Relieves torsional strain
(DNA Topoisomerse) generated by unwinding
 DNA Polymerases of Prokaryotes
DNA Polymerase-I
 The first DNA- dependent DNA
polymerase ( DNA Pol -I ) was
discovered in 1958 by Arthur
Kornberg who received Nobel
Prize in physiology & medicine in
1959.
 DNA Polymerase is considered
as Kornberg Enzyme.
 Later, DNA-Pol II and DNA-Pol III
were identified.
 All of them possess the following
biological activity.
1. 53 Polymerse activity
2. Exonuclease activity
Comparison of DNA Polymerases of E. coli
 Exonuclease functions
5´→3´ 3´→5´ exonuclease
exonuclease activity
activity excise mismatched
removes primer or nuleotides
excise mutated
segment
5' 3'
C T T C A G G A

G A A G T C C G G C G
3' 5'
 DNA Polmerase - I

 Mainly
responsible for
proofreading and
filling the gaps,
repairing DNA
damage

30
 N-end DNA-pol Ⅰ C-end

Cleavage
Small fragment Large fragment

 Small fragment (323 AA): having 5´→3´

exonuclease activity
 Large fragment (604 AA): called Klenow
fragment, having DNA polymerization and
3´→5´exonuclease activity
 DNA Polymerase - II
 Temporarily functional when DNA-pol I
and DNA-pol III are not functional.
 Still capable for doing synthesis on the
damaged template.
 Participates in DNA repair process.
 DNA Polymerase - III
 A heterodimer enzyme composed of
ten different subunits
 Having the highest polymerization
activity (105 nt/min)
 The true enzyme responsible for the
elongation process
 Structure of DNA-pol III

α： has 5´→ 3´
polymerizing activity

ε：has 3´→ 5´
exonuclease activity and
plays a key role to ensure
the replication fidelity.

θ: maintain heterodimer
structure
Nucleotides are always added to the growing
strand at the 3’ end – the end at which the
DNA strand has a free –OH group on the 3’
carbon of its terminal deoxyribose

Free 3’- hydroxyl group

 RNA Primase

• Also called DnaG

• Primase is able to synthesize primers
using free NTPs as the substrate and
the ssDNA as the template.
• Primers are short RNA fragments of a
several nucleotides long.
• Primers provide free 3´-OH groups to
react with the -P atom of dNTP to
form phosphodiester bonds.
• Primase, DnaB, DnaC and an origin
form a Primosome complex at the
initiation phase.
 Helicase
• Also referred to as DnaB.
• It opens the double strand DNA with
consuming ATP.
• The opening process with the assistance
of DnaA and DnaC
 SSB protein

• Single Strand DNA Binding protein.

• SSB protein maintains the DNA
template in the single strand form in
order to
• prevent the dsDNA formation;
• protect the vulnerable ssDNA from
nucleases.
 DNA Gyrase
• It cuts phosphoester bonds on both
strands of dsDNA, releases the
supercoil constraint, and reforms the
phosphodiester bonds.
• It can change dsDNA into the negative
supercoil state with consumption of
ATP.
 DNA Ligase
3' 5'
5' 3'
RNAase
3' 5'
5' OH P 3'

dNTP DNA polymerase

3' 5'
5' P 3'
ATP DNA ligase
3' 5'
5' 3'
• Connect two adjacent ssDNA strands
by joining the 3´-OH of one DNA
strand to the 5´-P of another DNA
strand.
• Sealing the nick in the process of
Replication, Repairing, Recombination,
and Splicing.
 Replication Fidelity

• Replication based on the principle of

base pairing is crucial to the high
accuracy of the genetic information
transfer.
• Enzymes use two mechanisms to
ensure the replication fidelity.
– Proofreading and real-time correction
– Base selection
 Proofreading and Correction
 DNA-pol I has the function to correct the
mismatched nucleotides.
 It identifies the mismatched nucleotide,
removes it using the 3´- 5´ exonuclease
activity, add a correct base, and continues
the replication.
DNA Replication
Process
STAGES

Initiation

Elongation

Termination
 Three Stages of replication
1). Initiation
 occurs at the origin of replication
 separates dsDNA, primer synthesis
2). Elongation
 involves the addition of new nucleotides
(dNTPs ) based on complementarity of the
template strand
 forms phosphoester bonds, correct the
mismatch bases, extending the DNA strand, …
3). Termination
 stops the DNA Replication occurs at a specific
termination site
 Genome of E. coli

ori-Site
 Initiation
 The replication starts at a particular
point called origin of Replication (or)
ori-Site.
 The structure of the origin is 248 bp long
and AT-rich.

13 mer- sequence 9 mer- sequence

Formation of Preprimosome
 Origin of Replication
Site where DNA synthesis
starts
 Formation of Replication fork
 DnaA recognizes ori C.
 DnaB ( Helicase ) and DnaC join the DNA-
DnaA complex, open the local AT-rich region,
and move on the template downstream
further to separate enough space.
 DnaA is replaced gradually.
 SSB protein binds the complex to stabilize
ssDNA.
 Primer synthesis
 Primase joins and forms a complex called
primosome.
 Primase starts the synthesis of primers on
the ssDNA template using NTP as the
substrates in the 5´- 3´ direction at the
expense of ATP.
 The short RNA fragments provide free
3´-OH groups for DNA elongation.
 Primase synthesizes PRIMER
3’

5’

Single stranded binding protein

5’

18-12-15 08:48:25
 Elongation
 dNTPs are continuously connected to the
primer or the nascent DNA chain by
DNA-pol III.
 The core enzymes (、、and  ) catalyze
the synthesis of leading and lagging
strands, respectively.
 The nature of the chain elongation is the
series formation of the phosphodiester
bonds.
 3’
Primer
5’ Leading strand
Primer is removed
Elongation byPolymerase
by DNA DNA Polymerase
III I

Parental DNA
5’ 3’
Okazaki fragments
5’
3’
5’

3’ Laging strand
Gap filled byDNA Ligase
5’

18-12-15 08:48:25
 Lagging strand synthesis
 RNA Primers on Okazaki fragments
are digested by the enzyme RNase.
 The gaps are filled by DNA-pol I in
the 5´→3´direction.
 The nick between the 5´end of one
fragment and the 3´end of the next
fragment is sealed by ligase.
 Okazaki fragments
• Many DNA fragments are synthesized
sequentially on the DNA template strand
having the 5´- end. These DNA
fragments are called Okazaki fragments.
They are 1000 – 2000 nt long in
prokaryotes and 100-150 nt long in
eukaryotes.
• The daughter strand consisting of
Okazaki fragments is called the
lagging strand.
63
Directionality of the DNA strands at a replication fork

Lagging strand

Leading strand Fork movement

 Lagging strand
(discontinuous)

Leading strand
(continuous)
3' 5'
5' 3'

RNAase
3' 5'
5' OH P 3'

dNTP DNA polymerase

3' 5'
5' P 3'
ATP DNA ligase
3' 5'
5' 3'
 Termination
 The replication of E. coli is
bidirectional from one origin, and
the two replication forks must meet
at one point called ter at 32.
 All the primers will be removed, and
all the fragments will be connected
by DNA-pol I and ligase.
Ter-binding proteins - will recognizes the
Termination sequences and helps to
achieve the termination process.
Nalidixic acid

Novobiocin

Ciprofloxacin
Adriyamycin

Etoposide

Doxorubicin
Eukaryotic DNA Replication
 DNA replication is closely related with
cell cycle.
Multiple origins on one chromosome, and
replications are activated in a sequential order
rather than simultaneously.
 DNA Polymerse of Eukaryotes
Eukaryotic Enzyme Prokakaryotic Enzyme
DNA-pol : initiate replication DnaG,
and synthesize primers primase
DNA-pol : replication with Repair
low fidelity

DNA-pol : Mitochondrial DNA synthesis

DNA-pol : elongation DNA-pol III

DNA-pol : lagging strand DNA-pol I
synthesis, proofreading and
gap filling
 Initiation
 The eukaryotic replication origins are shorter than
that of E. coli. The ori-sites in Eukaryotes called
ARS (Autonomously Replicating Sequences) (or)
Replicators.
 Requires DNA-pol  (primase activity) and DNA-
pol  (polymerase activity and helicase activity).
 DNA-pol  requires a protein called for its
activity Proliferating Cell Nuclear Antigen (PCNA).
 Needs Topoisomerase and Replication factors (RF)
to assist.
 Elongation
 DNA replication and nucleosome
assembling occur simultaneously.
 Overall replication speed is
compatible with that of prokaryotes.
 Termination
3' 5'
5' 3'
3' 5'
5' 3'

connection of discontinuous
segment
3' 5'
5' 3'
3' 5'
5' 3'

81
 Telomere
 The terminal structure of eukaryotic DNA
of chromosomes is called telomere.
 Telomere is composed of terminal DNA
sequence and protein.
 The sequence of typical telomeres is rich in
T and G.
 The telomere structure is crucial to keep
the termini of chromosomes in the cell
from becoming entangled and sticking to
each other.

82
 Telomerase
• The eukaryotic cells use telomerase to maintain
the integrity of DNA telomere.

• The telomerase is composed of

telomerase RNA
telomerase association protein
telomerase reverse transcriptase

• It is able to synthesize DNA using RNA as the

template. 83
 Step in Replication Prokaryotic cells Eukaryotic cells

Recognition of origin of Dna A protein RpA

replication (Replication Protein-A)
Unwinding of DNA double helix Helicase Helicase
(requires ATP) (requires ATP)
Stabilization of unwound Single-stranded DNA-binding Single-stranded DNA-binding
template strands protein (SSB) protein (SSB)
Synthesis of RNA primers Primase Primase
Synthesis of DNA
Leading strand DNA polymerase III DNA polymerase δ
Lagging strand DNA polymerase III DNA polymerase Ԑ
Removal of RNA primers DNA polymerase I RNAse-H
(5 3' exonuclease)
Replacement of RNA with DNA DNA polymerase I Unknown

Joining of Okazaki fragments DNA ligase DNA ligase

(requires NAD) (requires ATP)
Removal of positive supercoils DNA topoisomerase II DNA topoisomerase II
ahead of advancing (DNA gyrase)
replication forks
 BASE ANALOGUES
A base analog is chemical that can substitute for a
normal nitrogen base in Nucleic acids.

They are categorized in two separate

groups, purine analogues and pyrimidine
analogues.

Oncologists employ 5-fluoro- or 5- iodouracil,

3-deoxyuridine, 6-thioguanine and 6-
mercaptopurine, 5- or 6-azauridine, 5- or 6-
azacytidine and 8-azaguanine which are
incorporated into DNA prior to cell division.
 Intercalating agents
These are the molecules that can insert between
bases in DNA base pairs, causing mutation
during replication.

Examples: Ethidiumbromide, Proflavine and

Daunorubicin.

Ethidiumbromide
 Proflavine

It also called Proflavin and Diaminoacridine , is

an acriflavine derivative, a disinfectant
bacteriostatic against many gram-positivebacteria.
Daunorubucin is most commonly used to treat
specific types of leukemia such as Acute myeloid
leukemia , Acute lymphocytic leukemia) and also
for the treatment of Neuroblastoma.

Daunorubicin
 DNA Polymerase Inhibitors

Acyclovir Gancyclovir
Guanosine

Natuarlly occuring
Inhibitors of Viral DNA Polymerase
Nitorgen base
essential in DNA
Replication
 Aphidicolin

Inhibits DNA Polymerase-ε in Eukaryotes

 DNA DAMAGE
 DNA is easily damaged under normal physiological
conditions.

 The return of damaged DNA to its normal sequence

and structure is called Repair.

 Many different kinds of physical & chemical agents

damage DNA. Some of these are:-
1) Endogenous agents
2) Exogenous agents
Agents that damage DNA can be mutagenic, cytotoxic
or both.
DNA damaging agents that cause mutations are called
Mutagens.
 Types of DNA Damage
The damages done to DNA by physical, chemical and
environmental agents can be broadly classified into
four categories with different types.
Single Base Alterations
Double Base Alterations
 DNA damaging Agents
Spontaneous Agents
Highly reactive oxygen radicals produced as a
by products during normal cellular respiration as
well as by other biochemical pathways.
Reactive Oxygen Species (ROS) :
 Hydrogen peroxide (H2O2)
 Hydroxyl radicals (OH.- ) – Most potent
 Superoxide (O2 ) .-

 ROS causes DNA damage such as

Oxidation of Nitrogen Bases, deoxy Ribose
and Strand breaks.
Radiation can cause mutations
 Radiation
 The high energy electromagnetic radiation to the exposure of
which cell experience considerable damage to their DNA
are:
1. Ultraviolet light:
 The major type of damage caused by UV light is divided
into three bands:
I. UV-A (321-400 nm)
II. UV-B (296-320 nm)
III. UV-C (100-295 nm)
2. X- Rays
3. Gamma Rays
 Through these direct damage takes
place when DNA or water tightly bound
to it absorbs the radiation.
Indirect damage takes place
when water or other molecules
surrounding the DNA absorbs the
radiation & form reactive species that
then damage DNA.
Effect of UV on DNA structure
 Chemicals Agents
1) Deaminating Agents:
Sodium Nitrite (NaNO2)
Sodium Nitrate (NaNO3)
Nitrosamine
Nitrous Acid (HNO2)

2) Alkylating Agents:
Dimethyl sulfate (DMS)
Dimethyl nitrosamine
Nitrogen mustard
 Mutations
Mutation refers to a change in the DNA
structure of a gene. The substances
(chemicals) which can induce mutations are
collectively known as mutagens.
The changes that occur in DNA on mutation
are reflected in Replication, Transcription and
Translation .
Mutations occur in 2 ways:
1) Spontaneous mutations: Mistakes in DNA
replication.
2) Induced mutation: Caused by Mutagens.
1) Point mutations : A point mutation or single
base substitution, is a type of mutation that causes the
replacement of single base nucleotides with another
nucleotides of DNA .

Substitutions
(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.
Substitution
 Point Mutations
Silent Mutation :
UCA UCU
Serine Serine

Missense Mutation :

UCA ACA
Serine Threonine
Nonsense Mutation :
UGG UGA
Tryptophan Stop Codon

UAU UA A
Tyrosine Stop Codon

UAC UA G
Tyrosine Stop Codon
A point mutation can also change a codon so that a
different protein is specified, a non synonymous
change. . Sickle Cell Anemia

111
Normal red blood cell Sickle cell RBC
2). Frameshift mutations : These occur when
one or more base pairs are inserted in or deleted
from the DNA, respectively, causing insertion
(or) deletion mutations.

deletion
DNA REPAIR MECHANISMS
 Base Excision Repair
 For correction of specific Chemical damage in
DNA
 Uracil
 Hypoxanthine
 3-methyl Adenine
 Formamido pyrimidine
 5,6 - Hydrated Thymine
Base Excision Repair Deaminated
(BER) Cytosine

Variety of DNA
glycosylases, for different
types of damaged bases.

AP endonuclease
recognizes sites with a
missing base; cleaves sugar-
phosphate backbone.

Deoxyribose
phosphodiesterase removes
the sugar-phosphate lacking
the base.
Nucleotide Excision Repair (NER)
 Used by the cells to repair bulky DNA
damages
 Non specific DNA damage
 Chemical adducts …
 UV photoproducts
Xeroderma pigmentosum (XP) is a rare
autosomal recessive disease. The affected
Patients are photosensitive and susceptible to
Skin cancers.

It is due to a defect in the Nucleotide Excision

Repair of the damaged D NA.
Mismatch repair
Mismatch repair system is an excision/resynthesis
system that can be divided into 4 phases:

(i) recognition of a mismatch by MutS proteins

(ii) recruitment of Repair enzymes

(iii) excision of the incorrect sequence

(iv) resynthesis by DNA polymerase using the

parental strand as a template.
Parental Srand
DNA REPAIR DISORDERS
Xeroderma Pigmentosum
• Transmitted as autosomal recessive disorder.

• Genetic defect: DNA repair mechanisms are

defective.
• DNA damage produced by UV irradiation specially
thymine dimers, cannot be incised. Results from
inborn deficiency of the enzyme “nicking
endonuclease”.
Clinical Manifestations :
• Increased cutaneous sensitivity to UV rays of sunlight.
• Produces blisters on the skin.
• Dry keratosis, hyperpigmentation and atrophy of skin.
• May produce corneal ulcers.
Ataxia telangiectasia :
• A familial disorder.
• Inheritence: Autosomal recessive
• Increased sensitivity to X-rays and UV rays is
seen.
Clinical manifestations :
• Progressive cerebellar ataxia.
• Oculocutaneous telangiectasia.
• Frequent sin pulmonary infections.
• Lymphoreticular neoplasms are common in this
condition.
• IgE deficiency has been demonstrated in 67 per
cent of cases.
Bloom’s Syndrome
Chromosomal breaks and rearrangements are seen in
this condition.
• Genetic defect: Defective DNA-ligase.
Clinical Manifestations
– Facial erythema
– Photosensitivity
Fanconi’s Anaemia :

• An autosomal recessive anemia. Defective

gene is located in chromosomes 20q and 9q.

• Defect: Defective repair of cross-linking

damage.

• Characterized by An increased frequency of

cancer and by chromosomal instability.
 Hereditary Nonpolyposis Colon Cancer
(HNPCC)
• Most common inherited cancer.

• Defect: Faulty mismatch repair.

• Genetic defect has been located in chromosome 2,

The located gene is called hMSH-2.

• Mutations of hMSH-2 account for 50 to 60 per cent

of HNPCC cases.
 Recombination
Genetic diversity in a species is maintained
through both mutation and recombination.

Mutation alters single genes or small groups of

genes in an individual, whereas
recombination redistributes the contents
of a genome among various individuals
during reproduction.
 Recombination basically involves the exchange
of genetic information.

There are mainly two types of recombination.

- Homologous Recombination (Meiosis).
- Transposition.

 Recombination is mediated by the breakage

and joining of DNA strands.
 Recombination
ABCDEFGHIJKLMNOPQRSTUVWXYZ
abcdefghijklmnopqrstuvwxyz

Exchange of genes
between the
chromatids
of Chromosomes

ABCDEFGhijklmnoPQRSTUVWXYZ
abcdefgHIJKLMNOpqrstuvwxyz
 Homologous Recombination
In eukaryotes, homologous genetic
recombination can have several roles in
replication and cell division, including the
repair of stalled replication forks.

Recombination occurs with the highest

frequency during meiosis, the process by
which diploid germ-line cells with two sets of
chromosomes divide to produce haploid
gametes— sperm cells or ova in higher
eukaryotes—each gamete having only one
member of each chromosome pair.
Holliday Junction
Model
for
Homologous
Recombination
 Transposition
Transposition primarily involves the
movement of specific pieces of DNA in the
genome.

The mobile segments of DNA are called

transposons (or) transposable elements.

Types of Transposition : Two types

1). DNA transposition

2).Retrotransposition
DNA transposition :
Some transposons are capable of direct
transposition of DNA to DNA.

This may occur either by replicative transposition

or conservative transposition .

DNA transposition is less common than retro

transposition in case of eukaryotes.

However, in case of prokaryotes, DNA transposons

are more important than RNA transposons.
 Retrotransposition
Transposition involving RNA intermediate
represents Retrotransposition.

A copy of RNA formed from a transposon( also

called as retro transposon).

Then by the enzyme Reverse transcriptase, DNA

is copied from the RNA.
The newly formed DNA which is a copy of the
transposon gets integrated into the genome.
This integration may occur randomly on the same
chromosome or/ on a different chromosome.