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DNA

We start with DNA

Transformation, Transfection,
Chargaff’s Rules and the
Double Helix
History

• 1946 – DNA is the main constituent of genes (Avery)


• 1950 – First X-ray pictures of DNA (Franklin)
• 1953 – DNA structure revealed (Watson and Crick)
• 1970 - onwards - Multiple conformations and structures,
initially from fibers
• 1973 - X-ray structure confirms double helix (Rich)
• 1974 - t-RNA structure (Kim)
• 1980 – Structure of first complete turn of B DNA
(Dickerson)
1 Transformation

– Uptake of genetic material from an external source resulting in the


acquisition of new traits (phenotype is changed)
– Griffith’s experiment was the earliest document evidence of
transformation

• Experiment by Frederick Griffith – 1928


– Demonstrated first evidence that genes are
molecules
– Two different strains of Streptococcus pneumoniae
• Non-pathogenic = Avirulent = ROUGH cells (R)
• Pathogenic = virulent = SMOOTH (S)
– Smooth outer covering = capsule
– Capsule = slimy, polysaccharide
– Encapsulated strains escape phagocytosis
Transformation

– The capsule alone did not cause pneumonia


• Heat-killed S strain was avirulent
• Ability to escape immune detection and multiply
– When heat-killed S strain was mixed with living R strain  the
mouse dies of pneumoniae
• Encapsulated strain (S) recovered from dead mouse  Now a live
strain
• The R strain had somehow acquired the ability to produce the
polysaccharide capsule
– Transformation
– Ability to produce coat was an inherited trait  Daughter cells also
produced capsule
Transformation

• Avery, MacLeod and McCarty defined the transforming


agent of Griffith’s experiment as DNA (1944)
– Chemical components of heat-killed S strain bacteria were purified
and co-injected with live R strain
• Polysaccharide/Carbohydrate
• Lipids
• Protein
• Nucleic acids
– DNA
– RNA

Bacterial Transformation is
Mediated by DNA
2 Transformation

Viral DNA is Transferred into Cells During


Infection – The Hershey-Chase Experiment (1952)

• T2 Bacteriophage studies
– Bacteriophage = viruses that infect bacteria
– Major chemical components = DNA and protein
– Escherichia coli infected with T2 produce thousands of
new viruses in the host cell
• Host cell lyses and phage are released
Transformation

• Determination of whether DNA or protein was directing synthesis


of new phage particles
– Viral proteins were radioactively labeled with:
• 35S by growing T2-infected bacteria in 35S-methionine = 1st
Batch
– Amino acid labeling
– DNA does not contain any sulfur atoms
• 32P by growing T2-infected bacteria in 32-P = 2nd Batch
– Nucleic acid labeling
– Amino acids do not contain phosphorous
Transformation

– Radioactively labeled viruses were isolated from the


culture and used to REINFECT new host cells
• Batch 1 = protein labeled
• Batch 2 = DNA labeled
– Blender used to disrupt phage on surface of bacteria
from cells and their cytoplasmic components  then
centrifuged
• Supernatant?? (Protein never entered the cell)
• Pellet?? (DNA injected into the cell)
3 Chargaff’s Rules
• Erwin Chargaff (1947) provides more evidence that DNA = genetic
material
– Analysis of base composition of DNA compared between different
organisms
• Nitrogenous bases
– Adenine (A)
– Thymine (T)
– Guanine (G)
– Cytosine (C)
– Conclusions of Chargaff
• DNA composition is species specific
• The amounts of A,G,C and T are not the same between species
– Ratios of nitrogenous bases vary between species
Chargaff’s Rules

– This diversity strengthened argument that DNA


is the molecular basis of inheritance
– Chargaff’s Rules
• Amount of A = T
• Amount of G = C
4 X-Ray Crystallography

• The Race is On
– Linus Pauling
– Maurice Wilkins and Rosalind Franklin
– Watson and Crick
• X-ray Crystallography defined
– Diffracted X-rays as they pass through a crystallized
substance
– Patterns of spots are translated by mathematical
equations to define 3-D shape
X-ray Diffractometer
X-Ray Crystallography

• Rosalind Franklin’s data provide clues about DNA’s 3-D


shape
– Helix
– Width = 2 nm  probably two strands (DOUBLE
HELIX)
– Nitrogenous bases = 0.34 nM apart
– One turn every 3.4 nM (10 base pairs per turn)

X-Ray Crystallography Data Provides


James Watson and Francis Crick with
Insight into DNA Structure
X-Ray Crystallography

• The arrangement of the three major


components in nucleic acid polymers was
already well known – but the 3-D shape was
still unclear
– Sugar phosphate backbone
– Bases
Watson and Crick Deductions

• Putting the hydrophobic nitrogenous bases on the inside, and the sugar-
phosphate groups on the outside was a stable arrangement

• Base pairing was worked out by trial and error


– The distance between the sugar-phosphate backbone groups is
constant
• Therefore purine-purine or pyrimidine-pyrimidine were not
allowed because spacing would be in inconsistent with data
– Purines = A and G (two organic rings)
• Pyrimidines – C and T ( one organic ring)

• Hydrogen bonding between purines and pyrimidines established


the appropriate pairs and reinforced Chargaff’s Rules
– 2 hydrogen bonds between
A and T
– 3 hydrogen bonds between
G and C
Nature 171: 737-738 – April 1953

• Watson JD and Crick FC (1953) Molecular


Structure of Nucleic Acids: A Structure for
Deoxyribose Nucleic Acid.

• 1962 – Nobel Prize awarded to three men –


Watson, Crick and Wilkins
James Watson (L) and Francis Crick (R), and the model they

built of the structure of DNA


DNA and
RNA Structure

• Components
•Sugar
•Base
•Phosphate
• 5’ to 3’ direction
• T->U in RNA
• RNA - extra –OH at 2’
of pentose sugar
• DNA deoxyribose
The 5 Bases Purines

of DNA and RNA

• Pyrimadines and Purines


• T->U in RNA
• Names
• Numbering
• Bonding character
• Position of hydrogen
• Tautomers

Pyrimadines
BASE-PAIRINGS
H-bonds

G C

T A
DNA Double Helix
5 O 3

3 O 5
P P
5 O
1
G C 2 3
4 4
2 1
3 5
O
P P
3
5
O
T A
O 5
P 3 P
The diameter of the helix is 2 nm or 20 Å

2 nm
Geometry of
Watson Crick
Base Pairs
• A:T and G:C pairs are
spatially similar
• 3 H-bonds vs 2
• Sugar groups are attached
asymmetrically on the same
side of the pair
• Leads to a major and
minor grove
• Bases are flat but the
hydrogen bonding leads to
considerable flexibility
Backbone
Conformation
TAUTOMERIC FORMS OF
BASES

• TWO POSSIBILITIES
– KETO (LACTAM)
– ENOL (LACTIM)
• IMPORTANT IN ORDER TO SPECIFY
HYDROGEN BONDING RELATIONSHIPS
• THE KETO FORM PREDOMINATES
TAUTOMERIC FORMS OF BASES

KETO ENOL
Definition of Major
and Minor Groove

Hydrogen
bonding of WC
base pair

Mechanisms of
recognition The canonical Watson-Crick base pair, shown as the G-C pair. Positions of the
minor and major grooves are indicated. The glycosidic sugar-base bond is shown
by the bold line; hydrogen bonding between the two bases is shown in dashed
lines.
MAJOR AND MINOR
GROOVES
• MINOR
– EXPOSES EDGE FROM WHICH C1’ ATOMS
EXTEND
• MAJOR
– EXPOSES OPPOSITE EDGE OF BASE PAIR
• THE PATTERN OF H-BOND POSSIBILITIES IS MORE
SPECIFIC AND MORE DISCRIMINATING IN THE
MAJOR GROOVE
– STUDY QUESTION: LOCATE ALL OF THE
POSSIBILITIES FOR H-BONDING IN THE MAJOR
AND MINOR GROOVES FOR THE 4 POSSIBLE
BASE-PAIRS
STRUCTURE OF
THE DOUBLE HELIX

• THREE MAJOR FORMS


– B-DNA
– A-DNA
– Z-DNA
DNA double helix

major
groove
12 Å
one
helical
turn
34 Å minor
groove

backbone: deoxyribose and phosphodiester linkage


bases
B-DNA
B-DNA IS BIOLOGICALLY THE MOST COMMON
RIGHT-HANDED (20 ANGSTROM (A) DIAMETER)
COMPLEMENTARY BASE-PAIRING (WATSON-CRICK)
A-T
G-C
EACH BASE PAIR HAS ~ THE SAME WIDTH
10.85 A FROM C1’ TO C1’
A-T AND G-C PAIRS ARE INTERCHANGEABLE
“PSEUDO-DYAD” AXIS OF SYMMETRY

• IDEAL B-DNA HAS 10 BASE PAIRS PER TURN


• BASE THICKNESS
– AROMATIC RINGS WITH 3.4 A THICKNESS TO RINGS
• PITCH = 10 X 3.4 = 34 A PER COMPLETE TURN
• AXIS PASSES THROUGH MIDDLE OF EACH BP
• MINOR GROOVE IS NARROW
• MAJOR GROOVE IS WIDE
A-DNA
• RIGHT-HANDED HELIX
• WIDER AND FLATTER THAN B-DNA
• 11.6 BP PER TURN
• PITCH OF 34 A
• BASE PLANES ARE TILTED 20 DEGREES WITH
RESPECT TO HELICAL AXIS
– HELIX AXIS PASSES “ABOVE” MAJOR GROOVE
–  DEEP MAJOR AND SHALLOW MINOR GROOVE
• OBSERVED UNDER DEHYDRATING CONDITIONS
A-DNA
• WHEN RELATIVE HUMIDITY IS ~ 75%
– B-DNA  A-DNA (REVERSIBLE)
• MOST SELF-COMPLEMENTARY OLIGONUCLEO-
TIDES OF < 10 bp CRYSTALLIZE IN A-DNA CONF.
• A-DNA HAS BEEN OBSERVED IN 2 CONTEXTS:
– AT ACTIVE SITE OF DNA POLYMERASE (~ 3 bp )
– GRAM (+) BACTERIA UNDERGOING
SPORULATION
• RESISTANT TO UV-INDUCED DAMAGE
– CROSS-LINKING OF PYRIMIDINE BASES
Z-DNA
• A LEFT-HANDED HELIX
• SEEN IN CONDITIONS OF HIGH SALT
CONCENTRATIONS
– REDUCES REPULSIONS BETWEEN CLOSEST PHOSPHATE
GROUPS ON OPPOSITE STRANDS (8 A VS 12 A IN B-DNA)
• IN COMPLEMENTARY POLYNUCLEOTIDES WITH
ALTERNATING PURINES AND PYRIMIDINES
– POLY d(GC) · POLY d(GC)
– POLY d(AC)  POLY d(GT)
Z-DNA
• 12 W-C BASE PAIRS PER TURN
• A PITCH OF 44 DEGREES
• A DEEP MINOR GROOVE
• NO DISCERNIBLE MAJOR GROOVE
• REVERSIBLE CHANGE FROM B-DNA TO Z-
DNA IN LOCALIZED REGIONS MAY ACT AS
A “SWITCH” TO REGULATE GENE
EXPRESSION
THANK YOU

DNA