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The Structure and Function
of DNA - Part I
I. Bacterial Transformation is
Mediated by DNA
 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
– 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) recovedred 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
– Uptake of genetic material from an
external source resulting in the
acquisition of new traits (phenotype is
changed)
– Griffith’s expriment was the earliest
document evidence of 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
II. 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
 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 35Smethionine = 1st Batch
– Amino acid labeling
– DNA does not contain any sulfur atoms
 32P by growing T2-infected bacteria in 32-P
– Nucleic acid labeling
– Amino acids do not contain phosphorous
– 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)
III. 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
– This diversity strengthened argument
that DNA is the molecular basis of
inheritance
– Chargaff’s Rules
Amount of A = T
Amount of G = C
IV. X-Ray Crystallography Data Provides
James Watson and Francis Crick with Insight
into DNA Structure
 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
 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)
 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
 Putting the hydrophobic nitrogenous
bases on the inside, and the sugarphosphate groups on the outside was a
stable arrangement
 Base pairing was worked out by trial and
error
– The distance between the sugarphosphate backbone groups is constant
Therefore purine-purine or pyrimidinepyrimidine 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)
Purine-pyrimidine
base pairing
would be consistent with X-ray data
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
The Structure and Function
of DNA - Part II
DNA Replication: Utilization of
Numerous Enzymes, Proteins and
RNA Primers
I. The Molecular Mechanism of
DNA Replication
 The copying process of DNA is related
to nitrogenous base pairing rules
– Parent DNA molecule consists of two
strands
Complementary – A pairs with T; G
pairs with C
The two strands run in antiparallel
directions (DNA has polarity)
– The two DNA strands separate and
serve as templates to direct the
synthesis of “new” complementary
strands
– New nucleotides are inserted along
the template
– A pairs with T; G pairs with C
– Each nucleotide that is added is
covalently attached to the previous
one
Enzyme = DNA Polymerase
Sugar-phosphate backbone of new
strand is formed
 The linear DNA sequence exists in
many states
– Each gene has its own UNIQUE
sequence
– Knowing the sequence of one strand,
you can deduce the sequence of the
other (complementarity)
II. Three Models of DNA Replication
 Conservative model
– Parent molecule remains the same
– Completely new copy of the double
helix is made
 Semiconservative model
– Parent strands separate and serve as
templates for new strand synthesis
– Hybrid molecules are made
 Dispersive model
– New strands contain a mixture of old
molecules and newly synthesized
molecules
 Messelson and Stahl Experiment
Supports the Semi-Conservative Model
of DNA Replication
III. DNA Replication Involves a
Complex Assembly of Proteins and
Enzymes
 Human haploid genome = 3 x 109 bp
– ~>1000 X more complex than
Escherichia coli
– The Human Genome Project has
sequenced the entire genome of our
species
Worldwide effort – International
Collaborations
– The DNA synthesis phase
(interphase) during mitosis lasts only
a few hours despite its huge size
– Replication of the DNA sequence is
very accurate
Mutation rate ~1/109 errors
The Sequence of Events
1. Beginning of replication occurs at
the origins of replication
– Prokaryotic cells (e.g. E. coli) contain
only one origin
– Eukaryotic cells contain thousands of
orgins on each chromosome
– Proteins bind to origins and pry open
the two strands
– A replication bubble appears at the site of
strand separation and new DNA synthesis
Replication forks forma at the ends of
the bubbles
– Replication occurs in both directions on the
two strands
But ALWAYS in the 5’  3’ direction per
strand
– Replication bubbles fuse
2.
DNA polymerases add on new nucleotides
to the growing DNA strand
– One nucleotide is added at a time to the
3’-OH group of the previous nucleotide
– The 3’-OH group of the ribose sugar is
covalently linked to the nucleoside
triphosphate forming a phosphodiester
bond
– Two phosphate groups are liberated –
energy is released (PPi –
pyrophosphate)
3. How to resolve the replication fork
dilemma of antiparallel strands
– DNA strands have polarity
(antiparallel)
– DNA polymerase can only add new
nucleotides to the 3’ end of the
terminal deoxyribose
– Synthesis always progresses in the
5’ 3’ direction
– Leading strand is synthesized
continuously
DNA polymerase progresses as
DNA is unzipped
One continuous polymer is made
– Lagging strand is synthesized
discontinuously
Synthesized in opposite direction
Synthesized AWAY from the replication
fork
Initiated as a series of short segments
called Okazaki fragments (100-200
bases long)
Okazaki fragments are joined together
by DNA ligase (covalent phosphodiester
bonds between fragments)
4.
RNA primers are required for initiation of DNA
synthesis
– DNA polymerase can add nucleotides only to 3’OH group of an already existing nucleotide
paired to its complement on the other strand
– Q: How do things get started?
– A: RNA primers are made by an enzyme called
PRIMASE

~10 nucleotides long primers  H-bonds to
template and provides substitute for DNA
polymerase
Leading
strand requires only one
RNA primer
Lagging strand requires one RNA
primer for every Okazaki fragment
– RNA primers are removed by specific
enzymes and replaced with DNA
nucleotides
Gaps are sealed with DNA ligase
5.
Helicases and single-stranded binding
proteins are important components of DNA
synthesis
– Helicases unwind the double helix and
separate the two templates
 Smoothing of twists
 Breaking of H-bonds
– Single-stranded binding proteins stabilize
the DNA for replication
The Telomere Problem
 Eukaryotic cells have a
problem replicating the 5’
ends of daughter DNA
strands
– The ends of
chromosomes contain
100-1000 repeating
(TTAGGG)n segments
– Result: Ends of DNA get
shorter and shorter in
most dividing somatic
cells
 Older people have
shorter telomeres
 Some cells have a solution:
– Telomerase
Enzyme + RNA fragment –
catalyzes the extension of the ends
RNA = template for new telomere
pieces
– Examples:
Germ cells
Some cancerous cells
Immortalized cultured cells
The Structure and Function
of DNA – Part III
The Central Dogma
I. Breaking the Genetic Code – Finding
the Central Dogma
 An “RNA Club” organized by George
Gamow (1954)  assembled to
determine the role of RNA in protein
synthesis
 Radioactive tagging experiments
demonstrate intermediate between DNA
and protein = RNA
 Vernon Ingram’s research on sickle cell
anemia (1956) tied together inheritable
diseases with protein structure
– Link made between amino acids and
DNA
– RNA movement tracked from nucleus
to cytoplasm  site of protein
synthesis
DNA 
Transcription
RNA 
Protein
Translation
 Genetic code determined for all 20
amino acids by Marshal Nirenberg and
Heinrich Matthaei and Gobind Khorana
– Nobel Prize – 1968
3 base sequence = codon