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Chapter 2:
The Structure
of DNA
Imagine yourself inside one of those
cooling DNA solutions, observing the
rebirth of beautifully undulating, semirigid,
double-helical threads from the jumble of
billions of intertwisted single strands. It is
a mind reeling spectacle.
Christian de Duve, A Guided Tour of the Living Cell
(1984), p. 292
2.1 Introduction
Objectives
• Describe the structure of DNA
• Keep its function within living cells in
mind
2.2 Primary structure: the
components of nucleic acids
Components of nucleotides
• Five-carbon sugars
• Nitrogenous bases
• The phosphate functional group
Sugars
Ribose
Deoxyribose
5’
HO
CH2
4’
3’
OH
O
HO CH2
OH
O
1’
2’
OH OH
OH
lacks
2’-OH
• The carbons of the sugar are given numbers from 1’
to 5’.
• The sugar of DNA is deoxyribose, which lacks 2’-OH.
• The sugar of RNA is ribose.
Pyrimidines
Thymine
Cytosine
NH2
Uracil
O
O
4
5
N
H3C
3
NH
NH
2
6
NH
1
O
NH
O
NH
• The atoms of the pyrimidines are given
numbers from 1 to 6.
O
Purines
Guanine
Adenine
O
NH2
1
2
6
N
3
N
4
5
N
7
8
NH
N
HN
H2N
N
NH
9
• The atoms of purines are given numbers
from 1 to 9.
Edwin Chargaff’s “rules”
[A] = [T]
[G] = [C]
[A] + [G] = [T] + [C]
• %G+C differs among species but is
constant in all cells of an organism within a
species.
• Varies from 22 to 73%
Nucleosides and nucleotides
DNA and RNA chains are formed through a
series of three steps:
1. A base attached to a sugar is a nucleoside.
2. A nucleoside with one or more phosphates attached is a
nucleotide.
3. Nucleotides are linked by 5′ to 3′ phosphodiester bonds
between adjacent nucleotides to form a DNA or RNA
chain.
Structure of nucleosides:
base + sugar
β-N-glycosidic bond
• Nucleosides are
derivatives of purines
and pyrimidines that
have sugar linked to a
ring nitrogen:
N9 for purines
N1 for pyrimidines
via a β-N-glycosidic
bond.
Structure of nucleotides
• Nucleotides are composed of nucleosides (base + sugar) and 1, 2, or 3
groups of phosphate.
• Mononucleotide (nucleoside-monophosphate)= nucleoside + 1 phosphate
• Nucleoside-diphosphate = nucleoside + 2 phosphates
• Nucleoside-triphosphate = nucleoside + 3 phosphates
• The components of a DNA or RNA chain
are joined by covalent bonds.
• A covalent bond is a strong chemical bond
formed when electrons are shared
between two atoms.
• These bonds are very stable and do not
break spontaneously within cells.
Nomenclature of nucleotides
Example: the base cytosine (C)
DNA
deoxycytidine 5′-triphosphate (dCTP)
RNA
cytidine 5′-triphosphate (CTP)
Generic
deoxynucleoside 5′-triphosphate (dNTP)
nucleoside 5′-triphosphate (NTP)
Physiological functions of
nucleotides
• “Bricks” for nucleic acids synthesis
• Macroergic compounds which deliver energy
necessary to different biological processes (eg.
ATP, GTP)
• Allosteric regulators of different enzymes
• Methyl group donor (eg. Sadenosylmethionine)
• Signal transduction: intracellular messengers
of hormones (eg. AMPc, GMCc)
Free natural nucleotides: cAMP
NH2
N
N
O
O P
CH 2
O
O
O
AMPc
Adenine
N
N
Ribose
OH
• 3’,5’cyclic adenosine
monophosphate
(cAMP) serves as a
second messenger or
transducer of
information between
the extracellular and
intracellular medium.
The length of RNA and DNA
• RNA
The number of nucleotides (nt) or bases
is used as a measure of length.
• Double-stranded DNA
The number of base pairs (bp) is used as
a measure of length.
1000 bp = 1 kilobase pair (kb or kbp)
1,000,000 = 1 megabase pair (Mb or Mbp)
• Natural RNAs come in sizes ranging
from less than one hundred to many
thousands of nucleotides
• DNA can be as long as several kb to
thousands of Mb
• Oligonucleotides are short chains of
single-stranded DNA (< 50 bases)
Significance of 5′ and 3′
• The 5′-PO4 and 3′-OH ends of a DNA or
RNA chain are distinct and have different
properties
• By convention, a DNA sequence is
written with the 5′ end to the left and the
3′ end to the right
O
Polynucletide chain of DNA
(primary/covalent structure)
Guanine
N
HN
H2N
N
5' end HO
CH 2
N
O
NH2
1’
•
The polynucleotide chain of
DNA consist of nucleotides
joined together by 3’,5’
phosphodiester bonds.
•
Phosphodiester bonds link the
3’- and 5’-sugar carbons of
adiacent monomer.
•
Polynucleotides are directional
macromolecules: each end of a
polymer is distinct.
Cytosine
N
3’linkage
O
O P O
N
CH 2
O
O
O
5’linkage
O
O P O
O
O
H3C
Thymine •
NH
NH
O P O
CH 2
O
O
OH
3' end
•
3’ –end is one with a free 3’hydroxyl.
5’ –end is one with a free or
phosphorilated 5’-hydroxyl.
O
•
Polynucleotides bear a negative
charge at physiological pH
2.3 Secondary structure of DNA
What chemical forces hold (or drive)
the DNA strands together?
(also applies to double-stranded regions
of RNA)
1. Hydrogen bonds between bases
Also important that the
purine-pyrimidine base
pairs are of similar
size.
Hydrogen bonds form between
the bases
• Two common “Watson-Crick” or
“complementary” base pairs:
Adenine (A) is joined to thymine (T) by two
hydrogen bonds.
Guanine (G) is joined to cytosine (C) by three
hydrogen bonds.
Why aren’t there other stable base pairs
present in DNA?
• May not be able to form two or more hydrogen
bonds.
• Pairing of G with T produces a pair with a similar
shape to Watson-Crick base pairs.
• Fidelity of DNA replication: proofreading and
DNA repair mechanisms correct mistakes.
• GU base-pairing is of importance in RNA
structure.
Base stacking provides chemical stability to
the DNA double helix
• The hydrophobic nitrogenous bases stack
onto each other without a gap by means of
a helical twist.
• A double-stranded DNA molecule has a
hydrophobic core composed of stacked
bases.
• Hydrophobic bonding is an example of
weak van der Waals interactions.
• A large number of weak van der Waals
interactions can significantly increase the
stability of a structure, such as the DNA
double helix.
Structure of the Watson-Crick
DNA double helix
• Polarity in each strand: 5′ 3′
• Two strands are antiparallel
• Major and minor grooves
3 Ways of Depicting DNA Structure
Major and minor grooves
• The major groove carries a “message” that
can be read by DNA binding proteins..
• In the major groove, the pattern of hydrogenbonding groups is different for AT, TA, GC,
and CG base pairs.
• In the minor groove, there is only one
difference in the pattern between AT and GC
base pairs.
Distinguishing between features of
alternative double-helical structures
• B-DNA (Watson-Crick DNA)
• A-DNA
• Z-DNA
Higher Order RNA Structure
Stem-loops are common elements of secondary RNA structure.
Stems are double-stranded
regions of RNA that are A-form
helices. They usually follow
Watson-Crick base pairing rules
(U replaces T), but other pairs
occur (G – U is common).
(DNA is typically a B-form helix).
Stem
loop
• The predominant form of DNA in vivo is
B-DNA.
• But, there is evidence for a role of Z-DNA
in vivo:
– Z-DNA binding proteins.
– Short sections of Z-DNA within a cell are
energetically favorable and stable.
– Role in regulating gene expression?
• A region of Z-DNA is connected to B-DNA
through a junction in which one base pair
is flipped out, or extruded, from the DNA
helix.
• This process is called base flipping.
DNA can undergo reversible
strand separation
Significance of complementary base pairing:
•Fidelity of DNA replication, transcription,
and translation.
•Ability to manipulate in the lab by
denaturation, renaturation, and hybridization.
Denaturation or “melting”
of DNA
• Base stacking in duplex DNA quenches
the capacity of bases to absorb UV light.
• Hyperchromicity: As DNA “melts” its
absorption of UV light increases.
• Tm (melting temperature): The temperature
at which half of the bases in a dsDNA
sample have denatured.
Renaturation or “reannealing”
of DNA
• The capacity to renature denatured DNA permits
hybridization.
• Hybridization is the complementary base pairing
of strands from two different sources.
• The rate at which DNA reanneals is a function of
the length of the DNA and the initial
concentration in the sample.
A DNA renaturation Cot curve
• C/C0 = 1/[1 + KC0t]
• The expression C0t is called “Cot.”
• Cot ½ is when renaturation is half
completed.
• A plot of C/C0 versus C0t is called a Cot
curve.
Comparison of Cot curves for E.
coli and calf thymus DNA
• The Cot ½ of calf thymus DNA is greater
than the Cot ½ of E. coli DNA.
• Explanation: The larger the genome size,
the longer it will take for any one sequence
to encounter its complementary sequence.
Denaturation and Reannealing
DNA Denaturation Curve
Dependence of DNA denaturation on G + C content and
on salt concentration
• The E. coli DNA curve resembles an ideal
Cot curve.
• The calf thymus DNA curve is not smooth.
• Explanation: the rapidly reannealing
fraction in the calf DNA represents
repeated DNA sequences; the more
slowing reannealing fraction represents
unique sequences.
2.4 Unusual DNA secondary
structures
Slipped structures
• Occur at tandem repeats
• Found upstream of regulatory regions
• Formation of DNA slipped structures can
lead to repeat expansion during DNA
replication.
• A number of hereditary neurological
diseases are caused by the expansion of
simple triplet repeat sequences.
Cruciform structures
• Paired stem-loop formations
• Characterized in vitro for many inverted
repeats in plasmids and phage
• Role in vivo?
Triple helix DNA
• A third strand of DNA joins the first two to form
triplex DNA (intra- or inter-molecular).
• Favored by purine-pyrimidine stretches with
mirror repeat symmetry.
• The Watson-Crick duplex associates with the
third strand through Hoogsteen hydrogen bonds.
Friedreich’s ataxia and
triple helix DNA
• 5′-GAA-3′ trinucleotide repeat expansion in
first intron of Friedreich’s ataxia gene
(frataxin).
Normal individual: 8-30 repeats
Friedreich’s Ataxia:  1000
• Expanded GAA repeats form triple helix
DNA.
2.5 Tertiary structure of DNA
Supercoiling of DNA
• Supercoils form a twisted, 3-D structure
which is more favorable energetically.
• Less stable than relaxed DNA.
Negative (left-handed) supercoil: underwound
Positive (right-handed) supercoil: overwound
• The strain present within supercoiled DNA
sometimes leads to localized denaturation.
• B-DNA→ Z-DNA transitions may be
triggered by negative supercoiling.
• Topoisomerases are enzymes that
introduce transient breaks in DNA strands
and release the strain of supercoiling.
What is the significance of supercoiling in vivo?
• Virtually all DNA within prokaryotes and
eukaryotes is negatively supercoiled.
• Some architectural proteins, induce DNA
negative supercoiling upon binding.
• DNA is restrained when it is supercoiled around
DNA-binding proteins, such as in nucleosomes.
• Unrestrained supercoiled domains are in
equilibrium between tension and unwinding of
the helix.
DNA supercoiling plays an important role in
many processes, such as replication,
transcription, and recombination
• Genome of some viruses: small circle
Relaxed circle: reduced activity
Negatively supercoiled circle: increased activity
• Bacterial genome: very large circle
Form independent DNA loop domains
• Eukaryotic genomes: linear
Form independent DNA loop domains
• Negative supercoiling makes it easier to
separate the DNA strands during
replication and transcription.
• The DNA of thermophilic Archaea exists in
a positive supercoiled state that protects
the DNA from denaturation at high
temperatures.
ENOUGH !!!