Dr.

Maha Ismail

10/09/09 1
 Nucleic acids
 Nucleic Acids are macromolecules which store and
express all the information necessary for building and
maintaining life.

 The main function of DNA (Deoxyribonucleic Acid) is to

keep the genetic information which reside in the
sequence of nucleotides.

 Any change from the original sequence of nucleotides is

mutation

10/09/09 2
 DNA Structure

 The DNA Molecule consist of two polynucleotide

chains (strands) held together in an antiparallel
manner by hydrogen bonds formed between
specific pairs of bases [Adenine-Thymine]
[Guanosine-Cytosine].

 Thus the bases sequence (code) in one strands

determines the code of the other strands
(complementarity).

10/09/09 3
10/09/09 4
 DNA Structure

 The joined anti-parallel strands are twisted

about each other in the shape of a right-
handed double helix.

 Indeed DNA is often depicted as a twisted

ladder in which rungs are bases pairing and
sides are deoxyribose-phosphate chains.

10/09/09 5
 DNA Structure
 The double helix structure is mainly stabilized
by hydrogen bonds between bases pairs. Since
the hydrophobic bases are stacked inside and the
hydrophilic ribose-phosphate
chains are on the outside.
 In eukaryotic the DNA is found in
the nucleus, , not free. It is mixed
with proteins, while in prokaryotes
the DNA is found free in the
cytoplasm.
10/09/09 6
  There is normal turnover of the cells (cell
division), and the zygote must also
divide & differentiate to develop into a
human.
 In all these conditions the genetic
informations must be passed to the
daughter cell, & to the offspring

10/09/09 7
  So, all the cells of the body contain the
same genetic makeup, but certain cells
express different genes.
 Not all the of the genome is expressed
in the cell & different cells express
different genes.
 B- cell in express the gene responsible
for the synthesis of insulin

10/09/09 8
 DNA Replication

 Each time a cell divides into two daughter cells,

all the DNA molecule must be duplicated.
Duplication of an old DNA molecule into two new
DNA molecules is called Replication.

 DNA replication is the synthesis of DNA from

DNA – the reproduction of DNA.

 The objective of DNA Replication is to conserve

the genetic information of the same organism.
10/09/09 9
 DNA Replication
 The DNA replication is carried out with high accuracy
and fidelity.

 It is a semi conservative process (Each daughter DNA

molecule is an exact copy of its parent molecule,
consisting of one old and one new DNA strand).

 The whole process takes about 9 hours in mammals.

10/09/09 10
10/09/09 11
10/09/09 12
 DNA Replication Requirements
1. Template ( mother DNA molecule).
2. RNA –Primer.
3. The 4 Nucleotides (A,G,T,C) ( Building blocks),
which are used in the form of triphosphates,
dNTP.
4. Proteins, including enzymes & the other
accessory proteins.
The key enzyme in replication is DNA Polymerase
(Pol)
10/09/09 13
 Steps of DNA Replication
 There at least five steps in DNA replication:

1. Identification of the origin of replication .

2. Unwinding of DNA.
3. Formation of Replication fork.
4. Formation of Replication bubbles.
5. Reformation of chromatin.

10/09/09 14
 Identification of the origin of replication. 1

 The starting point / origin of replication should be defined.

 The origin of replication ( Ori) is usually near an area rich in

A – T bases.
 This (Ori) has a specific sequence & is recognized by a
specific protein called O –protein .

 This stage is only recognized in Prokaryotes, as in higher

animals is less well understood.

10/09/09 15
 .Unwinding of DNA. 2

 This stage is necessary in order for the DNA to

be accessible for replication.

 The unwinding process is a co-operative work

i.e. meaning different mechanisms work
cooperatively to unwind, separate the double
strands & to keep them separated:

10/09/09 16
 .Unwinding of DNA. 2
1. The binding of the O- protein to the origin result in
local unwinding of the adjacent A & T rich area.

2. The enzyme HELICASE is responsible for breaking

the helical structure, to provide a single stranded
template.

10/09/09 17
10/09/09 18
 Unwinding of DNA. 2

3. The Topoisomerase prevents supercoiling of

the strands.

10/09/09 19
 4. Single stranded binding proteins
(SSBP), they bind each single strand
separately.
They enhance the activity of helicase
and to bind to the single stranded
template until replication is
completed.

10/09/09 20
 Formation of Replication. 3
fork
 One of the new strand is formed from
5 to 3 & the other is from 3 to 5.

 The main enzyme needed in

polymerization is DNA polymerase,
which need RNA primer to start
polymerization.

10/09/09 21
 .Formation of Replication fork. 3

 DNA polymerase requires:

1. Energy.
2. RNA primer, with a free 3’OH group for
attachment of the new nucleotides.
3. DNA as a template.

10/09/09 22
 Formation of Replication. 3
fork
 Primer formation need PRIMASE enzyme.

 The Primase enzyme initiate the synthesis of an

RNA molecule.
 This RNA primer length varies between 20 -200
nucleotides.
The Primase + Helicase form a complex called
PRIMOSOME

10/09/09 23
10/09/09 24
 .Formation of Replication fork. 3

 The new nucleotide is added to the 3’ end of a primer & new

synthesis is said to occur in a 5’ to 3’ direction.

 The polymerization process involve the nucleophilic attack

by the 3’ hydroxyl group of the existing nucleotide, This
hydroxyl group serves as the acceptor of the α – phosphate
group of the first entering dNTP with the release of
pyrophosphate and forming phosphodiester bond.

10/09/09 25
10/09/09 26
10/09/09 27
 Replication fork

 Replication of the double – stranded

DNA is bidirectional, from the
replication origin

10/09/09 28
10/09/09 29
 DNA Replication

 Since the DNA strands are antiparallel, and

replication proceed only in the 5' to 3'
direction, one strand named the leading
strand forms a continuous copy, while the
other named lagging strand forms
discontinuous short DNA strings called
Okazaki fragments.

10/09/09 30
 . Okazaki fragments

 This lagging strands is synthesized in segments

and each segment is preceded by an RNA
primer.

 Each segment is called Okazaki fragment, its

length varies from 150-250 nucleotide and in
each replication fork there is up to 250 okazaki
fragments.

10/09/09 31
10/09/09 32
10/09/09 33
10/09/09 34
 .Formation of Replication bubbles. 4

 A number of replication forks form

replication bubbles.

10/09/09 35
10/09/09 36
 Action of DNA polymerase

1. Addition of nucleotides.
 DNA polymerase III initiate and elongate the chain
by adding nucleotides complementary to that in
the single stranded template, in 5 – 3 direction.
 All 4 deoxynucleotides should be present, if one
is absent synthesis will stop if the nucleotide is
depleted.

10/09/09 37
10/09/09 38
 Action of DNA polymerase

2. Proofreading of newly synthesized DNA:

 It edit its mistakes.

 Misreading can cause mutations.

10/09/09 39
 Action of DNA polymerase

3. The RNA primer sequence is not part

of the DNA structure so it must be
removed.
DNA polymerase I will remove the RNA
primer and fill the gap.

10/09/09 40
 Action of DNA Polymerase

 The DNA polymerase works on both strands

simultaneously

1. Addition or polymerization of nucleotides.

2. Proofreading.
3. Excision and repair activity.

10/09/09 41
10/09/09 42
 :In Summary

 The replication process is:

 Semi-discontinuous.
 Semi- conservative.
 Asymmetrical, two directions.

10/09/09 43
10/09/09 44
 Packaging of DNA, Reformation of. 5
.chromatin

 In Eukaryotic cells, the DNA is packaged in

Chromatin within the nucleus.
 DNA is surrounded by histones forming the basic
structural units of DNA [ Nucleosomes}

 Nucleosomes forms of chromatin (less densely

packaged) and then to chromosomes.

10/09/09 45
10/09/09 46
10/09/09 47
 Question 1

 What is the action of the following

drugs:
1. Adenine Arabinoside.
2. Azidothymidine (AZT).
3. Actinomycin D.
4. Nalidixic acid

10/09/09 48
 Question 2

 Compare and contrast between

Eukaryotic and prokaryotic replication.

10/09/09 49
10/09/09 50
 Substrate analogues:
 Adenine Arabinoside is an antiviral, Cystine
Arabinoside antileukemic, they alter the DNA
structure & it become more prone to breakage

2. Azidothymidine (AZT), antiviral, used for

treatment of HIV, the azido group in the 3
position will unacceptable primer

10/09/09 51
 3. Inhibitors that act directly with DNA
Actinomycin D, used for cancer, its structural ring
intercalate between the bases of DNA.

4. Inhibitors of replicative enzymes.

Nalidixic acid, inhibit topoisomerase, used in UTI.

10/09/09 52
10/09/09 53
10/09/09 54
10/09/09 55
10/09/09 56
10/09/09 57
10/09/09 58
10/09/09 59
 DeoxyriboNucleic Acid

 The Genome is the complete set of

instructions to create and maintain an
organism alive. DNA molecules are
support for the Genome of all living
organisms.

10/09/09 60
10/09/09 61
10/09/09 62