Chapter 2

DNA Replication
 Acknowledgement
• Centers for Disease Control and Prevention- Ethiopia
(CDC-E)
• American Society for Clinical Pathology (ASCP)

• Addiss Ababa University

• University of Gondar

• University of Hawassa

• Jimma University

• Haromaya University
 Learning objectives:

At the end of this lecture, students will be able to:

• Describe the composition, chemistry, and function of

nucleic acids

• List and describe the steps in DNA replication, including

initiation, elongation, and termination

 Outline

1. DNA Replication

2. DNA structure

alternative structure of DNA

3. DNA organization into chromosomes

4. Damage, mutability and Repair of DNA

5. Review questions

6. References
 Introduction to DNA replication

• DNA replicates normally whenever the cell replicates to

ensure equal division of information to the daughter cells.
• Occurs at S phase of the cell cycle.
• It is a complex activity involving many enzymes.
• Has three stages
– Initiation

– Elongation

– Termination
 Steps of replication
1. Initiation
• Involves recognition of origin of
replication by a complex of
proteins.
• The double strand must get
separated or unwind by the
action of helicase enzyme
• The single strand must be
stabilized transiently by single
strand binding protein (SSB).
• On the single strand priming will
be initiated by enzyme called
primase (The pol ) that
synthesizes short RNA sequence
(11-12 bases).
• Priming provides free 3'OH which
is required for elongation.

• Synthesis of the daughter strand is

initiated at the replication fork.

2. Elongation
• Undertaken by another complex of proteins called
replisome which contains DNA polymerase & other
enzymes.
• As the replisome moves along DNA, the parental

strands unwind and daughter strands are synthesized.

• Each of the parental strands serve as template.

• The elongation proceeds in 5'-3' direction.

• The leading strand is synthesized continuously but the

lagging strand is synthesized in discontinuous manner.
• The direction in which both strands are synthesized is
different.
• Priming is only once for the
leading strand but multiple
times for the lagging strand.
• Synthesis on the lagging
strand results in short
fragments called Okazaki
fragments.
• After DAN polymerase III(part
of replisome) extends RNA
primer to Okazaki fragments,
DNA pol I (5'-3' exonuclease
activity) uses nick translation to replace RNA primer by
DNA
• At the final stage of elongation, ligase enzyme seals the
nick.
3. Termination
• Complete unwinding of the mother strands

• End of the proof reading by polymerase enzyme

– DNA polymerases with proofreading nuclease activity

are capable of degrading DNA starting from a 3'-
end of the growing strand (exonuclease activity)
 Only ± 1 mistake in every 1010 bp synthesized

• arrest of replication and physical separation of the

daughter molecules
DNA replication is semi-conservative:
new/new + old/old
DNA polymerase(function and types)
 The structure of DNA
1. The poly-deoxynucleotide chain
• Deoxynucleic acids are polynucleotides in which the
phosphate groups link the C5' of one sugar to the C3' of the
next. By convention, we write down the sequence of such
a polymer starting with the nucleotide attached to the sugar
with the free C5' end.
• The molecule in the following figure can be abbreviated as
p-A-C-G-T. The ‘p’ indicates that a phosphate group is
present at the C5' end.
2. The double helix.
• DNA is a double helix molecule with the sugar-phosphate
backbone outside and its basis on the inside.
• The discovery of the double helix structure of the DNA is an
important milestone in the history of molecular biology.
• Two notions converged in the construction of this model
proposed by Watson & Crick in 1953 (Nature).
– First, X-ray diffraction data of extended DNA fibres
showed that DNA has the form of a regular helix,
– making a complete turn every 34 Å,

– and with a diameter of about 20 Å (10 Å = 1 nm).

– the distance between two adjacent nucleotides is 3.4 Å,

this means that there must be 10 nucleotides per turn.
• The second critical feature that explained how
this form of organization is accomplished lay in
the previous observation of Chargaff that,
irrespective of the actual amounts of each base,:
– the proportion of G and C is always the same in DNA,
– and the proportion of A and T is always the same.
– Thus any DNA can be characterized by its
(G+C)/(A+T) ratio,
– which ranges from 25 to 75 %, but is a characteristic
of each species.
• Watson and Crick proposed that the two polynucleotide
chains in the double helix are not connected by covalent
bonds,
– but associate through hydrogen bonding between the
bases.
• These hydrogen bonds can be formed because of the
proper location of the proton donor and proton acceptor
atoms.
• The structures of the thymine with adenine are
complementary in that they can be fitted together in the
same plane so that two hydrogen bonds can be formed
between them.
• The same situation holds for cytosine and guanine, except
that the base association in this case is accompanied by the
formation of three hydrogen bonds.
• The suggested base-pairing is in accordance with a content
of G = C and A = T
• The base pairs are essentially flat and
may be stacked one above the other
like a pile of plates
• so that the molecule is readily
represented as a spiral staircase with
the base pairs forming the treads
• The two polynucleotide strands of DNA are anti-
parallel
– That is the terminal nucleotide of one chain has a free 5'
end whereas the complementary chain has a free 3' end
at that same terminal end of the molecule.
– This means that the two strands are running in opposite
directions,
– and that the sequence of one strand determines
immediately the nucleotide sequence of the other,
complementary strand.
3. Why a helix?
• DNA inside a cell is surrounded by water.

– Sugars and phosphates are highly soluble in water,

– however the bases, adenine and thymine for example,

can be regarded as insoluble in water at neutral pH, they
are hydrophobic.
– Therefore the bases will try to escape from the water.
 When two polynucleotide chains encounter each other
they will tend to approach each other with the water
repellent parts of their molecule, leaving the sugar
phosphates at the outside.
Alternative DNA structures
• The existence of at least three different structural forms of
DNA has been observed by X-ray.
– A
– B
– Z
• Transitions between them occur when appropriate changes
are made in the environmental conditions.
• The B-DNA form for which Watson and Crick constructed
their model is found in fibers of very high relative humidity (92
%) and in solutions of low ionic strength.
• it was assumed that B form prevails in the living cell
• It has on average 10.5 bp per turn
• The A-DNA form is found in fibres at 75 % relative humidity
and requires the presence of sodium, potassium, or cesium
as the counter-ion.
• Instead of lying flat, the bases are tilted with regard to the
helical axis (20°).
• 11 bp/turn

• The A-form is biologically interesting because it is probably

very close to the conformation adopted by DNA-RNA
hybrids or by RNA-RNA double-stranded regions.
Z form DNA:
• Under condition of high salt concentration, DNA with
alternating purine-pyrimidine sequence such as (GC)n
apparently adopts a left-handed helical structure.
• It has the most base pairs per turn (12) in comparison to A
and B
• the name 'Z-DNA' is taken from the zigzag path of the
sugar-phosphate backbone along the helix.
DNA Topology / Supercoils
• Inside the cell a long DNA molecule is not free to rotate
about its long axis as a linear molecule in solution would be.
• Instead, rotation is constrained either by proteins holding
together a loop of DNA, as observed on the scaffolds of
eukaryotic chromosomes, or by the closure of a DNA
molecule into a circle as in the bacterial chromosome or
plasmids.
• Once rotation is constrained in this manner any physical
changes which favour an alteration of the helical twist of
DNA will place the molecule under torsional strain within the
limits of the region in which free rotation is restricted.
• In closed domains, any torsional stress will result in two forms of twisting:

– a change in the helical repeat, that is a change in the number of double

helical turns,
– and also a twisting of the long axis of the DNA molecule, or a supercoil.
• Supercoiling can be measured as the density of supercoils
per unit length of DNA, which is called σ.
• Supercoiling density has important effects in vivo, and that
specific enzymes are necessary to change it when the
structure of DNA requires manipulation

For instance:
• topoisomerase I- introduces transient single strand breaks
into substrate DNAs
• topoisomerase II- introduces transient double strand break
into substrate DNAs
• The information is within the unique base sequence of the
DNA.

• The double helix would have to be dissociated in order to

access the information
 Organization of DNA into chromosomes
• The total length of cellular DNA in prokaryotes & eukaryotes
is 1000s - 100,000x of the cell’s length. However, several
mechanisms operate to pack DNA sufficiently to fit inside the
cell.
• DNA is coiled at several levels. About 140 – 150 DNA is
wound around an eukaryyotic histone protein core to form a
nuclosome.
• The nuclosome in turn form a helical solenoid, each turn of
solenoid includes about six nuclosome
helical solenoid
• The solenoid themselves organized into chromatin loops
which contains approx. 100 kb of DNA.
• The end results of these coiling & looping is that DNA, at its
maximum stage of condensation, is only about 1/100,000 as
long as it would be if it were fully stretched
 Chromosomes
• Greek (chroma, color) & (soma, body) due to its capacity to
be stained
• It is a single large macromolecule of DNA which contains
many genes, regulatory elements & other intervening
nucleotide sequences physically organized in a cell.
 Composition of chromosomes
Eukaryotes contains:
• Double stranded single molecule of DNA

• Many copies of 5 kinds of positively-charged histones

proteins rich in lysine & arginine which shield phosphates
• Non-histone proteins: small copies of many different kinds
like transcription factors
• Duplicated chromosomes are held together at their
centromers common to call sister chromatids.
• The shorter of the two arms extending from the centromere
is called the p arm; the longer is the q arm.
 Sorting of chromosome
a. Sorting by size or length
• The ordered display of chromosomes is termed as its
karyotype, studied when the cell is at metaphase of
mitosis.
b. Sorting chromosome by position of Centromere
• Metacentric – centromer near to the middle

• Sub-metacentric- centromer between the middle & the tip

• Acrocentric- centromer near to the tip

 Summary
• DNA functions to serve as stable storage and transmission
of genetic information
• Semi-conservative is a type of DNA synthesis where
parental DMA stands act as templates for synthesis of new
DNA, resulting in the synthesis of complementary daughter
strands of DNA
• DNA synthesis includes initiation, elongation, and
termination steps
 Review questions
1. List the steps of DNA replication

2. Describe semi-conservative replication

3. Explain the structure of DNA

4. List the level of DNA organization

5. What are the components of chromosomes?

 References
• Robert F. weaver, Philip W. Hedrick. Genetics.

• Darnel, Lodish, Baltimore. Molecular Cell Biology

• James D. Watson: Recombinant DNA

• Robert F. Weaver. Molecular biology

• Richard J. Epistein: Human Molecular Biology

• P.K. Gupta: Cell and Molecular Biology

• Tarek H. EL-Metwally. Basic Medical Molecular Biology:

A Comprehensive update .