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.