DNA structure, function and

metabolism
 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 35S-methionine =
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)
Laying the Groundwork:
Levene Investigates the
Structure of DNA

Figure 1: Nucleotides have three components.

A nucleotide consists of a phosphate group, a pentose sugar (ribose or deoxyribose), and a nitrogen-
containing base, all linked together by covalent bonds. The nitrogenous bases have two different
chemical forms: purines have two fused rings, and the smaller pyrimidines have a single ring.
Strengthening the Foundation: Chargaff Formulates His "Rules"
• whether there were any differences in DNA among different spp.
• developed a new paper chromatography method for separating
and identifying small amounts of organic material
• Chargaff reached two major conclusions (1950).
• First, nucleotide composition of DNA varies among species (the
same nucleotides do not repeat in the same order, as proposed by
Levene).
• Second, almost all DNA-no matter what organism or tissue type it
comes from-maintains certain properties, even as its composition
varies.
– the amount of adenine (A) is usually similar to the amount of thymine
(T)
– the amount of guanine (G) usually approximates the amount of
cytosine (C).
– In other words, the total amount of purines (A + G) and the total
amount of pyrimidines (C + T) are usually nearly equal.
– (This second major conclusion
is known as "Chargaff's rule.“)

No idea about A-T and G-C combination

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

• 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
– 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)
 Putting the Evidence Together: Watson and Crick
Propose the Double Helix
• Chargaff's realization that A = T and C = G
• crucially important X-ray crystallography work by English researchers Rosalind
Franklin and Maurice Wilkins,
• contributed to Watson and Crick's derivation of the three-dimensional, double-
helical model for the structure of DNA.
• Watson and Crick's discovery was also made possible by recent advances in model
building
• the assembly of possible three-dimensional structures based upon known
molecular distances and bond angles, a technique advanced by American
biochemist Linus Pauling
• Using cardboard cutouts representing the individual chemical components of the
four bases and other nucleotide subunits, Watson and Crick shifted molecules
around on their desktops, as though putting together a puzzle.
• They were misled for a while by an erroneous understanding of how the different
elements in thymine and guanine (specifically, the carbon, nitrogen, hydrogen,
and oxygen rings) were configured.
• Only upon the suggestion of American scientist Jerry Donohue did Watson decide
to make new cardboard cutouts of the two bases,
Figure 3: DNA is a double helix.
(A) Francis Crick (left) and James Watson (right) proposed that the DNA molecule has a double-helical structure.
(B) Biochemists can now pinpoint the position of every atom in a DNA molecule.
• Although scientists have made some minor changes to the Watson and
Crick model, or have elaborated upon it, since its inception in 1953, the
model's four major features remain the same yet today. These features
are as follows:
• DNA is a double-stranded helix, with the two strands connected by
hydrogen bonds. A bases are always paired with Ts, and Cs are always
paired with Gs, which is consistent with and accounts for Chargaff's rule.
• Most DNA double helices are right-handed; that is, if you were to hold
your right hand out, with your thumb pointed up and your fingers curled
around your thumb, your thumb would represent the axis of the helix and
your fingers would represent the sugar-phosphate backbone. Only one
type of DNA, called Z-DNA, is left-handed.
• The DNA double helix is anti-parallel, which means that the 5' end of one
strand is paired with the 3' end of its complementary strand (and vice
versa). Nucleotides are linked to each other by their phosphate groups,
which bind the 3' end of one sugar to the 5' end of the next sugar.
• Not only are the DNA base pairs connected via hydrogen bonding, but the
outer edges of the nitrogen-containing bases are exposed and available
for potential hydrogen bonding as well. These hydrogen bonds provide
easy access to the DNA for other molecules, including the proteins that
play vital roles in the replication and expression of DNA
Figure 4 : Base
pairing in DNA is
complementary.
The purines (A and
G) pair with the
pyrimidines (T and
C, respectively) to
form equal-sized
base pairs
resembling rungs
on a ladder (the
sugar-phosphate
backbones). The
ladder twists into a
double-helical
structure.
 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
• Other scientists have elaborated on Watson and Crick's model
-identification of three different conformations of the DNA double helix.
• The most common conformation in most living cells (which is the one
depicted in most diagrams of the double helix, and the one proposed by
Watson and Crick) is known as B-DNA.
• A-DNA, a shorter and wider form that has been found in dehydrated
samples of DNA and rarely under normal physiological circumstances;
• Z-DNA, a left-handed confirmation.
• Z-DNA is a transient form of DNA, only
occasionally existing in response to certain types
of biological activity.
• Z-DNA was first discovered in 1979, but its
existence was largely ignored until recently.
• Scientists have since discovered that certain
proteins bind very strongly to Z-DNA, suggesting that
Z-DNA plays an important biological role in
protection against viral disease
(Rich & Zhang, 2003).
• During the cell cycle, replication of DNA occurs.

•The DNA double helix unwinds through the

work of an enzyme called a helicase.
The section that opens up is called a
replication fork, because as you can see
from this model, it resembles a fork.
Another enzyme, DNA polymerase adds
complementary nucleotides to the parent
strands of DNA
Then, DNA ligase helps to wind the
strands back up into a double helix.