Download DNA is Composed of Complementary Strands

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 proteins and drugs
NH3
Leucine zipper proteins bind DNA
major groove
H2N
H
N
NH3
NH2
4,6-diamidino-2-phenylindole (DAPI)
5’-ATT-3’
Others: netropsin, distamycin,
Hoechst 33258
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 (+) electrode (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
(cleave 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
Topotecan
OH
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
(cleave 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.