Download TEXT Components of DNA To understand the structure of DNA, it is

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Components of DNA
To understand the structure of DNA, it is important
to understand the individual components of DNA. It is
composed of pentose sugar, aromatic bases (a purine or
pyrimidine ring) and phosphate groups. The many
variations in the structures of the bases and sugars, and
in the structural relationship of the bases to the sugar,
give rise to differences in the helical structure of DNA.
A. Pentose sugar
Two kinds of pentoses are found in nucleic acids. The
recurring deoxyribonucleotide units of DNA contain 2’deoxy-D-ribose or simply, deoxyribose (Fig. 1), which is
the reason for the name deoxyribose nucleic acid. The
ribonucleotide units of RNA contain D-ribose, hence
ribose nucleic acid. In nucleotides, both types of pentoses
are in the β- furanose form, i.e. in a closed five-member
ring. The oxygen atom present at the second carbon of
ribose is missing in deoxyribose, hence its name 2’deoxyribose. The position of carbon atoms of pentose
sugars are denoted as 1’ , 2’ , 3’ , 4’ and 5’ in order to
differentiate them from the corresponding positions in
DNA bases which are not marked by a prime (‘).
The sugar moiety of DNA is one of the most flexible
and dynamic parts of the molecule. Fig. 2 shows
structures of the common sugar conformations that are
found in the various forms of DNA. The sugar ring is easy
to envision if one thinks of an envelope. In the envelope
form, the four carbons form a plane at the corners of the
body of the envelope. The oxygen is at the position
representing the top of the envelope flap. The oxygen
can be bent out of the plane of the body of the envelope
twisting the C2’ and C3’ carbons relative to the other
atoms, resulting in various twist forms of the sugar ring.
To form the C2’endo form of the ribose sugar, C2’ twists
up from the plane of the four carbons. To form the C3’
endo, C3’ twists down out of the plane of the four
carbons.
B.
Organic base
The organic bases found in nucleic acids are
heterocyclic compounds containing nitrogen in their
rings; hence they are called nitrogenous bases. The
bases found in nucleotides are substituted pyrimidines
and purines. Unsubstituted pyrimidines and purines are
not found in biological systems, but a number of
substituted derivatives are present, which are classified
as pyrimidines or purines, depending on the parent
molecule from which they are derived.
a.
Pyrimidine bases:
Pyrimidine bases consist of a six membered
pyrimidine ring, which is similar to the benzene ring
except that it contains nitrogen in place of carbon at
positions 1 and 3 (Fig. 3). Therefore, pyrimidines contain
four carbons and two nitrogen atoms. The carbons and
nitrogens of the purine rings are numbered 1 to 9.
DNA contain two major pyrimidines:
i.
Thymine: Thymine contains a methyl group at the
C5 position with the carbonyl groups at C4 and C2
positions (Fig.3 ), so it is 5-methyl, 2,4-dioxo pyrimidine.
ii. Cytosine: Cytosine contains a hydrogen atom at the
C5 position and an amino group at C4 (Fig.3 ), so it is 2oxo-4-amino pyrimidine.
It will be pertinent to mention that RNA contains
uracil in place of thymine. Only rarely thymine occurs in
RNA or uracil in DNA. In uracil, a keto group (=O) is
present at the 2nd and 4th carbon, so it is 2,4-dioxo
pyrimidine (Fig.3 ). The only difference between uracil
and thymine is the absence of a methyl group at position
C5 position.
b.
Purine bases
Purine is a bicyclic structure consisting of pyrimidine
fused to an five membered imidazole ring. The five
member ring of purine has nitrogen in the place of
carbon at positions 7 and 9 (Fig.4 ). Therefore, purines
contain five carbons and four nitrogen atoms. The
carbons and nitrogens of the pyrimidines are numbered 1
to 6.
The two major purine bases present in DNA are:
i.
Adenine: In adenine, an amino group is present at
position 6 (Fig.4 ), so it is 6-amino purine.
ii. Guanine: In guanine, an amino group is attached
with the 2nd carbon and a keto group is found at position
6 (Fig.4 ), therefore it is 2-amino-6-oxo purine.
Each commonly occurring pyrimidine and purines can
be drawn in two tautomeric forms. Adenine and cytosine
can exist as either amino- or imino- forms, and guanine,
thymine and uracil can exist as either lactum or lactim
forms (Fig.5 ). The two forms for each base exist in
equilibrium, but under the conditions found inside most
cells, the amino and lactum tautomers are more stable,
and therefore, predominate.
The nitrogenous base is linked to position 1 on the
pentose ring by a glycosidic bond (Fig.6 ). The purine
bases are bonded at the 9 nitrogen, while the pyrimidines
bond at the 1 nitrogen. This bond is said to be in the β
(up) configuration with respect to the ribose sugar, in
contrast to the α (down) position of the hydrogen. The
base is free to rotate around the glycosidic bond. The two
standard conformations of the base around the glycosidic
bond is syn and anti. The anti conformation reflects the
relative spatial orientation of the base and sugar as found
in most conformations of DNA, for example, B-form DNA.
The syn conformation is found (in conjugation with a
different sugar pucker) in Z-form DNA.
The purines and pyrimidines are well-suited to their
roles as the informational molecules of the cell. The
differential placement of hydrogen bond donor and
acceptor groups gives the bases the unique structural
identity that allows them to serve as the genetic
information. The hydrogen atoms of amino groups
provide hydrogen bond donors, where as the carbonyl
oxygen and ring nitrogen provide hydrogen bond
acceptors. All the carbon atoms of purine and pyrimidines
are sp2 hybridized (that is none are saturated); thus they
are planar. This flatness is important in the organization
of bases within the helix, since it allows the bases to
stack uniformly within the helix and this stacking helps to
protect the chemical identity of the bases.
C.
Phosphate group
Phosphoric acid has three reactive hydroxyl groups
of which two are involved in forming the sugar phosphate
backbone of DNA.
Nucleoside and nucleotide
A base linked to sugar is called nucleoside; when a
phosphate group is added, the base-sugar-phosphate is
called nucleotide. ATP and AMP are nucleotides, whereas
the unphosphorylated form, adenosine, is a nucleoside.
Fig.7 depicts the structures and names of the four major
deoxyribonucleotides
(deoxyribonucleoside
5’monophosphate).
Nucleotides provide the building blocks from which
nucleic acids are constructed. The nucleotides are linked
together into a polynucleotide chain by a backbone
consisting of an alternating series of sugar and phosphate
residues.
The 5’ position of one pentose ring is
connected to the 3’ position of the next pentose ring via a
phosphate group. So the sugar-phosphate backbone is
said to consist of 5’-3’ phosphodiester linkages (Fig.8 ).
Thus, the covalent backbones of nucleic acid consists of
alternating phosphate and pentose residues, and the
characteristic bases may be regarded as side groups
joined to the backbone
at regular intervals. It is
noteworthy that DNA is hydrophilic. The hydroxyl groups
of the sugar residues form hydrogen bonds with water.
The phosphate groups in the polar backbone are
completely ionized and negatively charged at pH 7, thus
DNA is an acid. These negative charges are generally
neutralized by ionic interactions with positive charges on
proteins (proteins that contain an abundance of the basic
residues arginine & lysine), metal ions (Mg2+) and
polyamines.
All the phosphodiester linkages in DNA strands have
the same orientation giving each linear nucleic acid
strand a specific polarity (Fig.9). The terminal nucleotide
at one end of the chain has a free 5’ group and the
terminal nucleotide at the other end has free 3’ group. It
is conventional to write nucleic acid sequence in the
5’→3’ direction, that is, from the 5’ terminus at the left to
the 3’ terminus at right.
When DNA is broken into its constituent nucleotides,
the cleavage may take place on either side of the
phosphodiester bonds. Depending on the circumstances,
nucleotides have their phosphate group attached to
either the 5’ or the 3’ position of the pentose (Fig.10).
Therefore, the two types of nucleotides released from
nucleic acids are nucleoside-3’monophosphates and
nucleoside-5’-monophosphates.
All the nucleotides can exist in a form in which there
is more than one phosphate group linked to the 5’
position. An example is shown in Fig.11). The bonds
between the first (α) and second (β), and between the
second (β) and third (γ), phosphate groups are energyrich and are used to provide an energy source for various
cellular activities. The 5’
triphosphates
are the
precursors for nucleic acid synthesis (Fig.12). Nucleic
acid synthesis occurs by a reaction in which the 5’ end of
the incoming triphosphate reacts with a 3’-OH group at
the end of the polynucleotide chain. A bond is formed
from the α phosphate to the 3’-OH of the sugar at the
end of the polynucleotide chain, and the two terminal
phosphate groups (γ and β) of the triphosphate are
released, in the form of a single molecule called
pyrophosphate.
The physical structure of DNA
DNA is a duplex molecule, i.e. it consists of two
chains arranged in anti-parallel manner with the
nitrogenous bases facing each other. However, there are
some examples of viral DNA which are single-stranded. It
is sometimes useful to describe nucleic acid structure in
terms of increase in levels of complexity like primary,
secondary and tertiary.
A.
Primary structure
The primary structure of a nucleic acid is its covalent
structure and nucleotide sequence (Fig.13). It is in these
sequences where the genetic information is stored and
because the skeleton is the same for all, the difference in
the information lies in the different sequence of
nitrogenous bases. This sequence has a code, which
determines an information or otherwise, as the order of
the bases.
B.
Secondary structure
Any regular, stable structure taken up by some or all
of the nucleotides in a nucleic acid can be referred to as
secondary structure.
It is a double helix structure
(Fig.14). Double helix model explain the storage of
genetic information and the mechanism of DNA
replication. It was postulated by Watson and Crick, based
on X-ray diffraction and the equivalence of bases,
whereby the sum of adenines and guanines is equal to
the sum of thymines and cytokines.
C.
Tertiary structure
Refers to how DNA is stored in a confined space to
form the chromosomes. Varies, depending on whether
the organism is prokaryote or eukaryote. In prokaryotes
the DNA is folded like a super-helix, usually in circular
shape and associated with a small amount of protein
(Fig.15). The same happens in cellular organelles such as
mitochondria and the chloroplasts. In eukaryotes, since
the amount of DNA in each chromosome is very large,
the packing is more complex and compact due to the
presence of proteins such as histones and non-histones.
Discovery of DNA structure:
The deoxyribonucleotides and their ability to form
polynucleotide were discovered by Levene in 1931. But
Levene postulated that the four deoxyribonucleotides
occurred in a regularly repeated tetra nucleotide
sequence like AGCTAGCT…. and so on.
A.
Erwin Chargaff’s Ratios
A most important clue to the structure of DNA came
from Erwin Chargaff & his colleagues in 1949. The data
collected by them from DNAs of many different species,
led to the following conclusions:
a. The base composition of DNA generally varies from one
species to another (Table-1 ).
b. DNA specimens isolated from different tissues of the
same species have same base composition.
c. The base composition of DNA in a given species does
not change with the organisms age, nutritional state or
changing environment.
d. In all DNAs, regardless of the species, the number of
adenine residue is equal to the number of thymine
residues (A=T), and the number of guanine residues is
equal to the number of cytosine residues (G=C). From
these relationships it follows that the sum of purine
residues equals the sum of pyrimidine residues; that is
A+G=T+C.
Table-1:Chargaff’s DNA database composition in various
species:
Species
A%
T%
G%
C%
Homo sapiens
31.0
31.5
19.1
18.4
Drosophila melongaster 27.3
27.6
22.5
22.5
Zea mays
25.6
25.3
24.5 24.6S
Neurospora crassa
23.0
23.3
27.1
26.6
Escherichia coli
24.6
24.3
25.5
25.6
Bacillus subtilis
28.4
29.0
21.0
21.6
These quantitative relationships, sometimes called
“Chargaff’s rules”’ were a key to establishing the three dimensional structure of DNA and yielded clues to how
genetic information is encoded in DNA and passed from
one generation to the next.
Therefore, tetranucleotide hypothesis of Levene
proposing that DNA has repeating units of one of these
four bases was disapproved.
B.
Francis Wilkin’s diffraction data
Rosalind Franklin, working with Maurice H.F. Wilkins at
King’s College (London, England) studied isolated fibres
of DNA by using the X-ray diffraction technique, a
procedure in which beam of parallel X-rays is directed on
a regular, repeating array of atoms (Fig.16). The beam is
diffracted (=broken up) by the atoms in a pattern that is
characteristic of the atomic weight and the spatial
arrangement of the molecules. The diffracted X-rays are
recorded on a photographic plate. X-ray diffraction
pattern revealed that DNA is a helical structure which had
two distinctive regularities of 0.34 nm and 3.4 nm along
the axis of the molecule.
C.
Watson & Crick’s double helix model
Watson and Crick received the Nobel Prize in 1962
for their model of DNA. Using information generated by
Chargaff and Franklin, Watson and Crick built a model of
DNA. Their model was consistent with both Chargaff's
rules and dimensions of DNA polymer provided by
Franklin's photograph of X-ray diffraction of DNA.
The double-helix model proposed by Watson and
Crick in 1953 consists of two helical DNA chains coiled
around the same axis to form a right-handed double helix
(Fig.17). The hydrophilic backbones of alternating
deoxyribose and negatively charged phosphate groups
are on the outside of the double helix, facing the
surrounding water. The purine and pyrimidine bases of
both strands are stacked inside the double helix, with
their hydrophobic and nearly planar ring structures very
close together and perpendicular to the long axis of the
helix. The spatial relationship between these strands
creates a major or larger groove and minor or smaller
groove between the two strands. In the Watson-Crick
structure, the two strands of the helix are anti-parallel
i.e., their 5’-3’ phosphodiester bonds run in opposite
directions. Each base of one strand forms hydrogen
bonds with a base of the other strand, forming a base
pair. Only the lactum and amino tautomers of each base
accommodate such hydrogen bonding. Guanine pairs
with cytosine, and adenine with thymine. These base
pairs maximize hydrogen bonding between potential
sites. Accordingly, G/C base pairs have three hydrogen
bonds and A/T base pairs have two. This feature of
double-stranded DNA accounts for Chargaff’s earlier
discovery that the ratio of A to T and G to C is 1:1 for a
wide diversity of DNA molecules. Because A in one strand
pairs with T in the other and G pairs with C, the strands
are complementary and can serve as a template for each
other.
During replication, the two strands of a DNA
molecule uncoil, and the unpaired bases in the single
stranded region of the two strands bind with their
complementary bases present in the cytoplasm as
nucleotides. These nucleotides become joined by
phosphodiester linkages generating complementary
strands of the old ones. This provides for almost errorfree high fidelity replication of the genetic material.
To account for the periodicities observed in the
X-ray diffraction pattern, Watson and Crick used
molecular models to show that the vertically stacked
bases inside the double helix would be 0.34 nm apart and
that the secondary repeat distance of about 3.4 nm could
be accounted for by the presence of 10 nucleotide
residues in each complete turn of the double helix.
DNA double helix or duplex is held together by two
sets of forces:
a.
Hydrogen bonding between complementary
base pairs
A hydrogen bond is a short, non-covalent,
directional interaction between covalently bound H+ atom
(donor) and a negatively charged acceptor atom. The
acceptor is provided by electrons on a carbonyl oxygen [C=O] or the lone pair electrons on nitrogen [N:] (Fig.18).
In the DNA double helix, the nitrogen and oxygen atoms
involved in hydrogen bonding are separated by 0.282 0.292 nm. In A=T base pair, two hydrogen bonds are
separated by 0.282nm and 0.291nm and in the G≡C
base pair, three hydrogen bonds are separated by
0.284nm and 0.292 nm. In DNA, hydrogen bonds have
2-3 kcal/mol weaker than most hydrogen bonds (3-7
kcal/mol), which is due to geometric constraints within
the double helix.
b.
Base-stacking interactions
Since the aromatic bases are planar, they can stack
nicely on one another. The stacking involves a
combination of van der Waals and dipole-dipole
interaction between the bases, which is estimated to be
4-15 kcal/mol per dinucleotide. These base stacking
interactions help to minimize contact with water and are
very important in stabilizing the three-dimensional
structure of nucleic acids.
The specificity that maintains a given base sequence
in each DNA strand is contributed entirely by the
hydrogen bonding between base pairs. The base-stacking
interactions, which are largely non-specific with respect
to the identity of the stacked bases, make the major
contribution to the stability of the double helix.
Under physiological conditions, double stranded DNA
is more stable than are separated DNA strands.
Consequently, duplex DNA predominates in vivo.
Sometimes, however, the structure of duplex DNA can be
disrupted, as occurs during DNA replication and
transcription. Complete unwinding of a double helix and
separation of the complementary single strands is called
denaturation. Denaturation only occurs in vitro.
Double stranded DNA can be disrupted
when solutions of DNA are heated above a certain
temperature or when sufficient concentrations of
chaotropic agents like urea or guanidinium are added.
When the temperature of the solution is raised, more and
more of the bases become unstacked and hydrogen
bonds between base pairs are broken. Eventually, the
two strands separate completely. The temperature at
which the populations of duplex of DNA and DNA with
separated strands are equal is known as melting point
(tm). The transition from double-stranded DNA to the
single-stranded denatured form can be detected by an
increase in the absorption of UV light (hyperchromic
effect) or the decrease in the viscosity of the DNA
solution. Each species of DNA has a characteristic melting
point; higher its content of G≡C base pairs, the higher
the melting point of the DNA. This is because ≡C
G base
pairs, with three hydrogen bonds are more stable and
require more heat energy to dissociate than A=T base
pairs.
Structural forms of DNA
Deoxyribonucleic acid is a remarkably flexible
molecule. Considerable rotation is possible around a
number of bonds in the sugar-phosphate backbone. In
the years that followed publication of the Watson - Crick
model for double-helical DNA, X-ray crystallographic
studies of various synthetic oligo–deoxyribonucleotides of
known sequence resulted in refinement to the structural
dimensions. It is now known that DNA inside the cell
does not exist only in a Watson-Crick conformation, but
rather as a dynamic molecule whose exact conformation
changes as the DNA strand bends in solution and is
complexed to protein.
DNA can assume different conformations like B-, Aand Z- forms under different physical conditions (Fig.19).
Some of the key features of each DNA type are given in
table 2.
Table-2: Properties of A-DNA, B-DNA and Z-DNA:
Property
A-DNA
B-DNA
Z-DNA
Condition
in Dehydrating General
Sequence of
which found
conditions
conditions of alternating
the cell
purines and
pyrimidines
1.Coiling
Right
Right handed Left handed
handed
2. Rotation / + 34.7º
+ 34.0 º
-30.0 º
base pair
3. Turn
2.54 nm
3.5 nm
4.56 nm
4. Rise / base 0.23 nm
0.34 nm
0.38 nm
pair
5. Base pair / 11
10.5
12
turn
6.
Helical 2.3 nm
1.9 nm
1.8 nm
diameter
7. Glycosyl
angle
C: anti
G: syn
C:C2'-endo,
8. Sugar pucker C3'-endo
C2’-endo
G: C2'-exo
9.
Overall Short
and Longer
and Elongated
morphology
wide
thinner
and thin
anti-
anti-
A.
Features of B-DNA
The Watson-Crick structure is also referred to as Bform DNA. The B-form represents the general structure
of DNA in the conditions of the living cell, and is
therefore, the standard point of reference in any study of
the properties of DNA. This form shows right handed
coiling and contains 10.5 base pairs per pitch.
B.
Features of A-DNA
A-DNA occurs in dehydrated conditions. Hybrids
between DNA and RNA, which form during transcription,
are also thought to adopt the A-conformation because
the hydroxyl group (2'-OH) of the ribose in RNA prevents
it from adopting a B conformation made possible in DNA
by the 2-deoxyribose moieties which lack the hydroxyl
group. A-form DNA is also right handed helix, but the rise
per base pair is 0.23 nm and the number of base pairs
per helical turn is 11. A-DNA is more tightly wound than
B-DNA, and the difference between the major and minor
grooves are reduced.
C.
Features of Z-DNA
Z-DNA occurs when certain sequences of base pairs
are present. Certain nucleotide sequences fold up into
left-handed Z-helices more readily than do others.
Prominent examples are sequences in which pyrimidines
alternate with purines, especially alternating C and G or
5-methyl C and G.
Z-DNA is even more different from B-DNA. There are
no grooves and the helix shows left-handed coiling. There
are 12 base pairs per helical turn, with a rise of 0.38nm
per base pair. In Z-DNA, the sugar-phosphate backbone
follows a zigzagged path giving it the name Z-DNA.