dna topology dna has to be coiled to fit inside the cell organismnumber of base pairs contour...
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DNA Topology
DNA has to be coiled to fit inside the cell
Organism Number of base
pairsContour length, m
E. Coli
bacteria
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).
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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)
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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
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Consider a 260 bp B-duplex:
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Connect the ends to make a circular DNA:
Tw = 260/10.4 = 25
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Stryer Fig. 27.20
An electron micrograph of negatively supercoiled and relaxed DNA
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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
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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
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Human DNA Topoisomerase I: DNA: side view
20Å
Stryer Fig. 27.21
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Mechanism of DNA Topoisomerases I
OH
P-Topo
Wr = 1
-O BaseO
HOH
HHHH
723
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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
9
10
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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
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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
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Yeast DNA Topoisomerase II
Stryer Fig. 27.23
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Topoisomerase II - mechanism
Stryer Fig. 27.24
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Drugs that inhibit bacterial Topoisomerase II (DNA gyrase)
N NH3C
O
COOH
Et
NN
O
COOHF
NH
Nalidixic acid
Ciprofloxacin
Interfere with breakage and rejoining DNA ends:
OH3CO
O OH
O NH2
CH3
CH3
O
O
CH3
OH
NH
CH3
CH3
O
O OH
Novobiocin
Inhibit ATP binding:
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HO O
HO
HH
H
PO
O
O-
O
HO
HH
H
PO O-
O
HOH
HH
HH
Base
Base
Base
Enzymes that cut DNA and RNA : nucleases (Dnases and Rnase)
HO
• Degrade DNA in a stepwise manner by removing deoxynucleotides in 5’ 3’ (A) or 3’ 5’ direction (B)• Require a free OH • Exonucleases can be active on both single- and double-stranded DNA• Used for degrading foreign DNA and in proofreading during DNA synthesis
5’
5’3’
3’
+ dNMPs
3’
5’A
Nucleobase
Phosphate group
2’-deoxyribose
Exonucleases
B
Examples: B: snake venom phosphodiesterase A : calf intestinal phosphodiesterase
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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)
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Recognition sequences of some common restriction endonucleases
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DNARestrictio
nEnzyme EcoR V
5’-GAT ATC-3’3’-CTA TAG-5’
Asn185Thr186
HN
N
O
O
NN
N
N NH2
N H
O
NO
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Applications of Restriction Endonucleases in Molecular Biology
1. DNA fingerprinting (restriction fragment length polymorphism).
2. Molecular cloning (isolation and amplification of genes).
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Southern blotting
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Restriction fragment length polymorphisms are used to compare DNA from different sources
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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
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DNA Cloning: recombinant DNA technology
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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
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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
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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.