1.

DNA STRUCTURE
Nucleotides the building blocks of DNA

- Each nucleotide monomer in DNA is composed of a nitrogenous base (either a

purine or a pyrimidine), a deoxyribose sugar, and phosphate (Figure 1).

Figure 1: Nucleotide building blocks of DNA

The mononucleotides are linked to each other by 3',5 phosphodiester bonds.
These bonds join the 5'-hydroxyl group of the deoxyribose of one nucleotide to the
3'-hydroxyl group of the sugar unit of another nucleotide through a phosphate
group (Figure 2).
- The primary structure of a nucleic acid is the sequence of residues that are
connected by 3', 5-phosphodiester bonds linkages.

Figure 2: DNA strand structure

- In a DNA strand the deoxyribonucleotide residues are connected to each other by

3'-5-phosphodiester linkages. The sequence of the illustrated strand is 5-pATGC3.
- Polynucleotide chains have directionality i.e., one end of a linear polynucleotide
chain is said to be 5' (because no residue is attached to its 5'-carbon) and the
other is said to be 3' (because no residue is attached to its 3'-carbon).

- Each phosphate group that participates in a phosphodiester linkage has a pKa of

about 2 and bears a negative charge at neutral pH. Consequently, nucleic acids
are polyanions under physiological conditions.

2.2

DNA is Doubled Stranded

- Erwin Chargaff deduced certain regularities in the nucleotide compositions of DNA

samples obtained from a wide variety of prokaryotes and eukaryotes. Chargaff
observed that in the DNA of a given cell, A and T are present in equimolar
amounts, as are G and C (Table.1). Note that the total mole percent of (G + C)
may differ considerably from that of (A + T). The DNA of some organisms, such as
yeast, is relatively deficient in (G + C), whereas the DNA of other organisms, such
as the bacterium Mycobacterium tuberculosis, is rich in (G + C). In general, the
DNAs of closely related species, such as cows, pigs, and humans, have similar
base compositions. Note that one consequence of Chargaff's observations is that
the ratio of purines to pyrimidines in DNA is always 1:1.
Table 1: Base composition of DNA (mole %) and ratios of bases
Source

*Purine/
Pyrimidine

A/T*

G/C*

G+C

26.0

24.9

25.2

23.9

1.09

0.99

50.1

1.04

Mycobacterium tuberculosis 15.1

34.9

35.4

14.6

1.03

0.99

70.3

1.00

Yeast

31.7

18.3

17.4

32.6

0.97

1.05

35.7

1.00

Cow

29.0

21.2

21.2

28.7

1.01

1.00

42.4

1.01

Pig

29.8

20.7

20.7

29.1

1.02

1.00

41.4

1.01

Human

30.4

19.9

19.9

30.1

1.01

1.00

39.8

1.01

Escherichia coli

*Deviations from a 1:1 ratio are due to experimental variations.

- The model of DNA proposed by Watson and Crick in 1953 was based on the
chemical equivalencies noted by Chargaff, on the known structures of the
nucleosides, and on X-ray diffraction patterns that Rosalind Franklin and Maurice
Wilkins obtained from DNA fibers. The Watson-Crick model accounted for the
equal amounts of purines and pyrimidines by suggesting that DNA was doublestranded and that bases on one strand paired specifically with bases on the other
strand, A with T and G with C. Watson and Crick's proposed structure is now
referred to as the B conformation of DNA, or simply B-DNA.
- An appreciation of DNA structure is important for understanding the processes of
DNA replication and transcription.
- Essentially DNA consists of two polynucleotide strands wound around each other
to form a right-handed double helix.

Figure 4: A) DNA double helix as a spiral ladder. B) Space filing model of the helix.
2.4 nm

- The antiparallel orientation of the two polynucleotide strands allows hydrogen

bonds to form between the nitrogenous bases that are oriented toward the helix
interior (Figure 5).
- There are two types of base pairs (bp) in DNA:
i. adenine (a purine) pairs with thymine (a pyrimidine)
ii. guanine (a purine) pairs with cytosine (a pyrimidine)
- Because each base pair is oriented at an angle to the long axis of the helix, the
overall structure of DNA resembles a twisted staircase.
- The dimensions of crystalline DNA have been precisely measured.

a) One turn of the double helix spans 3.4 nm and consists of approximately 10.4
base pairs. (Changes in pH and salt concentrations affect these values
slightly).
b) The diameter of the double helix is 2.4 nm. The interior space of the double
helix is only suitable for base pairing a purine and a pyrimidine. Pairing two
pyrimidines would create a gap, & pairing purines would destabilize the helix.
c) The distance between adjacent base pairs is 0.34 nm.

Figure 5: Structure of double-stranded DNA and hydrogen

bonding interactions. The two strands run in opposite directions.
Due to base pairing the order of bases in one strand determines
the sequence of bases of the other strand

- As befits its role in living processes, DNA is a relatively chemically inert and
several types of non-covalent bonding interactions contribute to the stability of its
structure namely;
i. Hydrophobic interactions. The base ring cloud of electrons between
stacked purine and pyrimidine bases is relatively nonpolar. The clustering of
the base components of nucleotides within the double helix is a stabilizing
factor in the three-dimensional macromolecule because it minimizes their
interactions with water.
ii. Hydrogen bonds. The base pairs, on close approach, form a preferred set of
hydrogen bonds, three between GC pairs and two between AT pairs. The
cumulative "zippering" effect of these hydrogen bonds keeps the strands in
correct complementary orientation.
iii. Base stacking. The stacked base pairs form weak van der Waals contacts.
Although the forces between individual stacked base pairs are weak they are
cumulative, so in large DNA molecules, van der Waals contacts are a
significant source of stability.
iv. Electrostatic interactions. DNA's external surface, referred to as the sugar
phosphate backbone, possesses negatively charged phosphate groups.
Repulsion between nearby phosphate groups, a potentially destabilizing force,
is minimized by the shielding effects of divalent cations such as Mg 2+ and
polycationic molecules such as the polyamines and histones.
- In B-DNA, the base pairs are stacked one above the other and are nearly
perpendicular to the long axis of the molecule. The cooperative, non-covalent
interactions between the upper and lower surfaces of each base pair bring the
pairs closer together and create a hydrophobic interior that causes the sugarphosphate backbone to twist. It is these stacking interactions that create the
familiar helix (Figure 4). Because the two hydrophilic sugar-phosphate backbones
wind around the outside of the helix, they are exposed to the aqueous
environment. In contrast, the stacked, relatively hydrophobic bases are located in
the interior of the helix, where they are largely shielded from water.
- The double helix has two grooves of unequal width because of the way the base
pairs stack and the sugar-phosphate backbones twist. These grooves are called
the major groove and the minor groove (Figure 4). Within each groove, functional
groups on the edges of the base pairs are exposed to water and are chemically
distinguishable. Because the base pairs are accessible in the grooves, molecules
that interact with particular base pairs can identify them without disrupting the
helix. This is particularly important for proteins that must bind to double-stranded
DNA and "read" a specific sequence.

- The size of a DNA molecule can be expressed in term of its molecular weight.
However, due to rapid buildup of the molecular weights, it is convenient to express
the size of DNA as the number of nucleotide base (nt) or base pairs (bp) per
molecule. The genome of E. coli is 4.6 megabase (million) pairs.
2.3
Conformations of Double stranded DNA
- Double-stranded DNA can assume different conformations under different
conditions because deoxyribose is flexible and the C1-N-glycosidic linkage rotates.
- X-ray crystallographic studies of various synthetic oligodeoxyribonucleotides of
known sequence have suggested that DNA molecules inside the cell do not exist
in a "pure" B conformation. Instead, DNA is a dynamic molecule whose exact
conformation changes as it bends in solution or binds to protein.
- Two important alternative conformations are A-DNA, which forms when DNA is
dehydrated, and Z-DNA, which can form when certain sequences are present
(Figure 6).
- A-DNA is more tightly wound than B-DNA, and the major and minor grooves of ADNA are similar in width. Z-DNA differs even more from B-DNA. There are no
grooves in Z-DNA, and the helix is left-handed, not right-handed. Both alternative
conformations exist in vivo, but they are confined to short regions of DNA.
Table 2: Comparison of some A-, B- and Z-DNA structural properties
Helix type

Parameter

A-DNA

B-DNA

Z-DNA

Shape

Broadest

Intermediate

Narrowest

Helix diameter

2.6 nm

2.4nm

1.8nm

Base pairs per turn of helix 11

10.4

12

Rise per base pair

2.3

3.4

3.8

Glycosidic bond

anti

anti

Alternating anti and syn

Pitch per turn of helix

25.3

35.4

45.6

Helix rotation

Right-handed Right-handed Left-handed

Minor groove

Minor groove

Major groove

Major groove

B-DNA

A-DNA

Z-DNA

Figure 6: A-DNA, B-DNA and Z-DNA

- Certain segments of DNA have been observed to have several higher order
structures, namely;
i. Cruciforms are cross-like structures and they form when a DNA sequence
contains a palindrome (Figure 7). In contrast to language palindromes, the
"letters" are read in one direction on one of the complementary strands of
DNA and in the opposite direction on the other strand. One-half of the
palindrome on each strand is complementary to the other half. The DNA
sequences that form palindromes, which may consist of several bases or
thousands of bases, are called inverted repeats. In one proposed mechanism,
cruciform formation begins with a small bubble, or protocruciform, and
progresses as intrastrand base pairing occurs. The mechanism by which
bubble formation is initiated is unknown. The function of cruciforms is unclear
but is believed to be associated with the binding of various proteins to DNA.
DNA palindrome also play a role in the function of an important class of
enzymes called the restriction enzymes.

Figure 7: Cruciform

Figure 8: H-DNA formation depends on the formation

of nonconventional (Hoogsteen) base pairing

ii. Triple Helices - In certain circumstances (e.g., low pH) a DNA sequence
containing a long segment consisting of a polypurine strand hydrogen-bonded
to a polypyrimidine strand can form a triple helix. The formation of the triple
helix also referred to as H-DNA, depends on the formation of nonconventional
base pairing (Hoogsteen base pairing, Figure 8), which occurs without
disrupting the Watson-Crick base pairs. The significance of H-DNA is not
understood and it may have a role in genetic recombination.
iii. Supercoils - A circular DNA molecule with the B conformation said to be
relaxed. Such a molecule would lie flat on a surface. However, the double
helix can be overwound or underwound if the strands of DNA are broken and

the two ends of the linear molecule are twisted in opposite directions. When
the strands are rejoined to create a circle, there are no longer 10.4 base pairs
per turn, as required to maintain the stable B conformation. The circular
molecule compensates for over or underwinding by forming supercoils that
restore 10.4 base pairs per turn of the double helix (Figure 9). A supercoiled
DNA molecule would not lie flat on a surface. Most circular bacterial
chromosomes are supercoiled in cells, but even long linear DNA molecules
contain locally supercoiled regions. All organisms have enzymes that can
break DNA, unwind or overwind the double helix, and rejoin the strands to
alter the topology. These enzymes are called topoisomerases, and they are
responsible for adding and removing supercoils. Bacterial chromosomes
typically have about five supercoils per 1000 base pairs of DNA. The DNA in
the nuclei of eukaryotic cells is also supercoiled.

Figure 9: Supercoiling of DNA. The DNA molecule on the left is a relaxed, closed circle and
has the B conformation. Breaking the DNA helix and winding it by two turns
before reforming the circle produces two supercoils. The supercoils compensate
for the over winding and restore the B conformation.

DNA REPLICATION
E. coli DNA replication begins at a site called the origin or ori C site . The sequence
of the origin spans 245 bp in length (Oka et al., 1980). It is rich in palindromes and
inverted repeat sequences which might play a role as recognition sites for various
proteins implicated in the replication process (Oka et al., 1980; Funnell et al., 1987;
Kornberg and Baker, 1992). Auxiliary proteins, designated dnaA and protein n(PriA),
bind to specific sites within the origin which must be negatively supercoiled by a
gyrase for this to occur. Two other proteins, dnaB and

Propositional knowledge statements on symbolism associated with DNA replication

___________________________________________________________________
Symbolism /
Propositional knowledge statements
Symbolic language
___________________________________________________________________
Origin

DNA sequence or site where replication begins.

Eye or bubble

Unravelling of the origin to give DNA the conformation

resembling a bubble or shape of an eye.

Palindrome

Palindromes are sequences of bases that read the same

in both directions on opposite strands of double-stranded
DNA.

Inverted repeat sequence This refers to the exact base sequence in opposite
directions in duplex DNA.
Gyrase

A gyrase or topisomerase II is an enzyme which unwinds

double-stranded DNA to result in a right-handed
orientation or negative supercoil.

SSB protein

SSB protein refers to a protein which binds singlestranded template DNA preventing re-annealing of
parental templates.

dnaA protein

DnaA protein guides the binding of the primosome

complex to the replication eye or bubble where it also
promotes opening of the double-stranded DNA.

dnaB protein

DnaB protein is the alternate nomenclature for a helicase

which requires ATP to unwind DNA.

DNA primase

DNA primase is the enzyme which synthesizes RNA

primers using the DNA template strand to determine the
complementary RNA nucleotide sequence.

Protein n(PriA),
Protein n(PriA), protein n (PriB), protein i (dnaT) and
Protein n (PriB),
protein n(PriC) are protein components of the
primosome Protein i(dnaT) and
complex which facilitates RNA primer
synthesis.
Protein n(PriC)
.
.

dnaC protein

A protein which complexes dnaB protein and delivers

dnaB
protein to DNA at the origin.

Primosome complex

A complex of several proteins which facilitate the

synthesis of RNA primer at various points along a DNA
template.

Replication fork

Bifurcated or Y- shape feature in helical DNA produced

during helicase action, separating the template strands of
double-stranded DNA for replicative DNA synthesis.

Helicase

Helicase is an enzyme which unwinds DNA using ATP to

do so, exposing the DNA template strand for RNA primer
and nascent cDNA synthesis.

Re-annealing

Rehybridization of single-stranded nucleic acid strands

based on nucleic acid base complementarity.

Negative supertwist
or negative supercoil

Negative supertwist or negative supercoil refers to a

relaxed right-handed twist in the conformation of doublestranded DNA induced by the action of gyrase or
topoisomerase II.

Unravelling

Unravelling refers to the unwinding of the helical DNA

structure at the origin to form an open structure
resembling the conformation of an eye or bubble.

Positive supercoil
or positive supertwist

Positive supercoil or positive supertwist refers to a tight

left-handed twist in the conformation of double-stranded
DNA induced by the action of topoisomerase I.

ATP

ATP is the abbreviation which refers to adenosine 5triphosphate.

rNTP

rNTP is the abbreviation which refers to ribonucleoside

triphosphate.

RNA primer

RNA primer refers to a short template-bound RNA

fragment which serves as a growth point for cDNA
synthesis.

dNTP

dNTP
is
the
abbreviation
deoxyribonucleoside triphosphate.

Leading strand

The leading strand refers to nascent DNA which is

continuous in its synthesis.

Lagging strand

The lagging strand refers to nascent DNA which is

discontinuous in its synthesis.

Okazaki Fragments

Okazaki Fragments are short discontinuous singlestranded DNA fragments synthesized from the 3 ends of
RNA primers and they make up the lagging strand of
DNA.

Rnase H

Rnase H is the abbreviation for ribonuclease H.

3OH function
or structure

The 3OH function or structure refers to the hydroxyl

attached at atom position 3 usually of a ribose or
deoxyribose sugar.

Endonucleolytic
property

Endonucleolytic property refers to that of an enzyme

capable of cleaving DNA at an internal position or
nucleotide sequence.

Ligase

Ligase
refers to an enzyme capable of joining a
nick in DNA

53

53 is the shorthand notation which indicates the

nucleotide sequence in a DNA or RNA strand or direction
of synthesis of nucleic acid strands such as nascent
cDNA and RNA primer.

Nick

A nick is a feature showing breakage or unjoined 3,5

phosphodiester structure in a strand of DNA or RNA.

Gap

which

refers

to

A gap is a feature characterized by the removal of

nucleotides usually by exonuclease action from one
strand of double-stranded DNA or RNA.
___________________________________________________________________

dnaC join dnaA and protein n in opening or unravelling the origin into an eye or
bubble. These proteins constitute a pre-priming complex (Funnell et al., 1987).
DnaB protein is a helicase, requiring ATP to unwind the DNA bi-directionally (Baker
et al., 1986). The unwound DNA is stabilized by the interaction of SSB protein
(single-stranded binding protein). Unravelling of the DNA generates a positive
supercoil which is relaxed to a negative (right-handed) supercoil by DNA gyrase.
This allows replication to continue. The process of priming and DNA synthesis are
followed once a replication eye is formed. The interaction of dnaB protein and DNA
primase with proteins n (PriB), i(dnaT), n(PriC) and dnaC generates a mobile,
multi-subunit primosome complex which is responsible for priming both continuous
and discontinuous replication. The binding of the primosome complex at opposite
ends of the replication eye is guided by protein n(PriA). As dnaB protein unwinds
the helix at the replication fork, an expanded replication eye develops, characterised
by single-stranded regions which are stabilized by SSB protein (Baker et al., 1986;
1987). A swivel or negative supertwist that prevents re-annealing of the parental
DNA strands (Baker and Kornberg, 1988) is formed by the gyrase. These singlestranded regions are the template to which RNA primers are synthesized by rNTPrequiring DNA primase. DNA polymerase III (pol III) uses dNTPs and Mg2+ to
synthesize cDNA 53 from the 3OH function of the RNA primers, releasing
pyrophosphate (PPi). DNA synthesis occurs in opposite directions. As the replication
forks advance (Cairns, 1963a; b), newly exposed template requires priming by the
primosome complex, followed by discontinuous DNA synthesis or generation of the
lagging strand. In each direction, the original RNA primer forms the growth point for
continued cDNA synthesis, generating the leading strand. This is the characteristic
pattern of semi-discontinuous DNA replication. The short discontinuous DNA
fragments, synthesized from RNA primers of the lagging strand, are called Okazaki
Fragments (Okazaki et al., 1968). RNA primers may be removed by the exonuclease
action of pol I, and digestion by Rnase H, followed by copy synthesis by the same
polymerase to fill in the gaps, stopping short of the 5 end of the next fragment.
Using ATP, DNA ligase facilitates the closure of the nicks between 3 and 5 ends of
adjacent fragments (Funnell et al., 1986). It is clear that the parental strand
templates are each associated with a complementary nascent strand of DNA,
depicting the semi-conservative nature of DNA replication (Figure).

Labelled structural DNA replication intermediates.

A model by Bohinski, 1987: E.coli REPLICATION BUBBLE MODEL

Figure . Flow diagram of a replication bubble model (Bohinski, 1987).

Reproduced with permission from Pearson Education Inc., Upper Saddle River, NJ,
USA.

X174 ds RFII RFI replication model

The native state genome of phage X174 is single-stranded (ss) circular DNA of
5386 nucleotides (Sanger et al., 1978). This strand is called the + strand which
replicates to form the - strand in bacterial hosts. Both strands generate the
replicative double-stranded (ds) circular form (RF I) DNA. RF I molecules can
replicate to form ds circular RFII molecules which will be the emphasis of this section
(Figure 3.7). RF II DNA molecules can give rise to the single-stranded + DNA which
is required for phage assembly (van Mansfield et al., 1986; Kornberg and Baker,
1992).
Phage X174 gpA protein of 60kd is probably the only phage-encoded enzyme
implicated in the replication of X174 DNA. Enzymes, of the bacterial host E.coli,
facilitate several steps in replication of the phage DNA. GpA protein has both
endonucleolytic and ligase properties (van Mansfield et al., 1986). At the ori site,
GpA protein introduces a nick on the + strand of negatively supercoiled ds RF I
DNA. The nick results in a 3OH function at a G residue at position 4305 and the
high energy, covalent attachment of gpA via an active tyrosyl group to the 5
phosphate moiety of an A residue at position 4306 (Eisenberg et al., 1977; Ikeda et
al., 1979). The 3OH function acts as a growth point for continuous 53 cDNA
synthesis facilitated by E.coli DNA pol III using the strand as template. No RNA
primer is required in this case (Ikeda et al., 1976; Kornberg and Baker, 1992). As the
+ strand is unwound by E.coli Rep helicase, SSB protein binds to it, preventing its
reassociation with the

X174 RF DNA ROLLING CIRCLE MODEL (GUPTHAR, 2008)

Rolling circle model continued

Rolling circle model continued

Figure 3.7

Flow diagram of a rolling circle model illustrating the replication of

X 174 RF I RF II DNA (Gupthar, unpublished original illustration).

strand template. E. coli Rep helicase requires two molecules of ATP for each base
pair melted (Kornberg et al., 1978). The action of the helicase gives the effect of the
rolling circle (Dressler and Wolfson, 1967; Gilbert and Dressler, 1968) as the +

strand is unwound from the inner strand circular template. As the length of the
unwound plus strand increases, it requires priming by the E.coli primosome complex
as described. Short RNA fragments are required for 53 discontinuous cDNA
synthesis opposite the plus strand template, generating Okazaki Fragments which
are separated by nicks . Following synthesis of the nascent + strand around the -
strand template, the original linearized + template circularizes and the second
active tyrosyl residue of the 5attached gpA protein cleaves the replication origin as
shown to release circular intermediates A and B. The energy generated by the
cleavage is stored in the gpA-DNA intermediate A and is used to sustain a ligation
between 3OH function and the 5phoshate group, displacing gpA protein (see Figure
) (van Mansfields et al., 1986; Kornberg and Baker, 1992). The RNA primer in
intermediate B is excised by the template exonuclease action of Pol I, the gaps filled
by Pol I - synthesis of DNA and nicks closed by DNA ligase as described in the
replication bubble model. The original strands are each associated with nascent
DNA in separate molecules A and B, a characteristic feature of semi-conservative
DNA replication (Meselson and Stahl, 1958).