TABLE OF CONTENTS

Table of Contents............................................................................................................1
A. Introduction about genetic material................................................................................2
B. DNA and its properties as a Genetic Material.................................................................2
C. The Structure of DNA.....................................................................................................3
1. The primary Structure...............................................................................................3
2. The nucleotide base composition of DNA................................................................4
3. Formation of the DNA Polynucleotide.....................................................................6
4. The secondary structure of DNA (Double Helix) .....................................................8
5. Complimentary Base Pairing in DNA Double Helix...............................................10
6. Tertiary structure of DNA......................................................................................11
7. Significance of Supercoiling of DNA In Vivo........................................................12
8. Relaxation of Supercoiled DNA.............................................................................13
a. Mechanism of action of a type I topoisomerase..........................................13
D. DNA Replication..........................................................................................................14
1. Mechanism of DNA Replication.............................................................................16
a. Initiation stage............................................................................................18
b. Elongation stage.........................................................................................18
c. Termination stage.......................................................................................19
2. The Structure and Functions of DNA Polymerases.................................................24
E. DNA Repair..................................................................................................................25
1. The Mechanism of DNA Repair.............................................................................26
a. Nucleotide Excision Repair..............................................................................26
b. Base Excision Repair........................................................................................28
c. Mismatch Repair (MMR).................................................................................30
d. Double-Strand Breakage Repair (DSBR).........................................................31
F. Introduction about Gene Expression............................................................................33
G. Gene Expression...........................................................................................................33
1. Transcription..........................................................................................................35
2. Translation..............................................................................................................39
H. Conclusion....................................................................................................................42
I. References....................................................................................................................43

1
 GENETIC MATERIAL

A. Introduction about genetic material

Although genetic analysis began with the rediscovery of the work of Gregor Mendel in
the early part of the twentieth century, subsequent elegant experimentation involving both
bacteria and bacteriophages elucidated the nature of genetic information, gene structure, the
genetic code, and mutations. Following Gregor Mendel’s discovery, the laws of inheritance
which explained that heredity is the result of discrete units of inheritance and every single unit
(or gene) is independent in its actions in an individual’s genome, several scientists began
carrying out experiments to prove, advance and discover more about what Mendel had already
observed.
These scientist include; Walter Sutton and Theodor Boveri; who discovered the
chromosome theory of inheritance, Thomas Morgan; who the chromosomes theory of
inheritance through his experiments on fruit flies; Frederick Griffith; who postulated that
information could somehow be transferred between different strains of bacteria; Oswald Avery
and his co-workers Colin MacLeod and Maclyn McCarty; that developed the “transforming
principle” postulating that “a nucleic acid of the deoxyribose (DNA) type is the fundamental
unit of the transforming principle of Pneumococcus Type III”, meaning that DNA is the
hereditary material; and Alfred Hershey and Martha Chase; whose experiment proved that the
hereditary information injected into the bacteria that specified the new generation of viruses
was DNA and not protein (Allison, 2007).

B. DNA and its properties as a Genetic Material:

Deoxyribose Nucleic Acid (DNA) is the genetic material in which factors of inheritance
are stored. It occurs in all cells; eukaryotes (animals and plants) and prokaryotes. In eukaryotic
cells, DNA is situated in the nucleus whereas in prokaryotes it is found in the cytoplasm. Also,
in eukaryotes the mitochondria contain mitochondrial DNA (Allison, 2007). According to
Hartl (1998), DNA is admirably suited to a genetic function because it satisfies the three
essential requirements of a genetic material.
1. First, any genetic material must be able to be replicated accurately, so that the
information it contains is precisely replicated and inherited by daughter cells. The basis
for exact duplication of a DNA molecule is the complementarity of the A–T and G-C

2
 pairs in the two polynucleotide chains. Unwinding and separation of the chains, with
each free chain being copied, results in the formation of two identical double helices.
2. A genetic material must also have the capacity to carry all the information needed to
direct the organization and metabolic activities of the cell. DNA as a genetic material
can direct the order in which amino acid units are. This can be observed from the fact
that the product of most genes is a protein molecule—a polymer composed of molecular
units called amino acids. The sequence of amino acids in the protein determines its
chemical and physical properties.
3. A genetic material must also can undergo occasional mutations in which the
information it carries is altered. Furthermore, the mutant molecules must be capable of
being replicated as faithfully as the parental molecule, so that mutations are heritable.

C. The Structure of DNA

1. The primary Structure

The ability of DNA to carry the genetic information required by a cell to reproduce
itself is closely related to the chemical structure of DNA molecules. Chemically, DNA is a
polymer consisting of a chain of monomers called nucleotides. A nucleotide is a complex
substance consisting of a sugar, base (nitrogen-containing ring-structure), and a phosphate
groups. In a DNA polynucleotide molecule, the sugar is called 2’-deoxyribose. The latter is a
five-carbon sugar in which the OH-group on carbon 2 of the ribose is replaced by a Hydrogen
atom. The DNA bases are guanine(G), adenine(A), thymine (T), and cytosine(C). These bases
are classified into two groups; Purines and Pyrimidines. Purines are nucleotide bases
consisting of two carbon-nitrogen rings. Purines include; adenine and guanine. Pyrimidines are
nucleotide bases composed of a single carbon-nitrogen ring. Pyrimidines include; cytosine and
thymine. The bases are attached to the 2’-deoxyribose sugars to form nucleosides. The
phosphate groups in DNA is attached on carbon 5’ of 2’-deoxyribose sugars. When the PO4
group is attached, the nucleoside becomes a nucleotide. There are 3 phosphate groups (alpha,
beta, and gamma) in a nucleotide with the alpha phosphate directly attached to the sugar
(Fletcher, 2013:2-3).

3
 The chemical components of DNA

Source:(Allison, 2007:14)

2. The nucleotide base composition of DNA

Following Levene’s discovery of the basic chemical structure of DNA, several
chemical analyses of DNA was repeated using more sensitive techniques quite a different result
was obtained. The experiment results obtained by Erwin Chargaff showed that the nucleotide
composition of DNA molecules varied in complex ways, depending on the source of the DNA.
A careful case study by Chargaff’s Analysis of DNA of different organisms, showed that the
four nucleotides were not present in equal proportions in DNA molecules after all.

4
A table showing the nucleotide base composition of some organisms as discovered by
Erwin Chargaff
Base Composition (Mole Percent)
No. Organism A G T C
1. Escherichia coli (K12) 26.0 23.9 24.9 25.2
2. Mycobacterium tuberculosis 15.1 14.6 34.9 35.4
3. Yeast 31.3 32.9 18.7 17.1
4. Herring 27.8 27.5 22.2 22.6
5. Rat 28.6 28.4 21.4 21.5
6. Human 30.9 29.4 19.9 19.8
Source: (Allison, 2007:15).

However, despite the irregularities in the nucleotide base composition of the DNA of
different organisms, Chargaff observed two important underlying regularities in double
stranded DNA; (a) the amount of adenine present in DNA always equals the amount of
thymine; (b) the amount of guanine always equals the amount of cytosine.
These findings are commonly referred to as Chargaff’s rules, which states that, ‘‘In the DNA
of an organism, the proportion of A always equals that of T, and the proportion of G always
equals that of C: A = T, and G = C’’. This means that, in the DNA of an organism there is
always an equal proportion of purines (A and G) and pyrimidines (C and T) (Allison, 2007).

5
 Adenine (A) is joined to thymine (T) by two hydrogen bonds, while guanine (G) is joined to
cytosine (C) by three hydrogen bonds.
Source: (Allison, 2007:19).

3. Formation of the DNA Polynucleotide

The DNA polynucleotide is formed by joining nucleotide triphosphates through the
process of polymerisation. There are four nucleotide triphosphates used in the synthesis of
DNA polynucleotides, 2’-deoxyadenosine 5’- triphosphate (dATP or A), 2’-deoxycytosine 5’-
triphosphate (dCTP or C), 2’-deoxyguanosine 5’- triphosphate (dGTP or G), and 2’-
deoxythymidine 5’- triphosphate (dTTP or T). During polymerisation, the β and γ phosphates
are lost and the nucleotides are joined together by the remaining α phosphate. The 5’ α
phosphate of one nucleotide and the 3’ carbon of the next nucleotide forms a 3’-5’
phosphodiester bond(C-O-P). Formation of the C-O-P bond leaves a free 5’ α phosphate
exposed and ready to bond with another 3’ carbon on the next nucleotide. Also, the formed
bond leaves the 3’ -OH group and the other end of the 3’ Carbon end, hence giving DNA
polynucleotide polarity so that a DNA molecule runs in 5’-3’ or 3’-5’ direction (Fletcher,
2013).

6
 The chemical structure of a small segment of a single DNA strand showing all four
nucleotides
Source: (Karp, 2013:393).

7
 A table showing DNA nucleosides and DNA nucleotides
No. Base DNA Nucleoside DNA Nucleotide
1. Adenine(A) Deoxyadenosine Deoxyadenosine 5’-triphosphate
(dATP)
2. Guanine (G) Deoxyguanosine Deoxyguanosine 5’-triphosphate
(dGTP)
3. Cytosine (C) Deoxycytidine Deoxycytidine 5’-triphosphate
(dCTP)
4. Thymine (T) Deoxythymidine Deoxythymidine 5’-triphosphate
(dTTP)
Source: (Allison, 2007:17).

4. The secondary structure of DNA (Double Helix)

DNA has a three-dimensional secondary structure known as the double helix. The DNA
double helix structure which was discovered by Watson and Crick in 1953 and by X-ray
diffraction, its pictures were taken by Franklin and Wilkins. The double helix is composed of
two polynucleotide chains wrapped around each other. In this structure, the sugar-phosphate
part of the molecule forms a spine or backbone which is on the outside of the helix. The flat
bases face inwards towards each other, slightly off-centre, and are stacked on top of each other
like a pile of plates. The pitch of the helix is 3.4nm, the space between bases on each strand is
0.34nm, and the diameter of the helix is 2nm.
The double helix executes a turn every 10 base pairs and it is said to be anti-parallel,
with one strand running in 5’-3’ direction while the other in the 3’-5’ direction. The double
helix is an irregularly shaped and when viewed from the outside, it consists of a major groove
and a minor groove. These grooves are important for interaction with proteins, for replication
of the DNA, and for expression of the genetic information. The double helix structure of DNA
can exist in various forms when crystals of the DNA molecule are formed under different
conditions. The form present in the cells is called the B form and it is said to be right handed.
Other forms of DNA double helix structure include the A form, C, D, E and Z forms (Fletcher,
2013: 4-5).

8
Schematic representation of the DNA double Space-filling model of the B form of DNA.
helix.

9
Source:(Karp, 2013:395).
A table showing the comparison between the forms of the secondary structure of DNA
FORMS OF DNA
No. A-DNA B-DNA Z-DNA
1. Orientation Right-handed Right-handed Left-handed
2. Major groove Deep and narrow Moderate depth, wide Very shallow,
virtually non-
existent,
sometimes
called a ‘‘single
groove’’
3. Minor groove Shallow and broad Moderate depth, narrow Very deep and
narrow
4. Turns 11 10.5 12
5. Conditions Low humidity (75%), High humidity (95%), High humidity,
high salt low salt low salt
Source (Allison, 2007:22)

5. Complimentary Base Pairing in DNA Double Helix

Within the double helix structure of DNA, the bases of one polynucleotide or DNA
strand are complimentarily paired to the bases of another polynucleotide in such a way that the
two-ring purine interacts with a single-ring pyrimidine. In the double helix, thymine(T)
interacts with adenine(A) while cytosine(C) pairs with guanine(G). During base pairing, three
hydrogen bonds form between G AND C, and two hydrogen bonds between A and T. The
formation of the hydrogen bonds helps to stabilise the interaction between the bases. Non-
complimentary bases cannot pair up, i.e. the combination of G to C, and A to T does not work
because they are too large or too small to fit inside the helix or the do not align correctly to
allow hydrogen bond formation between them. Since G must always pair with C and A to T,
the sequence of the two strands are related to each other are said to be complimentary with the
sequence of one strand predicting and determining the sequence of the other. Therefore, one
strand can be duplicated into the another, hence retaining the genetic information and passing
it on to the other cells during cell division.

10
 The ribbon-like strands represent the sugar–phosphate backbones, and the horizontal rungs
are the nitrogenous base pairs, of which there are 10.5 per complete turn. The major and
minor grooves are apparent. The inset highlights the antiparallel nature of the two strands of
the helix. The DNA sequence shown reads 5′-GCTA-3′.
Source: (Allison, 2007:21).

6. Tertiary structure of DNA

Due to the polarity of the strands of the DNA double helix, the 5’ end of one strand can
join its own 3’ end to covalently close a circle. (i) In prokaryotes, at tertiary level, the DNA is
organized in the form of a closed circle with no 5’ and 3’ end in almost all prokaryotes except
in Borrelia in which the chromosomes are linear. Such circular DNA molecules often become
overwound or underwound, with respect to the number of complete turns of the DNA double
helix. This DNA can then become supercoiled (under torsional stress). Supercoils are a twisted,
three-dimensional structure which is more favourable energetically (Allison, 2007).
This circular double helix is further twisted into supercoiled DNA and is associated with basic
proteins but not with the histones found complexed with almost all eukaryotic DNA. These

11
histone like proteins help to organize bacterial DNA into a coiled chromatin like structure
(Karp, 2013).
Diagram showing the tertiary form of DNA in most prokaryotic cells

The DNA molecule at the left is underwound; that is, it has more than an average of 10 base
pairs per turn of a helix. An underwound molecule spontaneously assumes a negatively
supercoiled conformation, as shown on the right
Source: (Karp, 2013:398)
(ii). In eukaryotic cells, the DNA is much more highly organized and is associated with a
variety of proteins, with the most prominent being histones. The histones are small, basic
proteins rich in the amino acids lysine and/or arginine. There are five types of histones in almost
all eukaryotic cells studied: H1, H2A, H2B, H3, and H4. Eight histone molecules (two each of
H2A, H2B, H3, and H4) form an ellipsoid about 11 nm long and 6.5 to 7 nm in diameter. DNA
coils around the surface of the ellipsoid approximately 1 ¾ turns or 166 base pairs before
proceeding on to the next. This complex of histones plus DNA is called a nucleosome. The
stretch of DNA between the beads or nucleosomes, the linker region, varies in length from 14
to over 100 base pairs. Histone H1 associate with the linker regions to aid the folding of DNA
into more complex chromatin structures (Karp, 2013).

12
 7. Significance of Supercoiling of DNA In Vivo
According to Allison (2007:33), experimental evidence suggests that DNA supercoiling
plays an important role in many genetic processes, such as replication, transcription, and
recombination.
a. Negative (left hand) supercoiling puts energy into DNA. Underwinding makes it
easier to pull the two strands of the double helix apart. Therefore, negative
supercoiling makes it easier to open replication origins and gene promoters.
b. The potential energy in the supercoils also promotes formation of unusual DNA
secondary structures, like cruciform.
c. Also, it is possible that a B-DNA → Z-DNA transition is triggered by increased
negative supercoiling. This is because switching a portion of the DNA from a right-
handed to left-handed helix releases the strain imposed by the negative supercoils,
since the twist (base pairs per turn) in a portion of the DNA has been reversed.
d. Positive (right hand) supercoiling which occurs ahead of replication forks and
transcription complexes, makes it much harder to open the double helix and
therefore blocks essential DNA processes.

8. Relaxation of Supercoiled DNA

The supercoiled state is inherently less stable than relaxed DNA. The stress present
within supercoiled DNA molecules sometimes leads to localized denaturation, in which the
complementary strands come apart in a short section. This has important implications for
cellular processes such as replication and transcription. To overcome the implications, in cells
there are conserved topoisomerases; the enzymes that are convert (isomerize) topoisomers
(forms of DNA that have the same sequence yet differ in their linkage number or number of
turns) of DNA to another. Topoisomers do so by changing the linking number (L). DNA
topoisomerases fall into two major categories, type I and type II. The two types can be further
subdivided into four subfamilies: IA, IB, IIA, and IIB. There are at least five different
topoisomerases have been reported to be present in higher eukaryotes, including humans
(Allison, 2007).

13
 A table showing the human DNA topoisomerases
No. DNA Type DNA Structural role Function
topoisomerase cleavage
1. I IB ssb Relax both negatively and Replication
positively supercoiled Transcription
DNA Recombination
2. IIIα IA ssb Relax only negatively Recombination
supercoiled DNA Transcription of
Ribosomal RNA
genes
3. IIIβ IA ssb Relax only negatively Recombination
supercoiled DNA
4. IIα IIA dsb Relax both positively and Chromosomal
negatively supercoiled condensation
DNA Chromosomal
Facilitate unknotting or segregation
decatenation of entangled Replication
DNA
5. IIβ IIA dsb Relax both positively and Not well defined
negatively supercoiled
DNA
Facilitate unknotting or
decatenation of entangled
DNA
Source: (Allison, 2007:31).

a. Mechanism of action of a type I topoisomerase.

According to Allison (200:327, the mechanism of action of type I topoisomerase includes
the following steps;
i. The enzyme binds to a circular DNA molecule with one negative supercoil and
unwinds the double helix.

14
ii. It nicks one strand and prevents free rotation of the helix by remaining bound to
each broken end.
iii. The 5’ broken end is covalently attached to the amino acid tyrosine. The oxygen of
the tyrosine hydroxyl group in the active site of the enzyme attacks a DNA
phosphorus, forming a covalent phosphotyrosine link between the DNA and the
enzyme, and breaking a DNA phosphodiester bond at the same time.
iv. Re-joining of the DNA strand occurs by the reverse when the oxygen of the free
DNA 3’ -OH group attacks the phosphorus of the phosphotyrosine link, breaking
the covalent bond between the protein and DNA, and reforming the phosphodiester
bond between adjacent nucleotides in the DNA chain.
v. and the 3’ end is noncovalently bound to another region of the enzyme.
vi. The enzyme passes the unbroken strand of DNA through the break and ligates the
cut ends, thereby increasing the linking number of the DNA by one. The enzyme
falls away and the strands renature, leaving a relaxed circle.

15
Source: (Allison, 2007:32).

D. DNA Replication
DNA replication is the process by which a DNA reproduces a new copy of itself (Karp,
2013:545). DNA replication simply involves the melting apart of the two strands of the double
helix followed by the polymerization of new complementary strands on the resulting single-
stranded templates (Allison, 2007). Following Watson-Crick’s discovery of the double helix
structure of DNA, to explain the mechanism of DNA replication, scientists developed three
hypotheses. These were;
a) The semi-conservative model stated that during DNA replication is
complementarity. It explained that, to produce a new DNA molecule, it requires
assembling appropriate complementary nucleotides on the exposed single strands
to form two daughter duplexes with the same sequence.
b) The conservative model stated that the parental double helix would remain intact
and generate DNA copies consisting of entirely new molecules.
c) The dispersive model predicted that parental DNA would become dispersed
throughout the new copy so that each strand of all the daughter molecules would be
a mixture of old and new DNA.
The three hypotheses of DNA replication were evaluated in 1958 by Matthew Meselson
and Franklin Stahl of the California Institute of Technology. This was through their experiment
carried out on the bacteria. During Meselson–Stahl experiment, bacteria were grown in a
medium containing the heavy isotope of nitrogen, 15N, which became incorporated into the
bases of the bacterial DNA. After several generations, the DNA of these bacteria was denser
than that of bacteria grown in a medium containing the lighter isotope of nitrogen, 14N.
Meselson and Stahl then transferred the bacteria from the 15N medium to the 14N medium and
collected the DNA at various intervals. By dissolving the DNA, they had collected in a heavy
salt called caesium chloride and then spinning the solution at very high speeds in an
ultracentrifuge, Meselson and Stahl were able to separate DNA strands of different densities.
The enormous centrifugal forces generated by the ultracentrifuge caused the caesium
ions to migrate toward the bottom of the centrifuge tube, creating a gradient of caesium
concentration, and thus of density. Each DNA strand floats or sinks in the gradient until it
reaches the position where its density exactly matches the density of the caesium there. Because
15N strands are denser than 14N strands, they migrate farther down the tube to a denser region
of the caesium gradient. The DNA collected immediately after the transfer was all dense.
16
However, after the bacteria completed their first round of DNA replication in the 14N medium,
the density of their DNA had decreased to a value intermediate between 14N-DNA and 15N-
DNA. After the second round of replication, two density classes of DNA were observed, one
intermediate and one equal to that of 14N-DNA. From the experiment, Meselson and Stahl
observed that; after the first round of replication, each daughter DNA duplex was a hybrid
possessing one of the heavy strands of the parent molecule and one light strand; when this
hybrid duplex replicated, it contributed one heavy strand to form another hybrid duplex and
one light strand to form a light duplex. Thus, this experiment clearly confirmed that DNA
replicates in a semiconservative manner (Allison, 2007:110, Karp, 2013:545-548).

Source: (Karp, 2013:546).

17
 1. Mechanism of DNA Replication
The replication of the DNA double helix is a complex process involving several
enzymes and proteins. The process of replication in prokaryotes and eukaryotic cells is
thought to be similar and it involves stages of initiation, elongation and termination;
which occur in 12 major steps:

a. Initiation stage
• Replication begins by removal of histones at the point of origin of replication. This
allows access to the replication machinery of DNA.
• Prereplication complex (pre-RC) formation at the origins of replication. The
assembly of the pre-RC is an ordered process that is initiated by the association of
the origin recognition complex (ORC) with the origin.
• Replication “licensing.” Once bound, ORC recruits at least two additional proteins,
Cdc6 and Cdt1. ORC and these two proteins function together to recruit the Mcm2-
7 helicase complex to complete the formation of the pre-RC.
• Duplex unwinding at replication forks and relaxing of positive supercoils. Cdc6 and
Cdt1 are released from the complex, and other replication factors are recruited (e.g.
replication protein A, replication factor A; RPA). The helicase activity of Mcm2-7
unwinds the DNA duplex. Topoisomerase I and/or topoisomerase II resolve
positive supercoils ahead of the replication fork.
• The separation of the double helix structure of DNA creates a ‘Y’ shape called a
replication ‘fork’. The later consists of two separated strands; the leading strand
oriented in the 3’ to 5’ direction (towards the replication fork) and the lagging strand
oriented in the 5’ to 3’ direction (away from the replication fork). These two strands
act as templates for making the new strands of DNA. Because of their different
orientations, the two strands are replicated differently
b. Elongation stage

i. For Leading Strand:

18
 • Once present at the origin, DNA pol α/primase synthesizes an RNA primer and
briefly extends it. The primer comes along and binds to the 3’ end of the leading
strand. The primer acts as the starting point for DNA synthesis.
• The resulting primer–template junction is recognized by the sliding clamp loader
(replication factor C; RFC), which assembles a sliding clamp (proliferating cell
nuclear antigen; PCNA) at these sites. Then DNA polymerase δ recognizes this
primer and begins leading synthesis. The DNA polymerase δ binds ‘walks’ along
it, adding new complimentary nucleotide bases (A, C, G and T) to the strand of
DNA in the 5’ to 3’ direction. During elongation, the DNA polymerase δ enzyme
is held in place by the protein called sliding clamp.

ii. For Lagging strand:

• Once present at the origin, DNA polymerase α/primase synthesizes RNA primers
which bind at various points along the lagging strand. The primers act as the starting
points for DNA synthesis.
• The resulting primers–template junction is recognized by the sliding clamp loader
(RFC), which assemble sliding clamps (PCNA) at these sites. Then DNA
polymerase ε recognizes the primers and begin lagging synthesis by adding chunks
of DNA, called Okazaki fragments, in the 5’ to 3’ direction. During elongation, the
DNA polymerase ε enzyme is held in place by the protein called sliding clamp.
c. Termination stage
• After elongation is complete, the RNA primer(s) degraded by the endonuclease
activity of FEN-1
• After the filling of gaps left by primer removal is mediated by either DNA pol δ for
(leading strand) or DNA pol ε (lagging strand).
• For the lagging strand, the Okazaki fragments are joined by formation of a
phosphodiester bond between adjacent Okazaki fragments by the action of DNA
ligase I in association with PCNA.
• Finally, there new DNA is deposited with Histone. Nucleosomes are reassembled
on nascent DNA via interaction with chromatin assembly factor-1 (CAF-1) and
PCNA.

19
20
21
22
Source: (Allison: 2007:120-123).

23
A comparison of the proteins involved in prokaryotes and eukaryotes DNA replication
Prokaryote (eg. E. Coli) Human (SV40 model) Function
DNA-B Mcm2-7 (T antigen) Helicase
DNA-C Mcm2-7 (T antigen) Loading helicase/primase
SSB RPA Single strand maintenance
DNA-G (primase) Polymerase α / primase Priming
PROTEIN

β PCNA Sliding clamp

Γδ complex RFC Clamp loading (ATPase)
Polymerase III Polymerase δ / Strand elongation
polymerase ε
Polymerase I FEN-1, RnaseH1 RNA primer removal
Ligase Ligase 1 Ligation of Okazaki
fragments
Source: (Allison, 2007:116).

2. The Structure and Functions of DNA Polymerases

DNA polymerase III, the enzyme that synthesizes DNA strands during replication in E.
coli, is part of a large “replication machine” called the DNA polymerase III holoenzyme.
One of the noncatalytic components of the holoenzyme, called the β clamp, keeps the
polymerase associated with the DNA template. DNA polymerases possess two somewhat
contrasting properties: (1) they must remain associated with the template over long stretches if
they are to synthesize a continuous complementary strand, and (2) they must be attached
loosely enough to the template to move from one nucleotide to the next. These contrasting
properties are provided by the doughnut-shaped β clamp that encircles the DNA and slides
along it. If it is attached to a β “sliding clamp,” a DNA polymerase can move processively from
one nucleotide to the next without diffusing away from the template.
The assembly of the β clamp around the DNA requires a multi-subunit clamp loader that
is also part of the DNA polymerase III holoenzyme. In the ATP-bound state, the clamp loader
binds to a primer-template junction while holding the β clamp in an open conformation. Once
the DNA has squeezed through the opening in the clamp wall, the ATP bound to the clamp
loader is hydrolysed, causing the release of the clamp, which closes around the DNA. The β
clamp is then ready to bind polymerase III. DNA polymerase I, which consists of only a single
subunit, is involved primarily in DNA repair, a process by which damaged sections of DNA

24
are corrected. DNA polymerase I also removes the RNA primers at the 5’ end of each Okazaki
fragment during replication and replaces them with DNA.

A diagram showing the Schematic representation of DNA polymerase III holoenzyme

The holoenzyme contains ten different subunits organized into several distinct components.
Included as part of the holoenzyme are (1) two core polymerases which replicate the DNA,
(2) two or more β clamps, which allow the polymerase to remain associated with the DNA,
and (3) a clamp loading γ complex, which loads each sliding clamp onto the DNA. The clamp
loader of an active replication fork contains two T subunits, which hold the core polymerases
in the complex and bind the helicase. Another term, the replisome, is often used to refer to
the entire complex of proteins that is active at the replication fork, including the DNA
polymerase III holoenzyme, the helicase, SSBs, and primase.
Source: (Karp, 2013:555).

E. DNA Repair
DNA is one of the molecules in a cell that is most susceptible to environmental damage.
Damages in DNA occur when; stuck by ionising radiation, which leads to breakage of the
backbone of DNA molecule; when exposed to a variety of reactive chemicals, many of which
are produced by a cell’s own metabolism, the bases of a DNA molecule may be altered
structurally; when subjected to ultraviolet radiation, adjacent pyrimidines on a DNA strand
have a tendency to interact with one another to form a covalent complex, that is, a dimer. Even
the absorption of thermal energy generated by metabolism is sufficient to split adenine and

25
guanine bases from their attachment to the sugars of the DNA backbone. Failure to repair such
lesions produces permanent alterations, or mutations, in the DNA. If the mutation occurs in a
cell destined to become a gamete, the genetic alteration may be passed on to the next generation
(Karp, 2013).
Considering the potentially drastic consequences of alterations in DNA molecules and
the high frequency at which they occur, it is essential that cells possess mechanisms for
repairing DNA damage so that the genetic information remain mostly unchanged as it is passed
from cell to cell and individual to individual. Both prokaryotic and eukaryotic cells possess a
variety of proteins that patrol vast stretches of DNA, searching for subtle chemical
modifications or distortions of the DNA duplex. In some cases, damage can be repaired
directly. Humans, for example, possess enzymes that can directly repair damage from cancer-
producing alkylating agents. Most repair systems, however, require that a damaged section of
the DNA be excised, that is, selectively removed. The repair of DNA damage in eukaryotic
cells is complicated by the relative inaccessibility of DNA within the folded chromatin fibres
of the nucleus, for example; in the case of transcription, DNA repair involves the participation
of chromatin-reshaping machines, such as the histone modifying enzymes and nucleosome
remodelling complexes (Karp, 2013).

1. The Mechanism of DNA Repair

a. Nucleotide Excision Repair
Nucleotide excision repair (NER) operates by a cut-and patch mechanism that removes
a variety of bulky lesions, including pyrimidine dimers and nucleotides to which various
chemical groups have become attached. There are two distinct NER pathways, i.e.
i. A transcription-coupled pathway in which the template strands of genes that are being
actively transcribed are preferentially repaired. Repair of a template strand is thought
to occur as the DNA is being transcribed, and the presence of the lesion may be
signalled by a stalled RNA polymerase. This preferential repair pathway ensures that
those genes of greatest importance to the cell, which are the genes the cell is actively
transcribing, receive the highest priority on the “repair list.”
ii. The global genomic pathway that corrects DNA strands in the remainder of the genome.
This is a slower, less efficient mechanism in DNA repair.
The transcription-coupled pathway and global genomic pathway of NER involve almost
the same steps during DNA repair but only differ in protein that recognises the lesion. The
steps involved in NER repair are; (1) damage recognition in the global pathway is mediated by
26
an XPC-containing protein complex, whereas damage recognition in the transcription-coupled
pathway is thought to be mediated by a stalled RNA polymerase in conjunction with a CSB
protein; (2) DNA strand separation (by XPB and XPD proteins, two helicase subunits of
TFIIH); (3) incision (by XPG on the 3’ side and the XPF–ERCC1 complex on the 5’ side); (4)
excision, (5) DNA repair synthesis (by DNA polymerase δ and/or ε); and (6) ligation (by DNA
ligase I) (Karp, 2013).

27
Source: (Karp, 2013: 566).

b. Base Excision Repair

This is a separate excision repair system that operates to remove altered nucleotides
generated by reactive chemicals present in the diet or produced by metabolism. The steps in
this repair pathway in Eukaryotes are; (i) BER is initiated by a DNA glycosylase that recognizes
the alteration and removes the altered base by cleavage of the glycosidic bond holding the base
to the deoxyribose sugar; (ii) A specific DNA glycosylase enzyme removes the altered base
and inserts a specific amino acid side chain into the DNA helix, for example; DNA glycosylase
that removes the highly mutagenic 8-oxoguanine (oxoG). It is indicated that this enzyme
diffuses rapidly along the DNA “inspecting” each of the G-C base pairs within the DNA
duplex. When the enzyme has come across an oxoG-C base pair, the enzyme inserts a specific
amino acid side chain into the DNA helix, causing the nucleotide to rotate (“flip”) 180 degrees
out of the DNA helix and into the body of the enzyme; (iii) If the nucleotide contains an oxoG,
the base fits into the active site of the enzyme and is cleaved from its associated sugar.
However, if extruded nucleotide contains a normal guanine, which only differs in structure by
two atoms from oxoG, it is unable to fit into the enzyme’s active site, and it is returned to its

28
appropriate position within the stack of bases; (iv) Once an altered purine or pyrimidine is
removed by a glycosylase, the “beheaded” deoxyribose phosphate remaining in the site is
excised by the combined action of a specialized (AP) endonuclease and a DNA polymerase.
AP endonuclease cleaves the DNA backbone, and a phosphodiesterase activity of polymerase
β removes the sugar-phosphate remnant that had been attached to the excised base; (v)
Polymerase β then fills the gap by inserting a nucleotide complementary to the undamaged
strand; (vi), and the strand is sealed by DNA ligase III.

29
 Source: (Karp, 2013:566)

c. Mismatch Repair (MMR)

This is a mechanism for repairing mismatched bases that are incorporated by the DNA
polymerase and escape the enzyme’s proofreading exonuclease. MMR is possible because a
mismatched base pair causes a distortion in the geometry of the double helix that can be
recognized by a repair enzyme. For successful MMR, the repair system distinguishes the newly
synthesized strand, which contains the incorrect nucleotide, from the parental strand, which
contains the correct nucleotide. In prokaryotes e.g.; E. coli, the two strands are distinguished

30
by the presence of methylated adenosine residues on the parental strand. In eukaryotes,
however due to lack of DNA methylation, the mechanism of identification of the newly
synthesized strand remains unclear (Karp, 2013).

d. Double-Strand Breakage Repair (DSBR)

Double-strand breaks (DSBs) are caused by ionisation radiation from X-rays, gamma rays,
and particles released by radioactive atoms. When these forms of radiation collide with a fragile
DNA molecule, they often break both strands of the double helix. Also, DSBs can be caused
by certain chemicals, including several (e.g., bleomycin) used in cancer chemotherapy, and
free radicals produced by normal cellular metabolism. DSBs are also introduced during
replication of damaged DNA. A single double- strand break can cause serious chromosome
abnormalities, and therefore require reparation.
DSBs can be repaired by several alternate pathways, however; the predominant pathway in
mammalian cells is called nonhomologous end joining (NHEJ), in which a complex of proteins
binds to the broken ends of the DNA duplex and catalyses a series of reactions that re-join the
broken strands. The major steps that occur during NHEJ are;
• the lesion is detected by a heterodimeric, ring-shaped protein called Ku, that binds to
the broken ends of the DNA;
• The DNA-bound Ku recruits another protein, called DNA-PKcs, which is the catalytic
subunit of a DNA-dependent protein kinase;
• Then substrates phosphorylated by DNA-dependent protein kinase bring the ends of the
broken DNA together in such a way that they can be joined by DNA ligase IV to
regenerate an intact DNA duplex (Karp, 2013).

31
Source: (Karp, 2013:568)

Another DSB repair pathway known as homologous recombination (HR) requires a

homologous chromosome to serve as a template for repair of the broken strand. Compared to
NHEJ pathway, homologous recombination is a more accurate pathway; that is, there are fewer
errors in the base sequence of the repaired DNA. However, because it requires that a
homologous chromosome be present in the nucleus, HR can only be employed during the cell
cycle after DNA replication takes place (i.e., during late S or G2 phase). Defects in both repair
pathways have been may lead to increased cancer susceptibility (Karp, 2013).

32
 GENE EXPRESSION

F. Introduction about Gene Expression

Life is characterized by the presence of metabolic processes that occur within the cell.
Metabolism is a process of chemical change from one form to another, for example from a
simple form to a more complicated form or vice versa. The metabolic process involves the
transformation of matter and energy.
Morphological appearance which is the phenotype of an organism is the result of metabolic
processes that occur within each cell composing the organism. The morphological diversity
among the individual members of a population is highly dependent on the variety of processes
and metabolic outcomes that occur in each individual. The difference in the colour of flowers
of a variety with other varieties depends on the metabolic processes occurring within the cells
and varieties concerned.
The process of metabolism in cells is a biochemical reaction catalysed by an enzyme, so that
the variety of processes and metabolic outcomes is determined by the enzymes involved in the
reaction. The diversity of the enzymes (both the structure and the amino acid structure) itself
is largely determined by the moulding arrangement of deoxyribonucleic acid (DNA). The DNA
segment that becomes the mould to synthesize an enzyme (protein) called a gene, so that the
gene is a controlling metabolic process or the controller of life. The morphological diversity of
an organism is the appearance of its genes.

G. Gene Expression
DNA is the biological information needed by an organism to reproduce itself. In
physical terms, gen is discrete segment of DNA with a base sequence that encodes the amino
acids sequence of a polypeptide (Fletcher, 2013). In higher organisms the genes are present on
a series of extremely long DNA molecules called Chromosomes.
The biological information in a DNA molecule is contained in its base sequence. That the
expression is the process made the information available to the cell. This information will be
describing by the Central Dogma and transferred from DNA to RNA to protein.

DNA RNA Translation Protein

Transcription

Reverse transcription

Figure 1. The central Dogma above and reverse transcription below

33
 Gene is the piece of DNA containing the information for a single specific polypeptide
(one gene, one polypeptide). Gene as being synonymous with “open reading frame” (ORF),
the region between the start and stop codons (Blackwell, 2012). In bacteria, usually a simple
uninterrupted sequence, but in eukaryotes, the presences of introns make this definition more
difficult, since the region of the chromosome that contains the specific information of
polypeptide may be many times longer than the actual coding sequences. DNA is a template
for non-coding RNA molecules, which play an important role in gene regulation and other
activities. Other areas of DNA are important in gene regulation because they act as binding
sites for regulating proteins.
After several years of research, Mendel draws conclusions in modern genetic
terminology: genes are the inheritance of the nature of an identical living thing that is not
identical with its parent. Alternative forms of genes are called alleles (Karp, 2006). In the
human body there are many genes that will be expressed into phenotypes (visible traits), such
as black hair, skin colour, and sharp nose. The central dogma of biology is a framework for
understanding the order of biopolymer information transfer (DNA, RNA, protein). Gene
expression is the process by which information encoded in a gene is translated into a sequence
of amino acids during protein synthesis, consisting of two stages: transcription and translation.

34
 Figure 2. Information Flow
1. Transcription
In the transcription process, the DNA strands provide information for the synthesis of
RNA strands. The enzyme responsible for both prokaryotic and eukaryotic cells is the RNA
polymerase present in DNA. This enzyme can combine nucleotides one by one into an RNA
strand complementing one strand of DNA. Sites in DNA where RNA polymerase molecules
bind before starting transcription are called promoters. Cellular RNA polymerase is unable to
recognize its own promoters but requires the help of an additional protein called transcription
factor. In addition to providing a binding site for polymerases, the promoter contains
information that determines which DNA strands of the two strands and the site where the
transcription starts.
A messenger RNA is assembled as a complimentary copy of one of the two DNA
strands that make up a gene. The synthesis of an RNA from a DNA template is called
transcription. Because its nucleotide sequence is complementary to that of the gene from which
it is transcribed, the mRNA retains the same information as the gene itself. An overview of the
role of mRNA in the flow of information through a eukaryotic cell is illustrated by figure below
(Karp, 2013).

Figure 3. An overview of the flow of information in a eukaryotic cell

Selected sites on the DNA are transcribed into pre-mRNAs (step 1), which are
processed into messenger RNAs (step 2). The messenger RNAs are transported out of the
35
nucleus (step 3) into the cytoplasm, where they are translated into polypeptides by ribosomes
that move along the mRNA (step 4). Following translation, the polypeptide folds to assume its
native conformation (step 5). mRNA as templates in amino acid combinations in certain
sequences encoded by DNA nucleotide sequences and mRNAs. The use of messenger RNA
also allows the cell to amplify its synthetic output. One DNA molecule can serve as a template
in the formation of many mRNA molecules, each of which can be used in the formation of a
large number of polypeptide chains.

As discovered in 1969 by Robert Roeder at the University of Washington, eukaryotic

cells have three distinct transcribing enzymes in their cell nuclei. Each of these enzymes is
responsible for synthesizing a different group of RNAs (Table 11.1). (Karp,2006).

Transcription is carried out by RNA polymerase. RNA polymerase recognizes and

binds to specific sequence (the promoter) and initiates the synthesis of mRNA from an adjacent
position (Blackwell, 2012). Eukaryotes have three different RNA polymerases. Only one of
these, RNA polymerase II, is involved in the transcription of protein- coding transcripts, plus
the transcription of a group of small non- coding RNAs called micro-RNAs. RNA polymerase
1 is responsible for the synthesis of large ribosomal RNAs, whilst RNA polymerase 3 makes
small RNAs such as transfer RNA (tRNA) and 5s ribosomal RNA
In the transcription process, the DNA strands provide information for the synthesis of
RNA strands. The enzyme responsible for both prokaryotic and eukaryotic cells is the RNA
polymerase present in DNA. This enzyme can combine nucleotides one by one into an RNA
strand complementing one strand of DNA. Sites in DNA where RNA polymerase molecules
bind before starting transcription are called promoters. Cellular RNA polymerase is unable to
recognize its own promoters but requires the help of an additional protein called transcription
factor. In addition to providing a binding site for polymerases, the promoter contains
36
information that determines which DNA strands of the two strands and the site where the
transcription starts.
RNA polymerase molecules that bind to DNA before the transcription process are
called promoters. the function of this promoter is to determine which DNA strands of the two
strands and the site where the transcription starts. RNA polymerase moves along the template
DNA strand to the 5 'end (direction 3’ to 5’), as polymerase progress, DNA temporarily
withdrawn, the polymerase assembles a complementary strand of RNA grown from tip 5 in
direction 3. Polymerase incorporates complementary nucleotides into the growing RNA chain.

Figure.4 Chain elongation during transcription. (b)Schematic drawing of an RNA

polymerase in the act of transcription elongation. The downstream DNA lies in a groove within
the polymerase, clamped by a pair of jaws formed by the two largest subunits of the enzyme.
The DNA makes a sharp turn in the region of the active site, so that the upstream DNA extends
upward in this drawing. The nascent RNA exits from the enzyme’s active site through a
separate channel.
Bacteria transcription, such as E. coli, contain one type of RNA polymerase consisting of five
closely related subunits forming the core enzyme. If the core enzyme is emitted from the
bacterial cell and added to the bacterial DNA molecule and ribonucleotide triphosphate, the
enzyme binds to DNA and synthesizes RNA. However, if so, a polypeptide called the sigma
factor () is added to the RNA polymerase prior to being attached to the DNA, the transcription
begins at the selected location (Fig. 11.6a, b). To repair and improve its affixes for DNA in
general. Checks, which have been completed glide freely throughout the DNA until he and the
promoter are appropriate.
37
 Schematic representation of the initiation of
transcription in bacteria. (a) In the absence of
the sigma factor, the core enzyme does not
interact with the DNA at specific initiation
sites. (b–d) When the core enzyme is
associated with the sigma factor, the
complete enzyme (or holoenzyme) is able to
recognize and bind to the promoter regions
of the DNA, separate the strands of the DNA
double helix, and initiate transcription at the
proper start sites. In the traditional model
shown here, the sigma factor dissociates
from the core enzyme, which is capable of
transcription elongation. Recent studies
suggest that, in at least some cases, sigma
may remain with the polymerase.

2. Translation
The synthesis of a polypeptide chain can be divided into three rather distinct activities:
initiation of the chain, elongation of the chain, and termination of the chain. After attaching to
the mRNA, the ribosome always moves along the mRNA from one of the codons to the next
38
codon, that is, in three consecutive nucleotide blocks. The ribosome attaches to the mRNA in
the right place, called the initiation codon, which is determined as the AUG. Tying codons
automatically puts the ribosome in the correct frame of reading. For example, the ribosome
movement from the initiation codon, AUG, to the next three nucleotides, CUC, then to CAG,
and so on along the lines. The basic steps in Fig.

Initiation of protein synthesis in bacteria. In

step 1, initiation of translation begins with the
association of the 30S ribosomal subunit with
the mRNA at the AUG initiation codon, a step
that requires IF1 and IF3.The 30S ribosomal
subunit binds to the mRNA at the AUG
initiation codon as the result of an interaction
between a complementary nucleotide
sequence on the rRNA and mRNA, as
discussed in the text. In step 2, the
formylmethionyl-tRNAfMet becomes
associated with the mRNA and the 30S
ribosomal subunit complex by binding to IF2-
GTP.In step 3, the 50S subunit joins the
complex, GTP is hydrolysed IF2-GDP is
released. The initiator tRNA enters the P site
of the ribosome, whereas all subsequent
tRNAs enter the A site

The basic step in the process of translational extension of bacterial cells. This series of steps is
repeated because the amino acids are polymerized into the growing polypeptide chain called
by Elongation.

39
 Steps in the elongation of the nascent
polypeptide during translation in bacteria.
(a) In step 1, an aminoacyltRNA whose
anticodon is complementary to the second
codon of the mRNA enters the empty A
site of the ribosome. The binding of the
tRNA is accompanied by the release of
EF-Tu-GDP. In step 2, peptide bond
formation is accomplished by the transfer
of the nascent polypeptide chain from the
tRNA in the P site to the aminoacyl-tRNA
of the A site, forming a dipeptidyl-tRNA
in the A site and a deacylated tRNA in the
P site. The reaction is catalysed by a part
of the rRNA acting as a ribozyme. In step
3, the binding of EF-G and the hydrolysis
of its associated GTP results in the
translocation of the ribosome relative to
the mRNA.

Translocation is accompanied by the movement of the deacylated tRNA and peptidyl-

tRNA into the E and P sites respectively. In step 4, the deacylated tRNA leaves the ribosome,
and a new aminoacyltRNA enters the A site. (b) Peptide bond formation and the subsequent
displacement of the deacylated tRNA. A ribosome can catalyse the incorporation of
approximately five amino acids into a growing polypeptide per second, which is roughly 10
million times greater than that observed in the uncatalyzed reaction using model substrates in
solution.
40
 Termination is the last step requires many protein factors. In bacterial cells, these
proteins include EF-G, IF3, and RRF (recycled factor ribosomes), which promote the
separation of ribosomal subunits.

3 of the 64 trinucleotide codons function as stop codons that terminate polypeptide

3 of the 64 trinucleotide codons function as stop codons that terminate polypeptide

assembly rather than encode an amino acid. No tRNAs exist whose anticodons are
complementary to a stop codon.10 When a ribosome reaches one of these codons, UAA, UAG,
or UGA, the signal is read to stop further elongation and release the polypeptide associated
with the last tRNA. Termination requires release factors. Release factors can be divided into
two groups: class I RFs, which recognize stop codons in the A site of the ribosome, and class
II RFs, which are GTP-binding proteins (G proteins) whose roles are not well understood.
Bacteria have two class I RFs: RF1, which recognizes UAA and UAG stop codons, and RF2,
which recognizes UAA and UGA stop codons. Eukaryotes have a single class I RF, eRF1,
which recognizes all three stop codons. Class I RFs enter the A site of the ribosome, where a
conserved tripeptide at one end of the release factor is thought to interact directly with the stop
codon in the A site not unlike the way that an anticodon triplet of a tRNA molecule would
interact with a sense codon in that site. The ester bond linking the nascent polypeptide chain to
the tRNA.
41
 H. Conclusion
Deoxyribose Nucleic Acid (DNA) is the genetic material in which factors of inheritance
are stored. It is situated in the nucleus (eukaryotes) and cytoplasm (prokaryotes). DNA is suited
to a genetic function because it satisfies the three essential requirements of a genetic material
which include; (i) being able to be replicated accurately, so that the information it contains is
precisely replicated and inherited by daughter cells; (ii) having the capacity to carry all the
information needed to direct the organization and metabolic activities of the cell; (iii) and
undergoing occasional mutations in which the information it carries is altered. Chemically,
DNA is a polymer consisting of a chain of monomers called nucleotides; a complex molecule
made up of deoxyribose sugar, base (nitrogen-containing ring-structure), and a phosphate
group.
DNA is a template for non-coding RNA molecules, which play an important role in
gene regulation and other activities. Other areas of DNA are important in gene regulation
because they act as binding sites for regulating proteins. Gene expression is the process by
which information encoded in a gene is translated into a sequence of amino acids during protein
synthesis, consisting of two stages: transcription and translation. In the transcription process,
the DNA strands provide information for the synthesis of RNA strands. The synthesis of a
polypeptide chain can be divided into three rather distinct activities: initiation of the chain,
elongation of the chain, and termination of the chain. After attaching to the mRNA, the
ribosome always moves along the mRNA from one of the codons to the next codon, that is, in
three consecutive nucleotide blocks. The ribosome attaches to the mRNA in the right place,
called the initiation codon, which is determined as the AUG.

42
References
1. Allison, A, Lizabeth. (2007). Fundamental Molecular Biology, Blackwell Publishing
Ltd.
2. Hartl, L, Daniel & Jones, W, Elizabeth. (1998). Genetics: Principles and Analysis,
Fourth Edition, Jones and Bartlett Publishers.
1. Fletcher, H & Hickey, I. (2013). Genetics- Bios Instant Notes, 4th Edition, Garland
Science, Taylor & Francis Group, LLC
2. Karp, G. (2013). Cell and Molecular Biology: Concepts and experiments, 7th Edition,
John Wiley and Sons, Inc

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