Chapter 1

An Introduction to DNA

Abstract
All living things comprise of one or more than one cells which are the basic units of
their structure and functions. These cells contain genetic material, controlling the life
of an individual. This genetic material is deoxyribose nucleic acid (DNA) that is
hereditary material possessing records or information of an organism’s life. Thus, the
transfer of genetic informations from parents to their off-spring is in the form of DNA
that has four ‘chemical’ alphabets at molecular level, i-e., Guanine (G), Cytosine (C),
Adenine (A), Thymine (T) or Uracil (U), all collectively forming DNA language or
the entire language of life. Hence, it is understandable that the implausible world of
cell is organized by this wonderful biological molecule which presents both a
glimpse into the secrets of life as well as a basic script of life’s theater. That is why,
the genetic material is said to be a life’s instruction book, having information for the
whole life of an individual. This introductory chapter will focus on and explain the
basics about DNA such as DNA structure, forms of DNA, DNA supercoiling and
DNA topology.
Keywords: DNA topology, DNA supercoiling, Linking number, Nucleotide,
Topoisomerases

1.1 Introduction
DNA is a long heteromorphic polynucleotide chain that consists of numerous copies
of four residues (nucleotides). A nucleotide is the basic unit of DNA, consisting of
three subunits; a sugar group, a phosphate group and a nitrogenous base, as shown
in Fig. 1.1.
In DNA, five carbons/pentose sugar, technically known as deoxyribose sugar, (in
which an oxygen group is absent at its 2́ Carbon position) is present in furanose form.
The other subunit comprises of nitrogenous bases which are of four types viz.,
Adenine, Guanine, Cytosine and Thymine/ Uracil (in case of RNA) as shown in Fig
1.2. On the basis of similarities in their structures, these nitrogenous bases are
grouped into purines and pyrimidines. Double ring molecules such as adenine and
guanine are purines whereas single ringed molecule such as cytosine, thymine and
uracil are pyrimidines. In addition to the difference in ring structures, amino group
is present at number 4 position of C-atom in Cytosine (pyrimidine) and at number 6
position of C-atom in Adenine (purine), whereas keto group (C=O) is present at
number 4 position of C-atom in Thymine as well as in Uracil (pyrimidine), and on

1
2 Chapter 1

C-atom of number 6 in Guanine (purine). For distinguishing the carbon atom (s)
present in nitrogenous base (s) and carbon atom (s) of pentose sugar, the position
number of C-atom is prime (΄) marked for the C-atom (s) of sugar in a nucleotide.

Fig. 1.1 Nucleotide:A basic unit of DNA molecule.

A nucleotide without phosphate group is a nucleoside, having deoxyribose sugar that

is linked with nitrogenous base by glycoside bond (Fig 1.1). Nucleosides of purines
are referred to as Adenosine and Guanosine while nucleosides of pyrimidines are
called Cytidine and Thymidine. Free nucleotides in a cell are present in triphosphate
form (Fig 1.3), whereas in a DNA molecule or in a polynucleotide chain, nucleotides
are in monophosphate form and are referred to as deoxyadenosine 5´ monophosphate
or deoxyadenylic acid, deoxyguanosine 5´ monophosphate or deoxyguanylic acid,
deoxycytidine 5´ monophosphate or deoxycytidylic acid, deoxythymidine 5´
monophosphate or deoxythymidylic acid (Griffiths et al. 1993; Nelson and Cox
2012). DNA molecule is double helical, wherein two antiparallel polynucleotide
chains are linked with each other by hydrogen bonding. Complementarity of
nitrogenous bases is a fundamental feature to carry out joining in the polynucleotide
chain.
An introduction to DNA 3

Fig. 1.2 Structure of Nitrogenous bases

Fig. 1.3 Structure of DNA Nucleotides

4 Chapter 1

1.2 Amount of DNA

The amount of DNA (in picogram) in a haploid genome is referred to be as its C
value. Erwin Chargaff, an Austrian-American biochemist, found an amazing equality
in the ratios of nucleotides in a DNA molecule. He gave an empirical rule about the
amount of DNA in 1950s. He stated that the amount of adenine is always in the same
proportion as thymine in a genome and similarly the amount of guanine is of equal
proportion to cytosine and vice versa (Griffiths et al. 1993).

1.3 Structure of DNA

The mystery of DNA structure was unraveled after the work of James Watson and
Francis Crick was published in 1953. Previously, Maurice Wilkins and Rosalind
Franklins in 1950-51 had presented data of X-ray diffraction analysis about DNA
structure. However, James Watson and Francis Crick described that DNA is in
double helical form (Fig 1.4). Hence, they proposed a DNA model, defining the DNA
structure in a very precise way. According to this model, DNA consists of two
polynucleotide chains which are coiled around an axis as a helical form, but both are
antiparallel. These polynucleotide chains are joined with each other by hydrogen
bonds present between two complementary nitrogenous bases (Watson and Crick
1953). In the joining of polynucleotide chain, complementarity of nitrogenous bases
is a fundamental feature. Guanine is complementary to cytosine, thus these bases
always make base pairing with each other. Similarly, adenine is complementary to
thymine, so base pairing between these nitrogenous bases always occurs. When
Guanine makes pair with Cytosine, triple H-bonds (G ≡ C) are present between them
and when Adenine makes pair with Thymine then double H-bonds (A ═ T) are
present between them. Nitrogenous bases are stacked inside of the helix and
perpendicular to the main fiber axis while sugar-phosphate backbone is outside the
helix (Calladine and Drew 1997; Nelson and Cox 2012).

Fig. 1.4 Structure of double stranded DNA molecule.

An introduction to DNA 5

In DNA, nucleotides are condensed into polynucleotide chain with the aid of
polymerases. Polymerases are the specialized group of enzymes that perform
condensation reaction for the synthesis of polynucleotide chain. In a condensation
reaction nucleotides are joined via phosphodiester bonds. Thus phosphodiester
linkage between each nucleotide in a chain is established when α phosphate group
(at 5́ C of pentose sugar) of later nucleotide, joins the hydroxyl group (-OH) present
on 3́C of pentose sugar of the former nucleotide. As a result of this, pyrophosphates
(β, γ) and water are released (Nelson and Cox 2012).

1.4 Forms of DNA

DNA exists commonly in three forms; A, B and Z form. In addition to these, some
alternative forms of DNA are also existed. Form B of DNA is more common in a
cell. The forms A and B of DNA have right-handed turns but form Z of DNA has
left-handed turns. Regarding compaction, water contents in form B of DNA are high
while form A of DNA is less hydrated; thus, it is more compact than form B of DNA.
Low water contents pull the bases to tilt away from the axis of double helix, making
the structure more compact. However, when two DNA strands interwind to form
helical structure then two types of grooves are formed i-e., major groove and minor
groove. These grooves serve as sites of interaction and access to DNA binding
proteins. However, the width of both grooves varies in all forms of DNA (Griffiths
et al. 1993; Lodish et al. 2012).

1.5 DNA Supercoiling

In a cell, DNA is present in extreme compact form with high degree of structural or
conformational organization. As already mentioned, a DNA molecule basically
consists of two complementary polynucleotide chains that are coiled around an axis
forming double helical structure. However, further coiling of a coiled double helical
DNA is referred to as DNA supercoiling. Hence it could be said that coiling of the
axis of the coiled DNA upon itself is supercoiling (an important characteristic of
DNA structure); otherwise DNA is relaxed. There are two principal types of
supercoiled DNA, called plectonemic form and solenoidal form. In plectonemic form
of negative supercoiling, there is right-handed superhelix and DNA molecules are
interwound (Fig 1.5a) while in case of solenoidal form, superhelix is left-handed and
DNA is wrapped around nucleosome (Fig 1.5b). Both these forms are
interconvertable. Generally, plectonemic form is said to be the property of
prokaryotic DNA while the solenoidal form is the property of eukaryotic DNA
(Sergei 2001; Nelson and Cox 2012).
On unwinding of DNA molecule, negative and positive supercoiling waves are
induced ahead and behind of the unwind DNA portion respectively. Negative
supercoiling is induced due to the underwinding of DNA molecule whereas positive
supercoiling is induced by the overwinding of DNA molecule. Negative supercoiling
facilitates the local and global DNA strand separation that is an indispensable
biological process, necessary and even pre-requisite for the execution of all
biological phenomena. Generation of negative supercoiling is basically the activity
6 Chapter 1

of DNA gyrase that executes this activity at the cost of ATP hydrolysis. The removal
of negative supercoiling is the activity of topoisomerase I and this enzyme performs
its duty in juxtaposition with DNA gyrase and plays a role in the regulation of supper
helical density in bacteria. Decatenation of DNA supercoiling and unknotting are the
activities of type II class of topoisomerases and among these, topoisomerase IV
exhibits marvelous roles in genome untangling and unknotting. In contrary, another
enzyme reverse gyrase does not induce negative supercoiling rather it induces
positive supercoiling in closed circular DNA molecule (Leroy and Wang 1987;
Guillaume and Andrzej 2010).

(a) Plectonemic form (b) Solenoidal form

Fig. 1.5 Principal types of negative supercoiled DNA (a) Conceived from Nelson
and Cox 2012 (b) Conceived from Sergei 2001.

1.6 DNA Topology

Topology is a conformational or structural state. The term DNA topology is basically
derived from topology, i.e., a branch of mathematics that deals with the object
properties which do not alter due to continuous deformation but discontinuous
deformation brings changes in it. This explains that the property of a DNA molecule
cannot be changed by continuous deformation such as conformational changes and
interaction with other biomolecules/ molecules (Andrew and Anthony, 2005). But
this can be changed only due to the discontinuous deformation i-e, breaking of
phosphodiester bonds either in one strand or in both DNA strands. These are basically
the topological aspects of DNA molecule; hence this property of DNA molecule is
called DNA topology. Thus, circular DNA, DNA bounded with membrane and DNA
attached with proteins, all are in their topological domains because there is no free
end rotation in all cases and they all are topological closed. Their topology would not
be changed until or unless a break introduces (Sergei 2001; Guillaume and Andrzej,
2010).

1.6.1 Characteristics of DNA topology

Referring to topological characteristics, linking number is the basic topological
property. Linking number (Lk) is the cumulative effect of writhe (Wr) and twist
(Tw) that are geometric descriptors of DNA configuration. Turning of DNA strands
An introduction to DNA 7

around each other is called twists (Tw) and turning of double helical DNA about
itself is called writhe (Wr). These characteristics (writhe & twist) are not the
topological parameters and are topologically variants, because these can easily be
changed by altering any condition, while the linking number is topologically
invariant because it cannot be changed by any continuous deformation. However, a
discontinuous deformation in the form of breakage of one or both DNA strands and
rejoining of these strands in another orientation by covalent closure can bring a
change in linking number (Crick 1976; Sergei 2001; Andrew and Anthony 2005).
In addition to linking number, knots and catenanes are also topological parameters
which are invariables too. Knots of various kinds and complexities are formed during
cyclization process of circular DNA molecule. If axial trajectory of covalently closed
circular DNA is isomorphic, then this molecule is unknotted otherwise it is knotted.
When DNA molecule is covalently closed, the knot characteristics can not be altered
by bringing any change in its conformation except breakage of DNA strands.
Commonly find knot types are simple knots (like trefoils) (Fig 1.6a) and Figure of
eight knot (Fig 1.6b). But catenanes is the state of circular DNA molecules when
they are interlinked by covalent closure. This is also another important topologically
invariant property and found commonly in cells (Fig 1.6c) (Joseph et al. 2008;
Guillaume and Andrzej 2010; Nelsonand Cox 2012).

(a) Simple knot (b) Figure of eight knot (c) Catenanes

Fig. 1.6 Knots and Catenanes: Topological parameters of DNA (a) Modified from,
Guillaume and Andrzej 2010 (b) Modified from Andrew and Anthony 2005 (c)
Modified from Nelson and Cox 2012.

1.6.2 Role of DNA topoisomerases in DNA topology

The change in a topological domain of DNA is the activity of a group of enzymes
called DNA topoisomerases. All topological problems that are related to the
execution of biological processes in a cell, like those of the DNA replication,
transcription, remodeling of chromatin material and genome recombination etc are
tackled by DNA topoisomerases; that are known as “the magicians of DNA world”.
The stabilization of genome geometry during these processes is the marvelous task
of topoisomerases. This molecular machinery has a potential role in biological
information flow by generating transient breaks in one or both strands of DNA
8 Chapter 1

molecule that make DNA accessible to cellular machineries for performing specific
biological tasks (James 2001; Goodsell 2002)
Topoisomerases are categorized into two main classes; Type I and Type II class of
topoisomerases. Cleaving of one DNA strand is the attribute of the enzymes that
belongto Type I class of topoisomerase while staggered double stranded break in a
DNA molecule is introduced by Type II class of topoisomerases that cleave both
strands of DNA molecule. Topoisomerases of type I class are monomeric with no
energetic cost because they do not require high energy co-factor. This group of
enzymes performs the job of generating transient single stranded cleavage followed
by the passing of second strand of double stranded DNA through this opening and
then reseals the break. Type I class of topoisomerases is divided into two sub-families
that are Type IA and Type IB subfamilies of topoisomerases. Enzymes of Type IA
subfamily assist the passage of DNA strand by “enzyme bridging” mechanism. In
this reaction, Type IA enzymes make bridge between the two termini of cleaved
DNA strand and the movement of both topoisomerases enclosed DNA termini, abet
the opening and closing of DNA door. In contrast to this, type IB enzymes take action
by a different swivel mechanism called DNA rotation/ /controlled rotation. In this,
the covalent linkage is established between enzyme and DNA in such a way that
protein is affixed at 3 ́ terminal of cleaved DNA strand and both termini are rotated
relatively to each other and then rejoined (Wang 1996; Satoshi and Akihiko 1998;
James 2001).
All enzymes of Type IA subfamily need divalent metal ions for their catalytic activity
while the enzymes that belong to Type IB subfamily do not need divalent metal ions
for their catalytic activity. Active sites of all types of topoisomerases have tyrosyl
residue that instigate DNA breakage by doing nucleophilic attack on phosphate group
of DNA backbone. This transestrification reaction results in the establishment of
transient phosphodiester linkage. This linkage is covalent phosphotyrosyl bond that
connects this enzyme with one terminus of cleaved DNA strand and also forms
hydroxyl moiety on the second terminus of broken DNA strands. Type IA
topoisomerases covalently join the phosphate of 5́ terminal of DNA backbone while
type IB topoisomerases make covalent linkage to phosphate group at 3́ terminal of
DNA molecule. Type II class of topoisomerases are multimeric in nature and demand
ATP for their catalytic activity. This class is further subdivided into subfamilies;
Type IIA subfamily and Type IIB subfamily. The enzymes of Type II class of
topoisomerases have the requirement of divalent metal ions for their catalytic
activity. The enzymes belonging to this class tailor DNA topology and even DNA
geometry by introducing transient double stranded cleavage in DNA molecule. As a
result of this, another double helical DNA is passed through this gate followed by
covalent closure of this opening by re-ligation of the break (Leroy et al. 1980;
Kikuchi and Asai 1984; Goodsell 2002).
In [Link] four DNA topoisomerases have been found. Of these four, two belong to
TypeIA subfamily; that are DNA topoisomerase I and DNA topoisomerase III. Other
two Topoisomerases belong to Type IIA subfamily; that are DNA gyrase and DNA
topoisomerase IV. Recent reports claim the finding of Type IB topoisomerases in a
few species of bacteria and not in all. DNA gyrase and Topoisomerase IV have amino
acids homology with Topoisomerase II (Topo II) of eukaryotes. DNA gyrase has
An introduction to DNA 9

two parts; gyrA, gyrB and topoisomerase IV also has two parts; parC, parE whereas
their eukaryotic counterpart, topoisomerase II has just one polypeptide chain having
two domains analogous to these enzymes. As already mentioned, the DNA gyrase
catenates negative supercoiling and decatenates positive supercoiling whereas its
eukaryotic analogue Topo II neither generates negative supercoiling nor positive
supercoiling; instead this enzyme relaxes th supercoiling (Wang 2002; Joseph et al.
2008; Guillaume and Andrzej 2010).

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