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).

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

OH

P-Topo

Wr = 1

-O BaseO

HOH

HHHH

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

9

10

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

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:

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

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

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

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.