DNA is Composed of Complementary Strands
Transcript of DNA is Composed of Complementary Strands
DNA is Composed of Complementary Strands
NH
N
N O
NH2
N NN
H2N
O
HNN
O
O
NN
N
N NH2
G•C
A•T
DNA
NH
N
N O
NH2
N NN
H2N
O
HNN
O
O
NN
N
N NH2
G•C
A•U
RNA
A ::
G :::
T ::
T
C
A
3'
3' 5'
5'
Anti-parallel Strands of DNA
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.
right handed helix
• planes of bases are nearlyperpendicular to the helix axis.
•Sugars are in the 2’ endo conformation.
•Bases are the anti conformation.
•Bases have a helical twist of 36º (10.4 bases per helix turn)
• Helical pitch = 34 A
B-DNA
• 3.4 A rise between base pairs
Wide and deep
Narrow and deep
HOO
OH
N
N
NH2
O
HO
OH (OH)
HO
BASE
1'3'
2'5'
7.0 A
• helical axis passes throughbase pairs
23.7 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
Wide and deep
Narrow and deep
Major groove
NHN
N O
NH2
N NN
H2N
O
Minor grooveTo deox
yribose
C-1’C-1’
HNN
O
O
NN
N
N NH2
C-1’
C-1’
Major groove and Minor groove of DNA
Major groove
Minor groove
NHN
N O
NH 2
N NN
H 2N
OC-1’C-1’
HNN
O
O
NN
N
N NH 2
C-1’
C-1’
Major groove
Minor groove
Base BaseTo deoxyribose-C1’ C1’ -To deoxyribose
Hypothetical situation: the two grooves would have similar size if dR residues were attached at 180° to each other
B-type duplex is not possible for RNA
steric “clash”
O
OH
HH
CH2
HO
HOBase
ribose
H
A-form helix: dehydrated DNA; RNA-DNA hybrids
Top View
Right handed helix
• planes of bases are tilted20 ° relative the helix axis.
• 2.3 A rise between base pairs
•Sugars are in the 3’ endo conformation.
•Bases are the anti conformation.
•11 bases per helix turn
• Helical pitch = 25.3 A
25.5 A
The sugar puckering in A-DNA is 3’-endo
O
OH (OH)
O
BASE O
O
H (OH)
OBASE
2' endo (3' exo) B-DNA
1'
3' endo (A-DNA)
3'
1'3'
2'5'
5'
2'
7.0 A
5.9 A
Living Figure – A-DNA
http://bcs.whfreeman.com/biochem5
A-DNA has a shallow minor groove and a deep
major groove
N
NHN
N
O
NH2
NN
H2N
O
To deoxy
ribose To deoxyribose
Major groove
Minor groove
B-DNA
N
NHN
N
O
NH2
NN
H2N
O
To deoxyribose To deoxyribose
Major groove
Minor groove
Helix axis
A-DNA
• •
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)
• Bases alternate between anti (pyrimidines) and syn conformation (purines).
•12 bases per helix turn
• Helical pitch = 45.6 A
• planes of the bases are tilted 9° relative the helix axis.
• Flat major groove• Narrow and deep minor groove
18.4 A
• 3.8 A rise between base pairs
Sugar and base conformations in Z-DNA alternate:
N
N
NH2
ON
HN
NN
O
H2NHO
OH
HO
HO
O
H
HO1' 3'
1'3'
2'
5'
5'
GC
5’-GCGCGCGCGCGCG3’-CGCGCGCGCGCGC
C: sugar is 2’-endo, base is antiG: sugar is 3’-endo, base is syn
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 5-methylcytosine = Z-like duplex
N
NH
NH2
O
5-methylcytosine (5-Me-C)
H3C
• RNA-DNA hybrids and ds RNA have an A-type structure• Structural variations play a role in DNA-protein interactions
Hydrogen bond donors and acceptors in DNA grooves
facilitate its recognition by proteins
N H2NOH2N
hn ho
n= Nitrogen hydrogen bond acceptoro= Oxygen hydrogen bond acceptorh= Amino hydrogen bond donor
The edges of base pairs displayed to DNA major and minor groove contain potential H-bond donors and acceptors:
N
NH
N
N
O
NH2
NN
H2N
OTo d
eoxyri
bose To deoxyribose
Major groove
Minor groove
Hydrogen bond donors and acceptors on each edge of a base pair
NHN
N O
NH2
N NN
H2N
O
HNN
O
O
NN
N
N NH2
G•C A•T
NO
H
OH
N HO
NN
O
Major groove
Minor groove
To deox
yribose To deoxyribose
Structural characteristics of DNA facilitating DNA-Protein Recogtnition
1. Major and major groove of DNA contain sequence-dependent 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.
HN NH3
NH2
NH3
H2N
DAPI
Groove binding drugs and proteins
Leucine zipper proteins bind DNA major groove
5’-ATT-3’
Others: netropsin, distamycin,Hoechst 33258
Triple helix and Antigene approach
Hoogsteen base pairing = parallelReversed Hoogsteen = antiparallel
N
NH
N
N
O
NH2
NN
H2N
O
G:GC
N
NH
N
N
O NH2
G
G C
Biophysical properties of DNA
T, C70 80 90 100
A260
TM
• 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.
• 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
T°
Single strands
duplex
H2C CH-C-NH2
O
SO4-
H2C CH-C-NH-CH2
OCH2HN-C-C
OH2C CH-
CO
H2N
Separation of DNA fragments by gel electrophoresis
• DNA strands are negatively charged – migrate towards the anode• Migration time ~ ln (number of base
pairs)
Polyacrylamide gel:
DNA Topology
DNA has to be coiled to fit inside the cell
Organism Number of base
pairsContour length, m
E. Colibacteria
4,600,000 1,360
SV40 virus 5,100 1.7
Human chromosomes
48,000,000-240,000,000
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
Stryer Fig. 27.20
An electron micrograph of negatively supercoiled and relaxed DNA
Organization of chromosomal DNA
• Chromosomal DNA is organized in loops (no free ends)• It is negatively supercoiled: 1 (-) supercoil per 200 nucleotides
Histone octamer (H2A, H2B, H3, H4)2
145 bp duplex
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 IRelax DNA supercoiling by
increments of 1 (cleave one strand)
Topoisomerases IIChange 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 BaseO
HOH
HHHH
OHP-Topo
Wr = 1
723
Drugs that inhibit DNA Topoisomerase I
• 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
N
N
O
O
OCH3CH2
OH
C-10 C-9
CamptothecinTopotecan
H HOH (CH3)2NHCH2
910
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 IRelax DNA supercoiling by
increments of 1 (cleave one strand)
Topoisomerases IIChange DNA supercoiling by
the increments of 2 (break both strands)
Usually introduce negative supercoiling
Topoisomerases II
OP
O
O(-)
O
O
O
DNA Chain
BASE
ENZYME
• 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)
N NH3C
OCOOH
Et
NN
OCOOHF
NH
Nalidixic acid
Ciprofloxacin
Interfere with breakage and rejoining DNA ends:
OH3CO
O OH
O NH2
CH3
CH3
O
OCH3
OHNH
CH3
CH3
O
O OH
Novobiocin
Inhibit ATP binding:
Enzymes that cut DNA: exonucleases
HO
• 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
5’5’3’3’
+ dNMPs
H
OH
3’
5’
HOB
A
Nucleobase
Phosphate group
2’-deoxyribose
DNA Endonucleases
G A A T T C
C T T A A G
Cleavage Site
Cleavage Site
EcoRI recognition site:
• Cleave internal phosphodiester bonds resulting in 3’-OH and 5’-phosphate ends
3’-OH3’-OH
5’5’-P
5’-P
• Type II Restriction endonucleases are highly sequence specific
• RE are found in bacteria where they are used for protection against foreign DNA
• some endonucleases cleave randomly (DNase I, II)
Palindromic site(inverted repeat)
Recognition sequences of some common restriction endonucleases
DNARestrictionEnzyme 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
OH P
O
-O O
O-
O P
O
O
O-DNA Ligase + (ATP or NAD+)
AMP + PPi
• 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
Enzyme substrate examples DNA regions involvedcytochrome 2B6 cyclophosphamide exons 1,4,5, and 9
tamoxifenbenzodiazepines
cytochrome 2D6 debrisoquine internal base changes cytochrome 1A2 caffein 5' flanking region
phenacetin
N-acetyltransferase aromatic amines
Examples of genetic polymorphisms of drug metabolizing enzymes
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. Inter-individual differences are observed at about 1 per 1,000 nucleotides.