DNA is Composed of Complementary
Strands
Base Pairing is Determined by Hydrogen Bonding
same size
Forces stabilizing DNA double helix
1. Hydrogen bonding (2-3 kcal/mol per base pair)
2. Stacking (hydrophobic) interactions
(4-15 kcal/mol per base pair)
3. Electrostatic forces.
B-DNA
right handed helix
• helical axis passes through
base pairs
23.7 A
•Sugars are in the 2’ endo
conformation.
HO
O
3'
7.0 A
1'
H (OH)
HO
• planes of bases are nearly
perpendicular to the helix axis.
BASE
2'
5'
•Bases are the anti conformation.
NH2
• 3.4 A rise between base pairs
N
Wide and deep
N
HO
O
O
OH
•Bases have a helical twist of 36º
Narrow and deep
(10.4 bases per helix turn)
• Helical pitch = 34 A
DNA can deviate from Ideal Watson-Crick structure
• Helical twist ranges from 28 to 42°
• Propeller twisting 10 to 20°
•Base pair roll
Major and minor groove of the double
helix
Major groove
O
N
H2N
NH
N
N
N
To
de
y
ox
N
NH2
C-1’
se
o
rib
O
C-1’T
od
Minor groove
N
Wide and deep
NH 2
O
N
N
C-1’
N
HN
N
O
Narrow and deep
C-1’
eox
yri
bo
se
Major groove and Minor groove of DNA
Hypothetical situation: the two grooves would have similar size if dR residues
were attached at 180° to each other
To deoxyribose-C1’
Base
C1’ -To deoxyribose
Base
Major groove
Major groove
N
O
N
H 2N
NH
N
C-1’
y
ox
e
d
To
os
rib
e
N
C-1’
N
NH 2
Minor groove
O
O
N
N
N
NH 2
C-1’T
od
N
HN
N
O
eox
yri
bo
se
Minor groove
C-1’
B-type duplex is not possible for RNA
HO
CH2
O
Base
H
H
O
OH
H
ribose
steric “clash”
H
A-form helix: dehydrated DNA; RNA-DNA hybrids
Right handed helix
•Sugars are in the 3’ endo
conformation.
• planes of bases are tilted
20 ° relative the helix axis.
•Bases are the anti conformation.
• 2.3 A rise between base pairs
25.5 A
•11 bases per helix turn
• Helical pitch = 25.3 A
Top View
The sugar puckering in A-DNA is 3’-endo
5.9 A
O
2'
5'
7.0 A
O
3'
BASE
1'
H (OH)
O
2' endo (3' exo) B-DNA
O
O
5'
3'
BASE
1'
O
2'
H (OH)
3' endo (A-DNA)
Living Figure – A-DNA
http://bcs.whfreeman.com/biochem5
A-DNA has a shallow minor
groove and a deep major groove
Major groove
O H2N
N
To
e N
os
b
i
yr
x
o
de
•
NH
N
N
NH2
O
Minor groove
Major groove
N To d
eo
xy
rib
os
e
•
Helix axis
N
To
B-DNA
O H2N
e
os
ir b
y
ox
e
d
N
NH
N
N
NH2
O
Minor groove
A-DNA
N To d
eo
xy
rib
os
e
Z-form double helix: polynucleotides of alternating
purines and pyrimidines (GCGCGCGC) at high salt
Left handed helix
• Backbone zig-zags because sugar
puckers alternate between 2’ endo
pyrimidines and 3’ endo (purines)
• planes of the bases are
tilted 9° relative the helix
axis.
• Bases alternate between anti
(pyrimidines) and syn conformation
(purines).
• 3.8 A rise between base pairs
•12 bases per helix turn
18.4 A
•
•
Flat major groove
Narrow and deep minor groove
• Helical pitch = 45.6 A
Sugar and base conformations in Z-DNA alternate:
5’-GCGCGCGCGCGCG
3’-CGCGCGCGCGCGC
C: sugar is 2’-endo, base is anti
G: sugar is 3’-endo, base is syn
NH2
O
N
HO
2'
5'
O
3'
N
1'
H
HO
C
HN
O
N
H2N
HO HO
5'
N
3'
N
O
1'
G
H
Living Figure – Z-DNA
http://bcs.whfreeman.com/biochem5
Biological relevance of the minor types of DNA
secondary structure
•Although the majority of chromosomal DNA is in B-form,
some regions assume A- or Z-like structure
• Runs of multiple Gs are A-like
•The upstream sequences of some genes contain
NH
5-methylcytosine = Z-like duplex
2
H3C
N
N
H
O
5-methylcytosine (5-Me-C)
• Structural variations play a role in DNA-protein interactions
• RNA-DNA hybrids and ds RNA have an A-type structure
Hydrogen bond donors and acceptors in
DNA grooves facilitate its recognition
by proteins
The edges of base pairs displayed to DNA major and minor groove
contain potential H-bond donors and acceptors:
Major groove
N
n
O
H 2N
h
o
h
To
n= Nitrogen hydrogen bond acceptor
o= Oxygen hydrogen bond acceptor
h= Amino hydrogen bond donor
O
N
H 2N
o
de
xy
N
se
o
rib
H2N
NH
N
N
N
NH2
O
Minor groove
To
d
eox
yri
bo
se
Hydrogen bond donors and acceptors on each
edge of a base pair
Major groove
To
o
de
xy
os
b
i
r
e
To
d
Minor groove
eox
yri
bo
se
Structural characteristics of DNA
facilitating DNA-Protein Recogtnition
1. Major and major groove of DNA contain sequencedependent patterns of H-bond donors and acceptors.
2. Sequence-dependent duplex structure (A, B, Z, bent
DNA).
3. Hydrophobic interactions via intercalation.
4. Ionic interactions with phosphates.
Groove binding drugs and proteins
NH3
H
N
H2N
NH3
NH2
DAPI
5’-ATT-3’
Others: netropsin, distamycin,
Hoechst 33258
Leucine zipper proteins bind DNA
major groove
Triple helix and Antigene approach
N
N
G
O
N
N
H
NH2
O
N
N
G
H2N
NH
N
C
N
N
NH2
O
G:GC
Hoogsteen base pairing = parallel
Reversed Hoogsteen = antiparallel
Biophysical properties of DNA
•
Facile denaturation (melting) and re-association of the duplex
are important for DNA’s biological functions.
In the laboratory, melting can be induced by heating.
•
A260
Single strands
T°
TM
duplex
70
•
80
90
100
T, C
Hybridization techniques are based on the affinity of complementary
DNA strands for each other.
• Duplex stability is affected by DNA length, % GC base pairs, ionic strength, the
presence of organic solvents, pH
•
Negative charge – can be separated by gel electrophoresis
Separation of DNA fragments by gel electrophoresis
Polyacrylamide gel:
O
H2C CH-C-NH2
O
O
H2C CH-C-NH-CH2 HN-C-C CH2
SO4-
H2N
O
C
H2C CH-
• DNA strands are negatively charged –
migrate towards the anode
• Migration time ~ ln (number of base
pairs)
DNA Topology
DNA has to be coiled to fit inside the cell
Organism
Number of base
pairs
Contour length,
m
E. Coli
bacteria
4,600,000
1,360
SV40 virus
5,100
1.7
Human
chromosomes
48,000,000240,000,000
1.6 – 8.2 cm
DNA polymers must be folded to fit into the cell or nucleus (tertiary structure).
DNA Topology:
• Negative supercoiling: DNA is twisted in the
direction opposite to the direction of the double
helix (underwound)
• Positive supercoiling: DNA is twisted in the same
direction as the direction of the double helix
(overwound)
DNA Topology: linking number
• Topoisomers
can be quantitatively
defined by the linking number (Lk).
• Lk is the number of times a strand of
DNA winds in the right handed
direction around the helix axis when
the axis is constrained.
• Tw (twist) is the helical winding of the
strands around each other (# b.p./10.4
for B form DNA).
• Wr (writh) is the number of
superhelical turns
Lk = Tw + Wr,
if Lk = const., Tw = - Wr
Consider a 260 bp B-duplex:
Connect the ends to make a circular DNA:
Tw = 260/10.4 = 25
An electron micrograph of negatively supercoiled and relaxed DNA
Stryer Fig. 27.20
Organization of chromosomal DNA
• Chromosomal DNA is organized in loops (no free ends)
• It is negatively supercoiled: 1 (-) supercoil per 200 nucleotides
145 bp duplex
Histone octamer (H2A, H2B, H3, H4)2
H1 is bound to the linker region
Enzymes that control DNA
supercoiling: DNA Topoisomerases
Change the linking number (Lk) of DNA duplex by
concerted breakage and re-joining DNA strands
Topoisomerase enzymes
Topoisomerases I
Topoisomerases II
Relax DNA supercoiling by
increments of 1 (cleave one strand)
Change DNA supercoiling by
the increments of 2
(break both strands)
Usually introduce negative supercoiling
Human DNA Topoisomerase I: DNA: side view
20Å
Stryer Fig. 27.21
Mechanism of DNA Topoisomerases I
-O
O
Base
H
H
H
H
OH H
723
OH
P-Topo
 Wr = 1
Drugs that inhibit DNA Topoisomerase I
9
O
10
C-10 C-9
N
Camptothecin H
OH
Topotecan
N
H
(CH3)2NHCH2
O
CH3CH2
OH
O
• Camptothecin, topotecan and analogs
• Antitumor activity correlates with interference with
topoisomerase activity
• Stabilizes topoisomerase I-DNA intermediate, preventing
DNA strand re-ligation
• Used in treatment of colorectal, ovarian, and small cell
lung tumors
Enzymes that control DNA
supercoiling: DNA Topoisomerases
Change the linking number (Lk) of DNA duplex by
concerted breakage and re-joining DNA strands
Topoisomerase enzymes
Topoisomerases I
Topoisomerases II
Relax DNA supercoiling by
increments of 1 (cleave one strand)
Change DNA supercoiling by
the increments of 2
(break both strands)
Usually introduce negative supercoiling
Topoisomerases II
•
•
•
•
•
Most of Topoisomerases II introduce
negative supercoils (e.g. E. coli DNA Gyrase)
Require energy (ATP)
Each round introduces two supercoils ( Wr
= - 2)
Necessary for DNA synthesis
Form a covalent DNA-protein complex
similar to Topoisomerases I
Yeast DNA Topoisomerase II
Stryer Fig. 27.23
Topoisomerase II - mechanism
Stryer Fig. 27.24
Drugs that inhibit bacterial Topoisomerase II (DNA
gyrase)
Interfere with breakage and rejoining DNA ends:
H3C
N
Et
N
NH
N
N
COOH
F
O
Nalidixic acid
COOH
O
Ciprofloxacin
Inhibit ATP binding:
CH3
O
CH3
H3CO
O
CH3
O
O
OH CH3
CH3
O
O
OH
N
OH H
NH2
Novobiocin
O
Enzymes that cut DNA: exonucleases
5’
HO
A
5’
3’
3’
5’
OH
+ dNMPs
• Degrade DNA in a stepwise manner by removing
deoxynucleotides in 5’  3’ (A) or 3’  5’ direction (B)
• Require a free OH
• Most exonucleases are active on both single- and
double-stranded DNA
• Used for degrading foreign DNA and in proofreading
during DNA synthesis
B HO H
3’
Phosphate group
Nucleobase
2’-deoxyribose
DNA Endonucleases
• Cleave internal phosphodiester bonds resulting in 3’-OH and 5’-phosphate
ends
5’
3’-OH 5’-P
5’-P 3’-OH
• some endonucleases cleave randomly (DNase I, II)
• Type II Restriction endonucleases are highly sequence specific
EcoRI recognition site:
Cleavage Site
G
A
A
T
T
C
C
T
T
A
A
G
Palindromic site
(inverted repeat)
Cleavage Site
• RE are found in bacteria where they are used for protection against foreign
DNA
Recognition sequences of some common
restriction endonucleases
DNA
Restriction
Enzyme
EcoR V
Applications of Restriction Endonucleases in Molecular
Biology
1. DNA fingerprinting (restriction fragment length
polymorphism).
2. Molecular cloning (isolation and amplification of
genes).
Southern blotting
Restriction fragment length polymorphisms are used to
compare DNA from different sources
DNA Ligase
AMP + PPi
O
O
OH
-O
P
O-
O
O
DNA Ligase +
(ATP or NAD+)
P
O
O-
• Forms phosphodiester bonds between 3’ OH and 5’ phosphate
• Requires double-stranded DNA
• Activates 5’phosphate to nucleophilic attack by transesterification
with activated AMP
DNA Cloning: recombinant DNA technology
Human Genetic Polymorphisms
• Human genome size: 3.2 x 109 base pairs
• 30,000 genes
• 2-4 % of total sequence codes for proteins
• Human genetic variation:
 1 sigle nucleotide polymorphism (SNP) per 1,300 bp
Examples of genetic polymorphisms of drug
metabolizing enzymes
Enzyme
cytochrome 2B6
substrate examples
cyclophosphamide
tamoxifen
benzodiazepines
DNA regions involved
exons 1,4,5, and 9
cytochrome 2D6
cytochrome 1A2
debrisoquine
caffein
phenacetin
internal base changes
5' flanking region
N-acetyltransferase
aromatic amines
DNA Structure: Take Home Message
1. Genetic information is stored in DNA.
2. DNA is a double stranded biopolymer containing repeating
units of nitrogen base, deoxyribose sugar, and phosphate.
3. DNA can be arranged in 3 types of duplexes which contain
major and minor grooves.
4. DNA can adopt several topological forms.
5. There are enzymes that will cut DNA, ligate DNA, and
change the topology of DNA.
6. Human genome contains about 3.2 billion base pairs. Interindividual differences are observed at about 1 per 1,000
nucleotides.