Deoxyribonucleic Acid (DNA). The double helix Nitrogenous Bases and Pentose Sugars.

Deoxyribonucleic Acid

(DNA)

The double helix

Nitrogenous Bases and Pentose Sugars

Purine and Pyrimidine Structure

(1)  Pyrimidines are planar

(2)  Purines are nearly planar

(3) Numbering is different

Numbering Is Different

Bases Have Tautomeric Forms

Uracil

Nucleosides vs. Nucleotides

Glycosidic bond

Nucleotides formed by condensation reactions

Monophosphates

Deoxyribonucleotides

Ribonucleotides

Only RNA Is Hydrolyzed by Base

Nucleoside Diphosphate and Triphosphate

Dinucleotides and Polynucleotides

Ester bonds

Watson-Crick Base Pairs

A=T

G=C

Hoogsteen Base Pairs

Other Base Pairs Are Possible

Homo Purines Hetero PurinesWatson-Crick,

Reverse Watson-Crick, Hoogsteen,

Reverse Hoogsteen, Wobble,

Reverse Wobble

Base Pairing Can Result in Alternative DNA Structures

Triplex Tetraplex

Hairpin Loop Cruciform

• Periodicity: A pair of strong vertical arcs (C & N atoms) indicate a very regular periodicity of 3.4 Å along the axis of the DNA fiber.

• Astbury suggested that bases were stacked on top of each other "like a pile of pennies".

• Helical nature: Cross pattern of electron density indicates DNA helix and angles show how tightly it is wound.

• Diameter: lateral scattering from electron dense P & O atoms.

DNase can only cleave external bond demonstrating periodicity

Watson and Crick Model (1953)

• 2 long polynucleotide chains coiled around a central axis

• Bases are 3.4 Å (0.34 nm) apart on inside of helix

• Bases flat & lie perpendicular to the axis

• Complete turn = 34 Å • 10 bases/turn• Diameter = 20 Å• Alternating major and

minor grooves

Hydrophobic

Hydrophilic

Complementarity

Base Pairing Results from H-Bonds

Only A=T and GC yield 20 Å Diameter

A:C base pair incompatibility

Bases Are Flat

Chains Are Antiparallel…

Base Pairs and Groove Formation

Base flipping can occur

Helix Is Right-Handed

Biologically Significant Form = B-DNA

Low Salt = Hydrated, 10.5 bp/turn

A- DNA Exists Under High Salt Conditions

Side-view Top-view

Base pairs tilted, 23 Å, 11bp/turn

Z-DNA Is a Left-Handed Helix

Zig-zag conformation, 18 Å, 12 bp/turn, no major groove

Propeller Twist Results from Bond Rotation

Reassociation Kinetics

Denaturation of DNA Strands and the Hyperchromic Shift

• Denaturation (melting) is the breaking of H, but not covalent, bonds in DNA double helix duplex unwinds strands separate

• Viscosity decreases and bouyant density increases• Hyperchromic shift – uv absorption increases with

denaturation of duplex• Basis for melting curves because G-C pairs have three

H bonds but A-T pairs have only two H bonds• Duplexes with high G-C content have a higher melting

temperature because G-C pairs require a higher temperature for denaturation

Molecular Hybridization

• Reassociation of denatured strands• Occurs because of complementary base pairing • Can form RNA-DNA Hybrids• Can detect sequence homology between species• Basis for in situ hybridization, Southern and

Northern blotting, and PCR

Hybridization

Reassociation Kinetics• Derive information about the complexity of

a genome• To study reassociation, genome must first

be fragmented (e.g. by shear forces)• Next, DNA is heat-denatured• Finally, temperature is slowly lowered and

rate of strand reassociation (hybridization) is monitored

• Initially there is a mixture of unique DNA sequence fragments so hybridization occurs slowly. As this pool shrinks, hybridization occurs more quickly

• C0t1/2 = half-reaction time or the point where one half of the DNA is present as ds fragments and half is present as ss fragments

• If all pairs of ssDNA hybrids contain unique sequences and all are about the same size, C0t1/2 is directly proportional to the complexity of the DNA

• Complexity = X represents the length in nucleotide pairs of all unique DNA fragments laid end to end

• Assuming that the DNA represents the entire genome and all sequences are different from each other, then X = the size of the haploid genome

The Tm

The Hyperchromic Shift (Melting Curve Profile)

Tm = temperature at which 50% of DNA is denatured

Maximum denaturation = 100% single stranded

Double stranded

50% double, 50% single stranded

High G-C Content Results in a Genome of Greater Bouyant Density

Ideal C0t Curve

100% ssDNA

100% dsDNA

Larger genomes take longer to reassociate because there are more DNA

fragments to hybridize

Largest genomeSmallest genome

C0t1/2 Is Directly Proportional to Genome Size

Genomes are composed of unique, moderately repetitive and highly repetitive

sequences

Highly repetitive DNA

Moderately repetitive DNA

10-4 10-2 100 102 104

Fra

ctio

n r

emai

nin

gsi

ngl

e-st

ran

ded

(C

/C0)

Unique DNA sequences

0

100

C0t (moles x sec/L)

More complex genomes contain more classes of DNA sequences

G-C Content Increases Tm