FCH 532 Lecture 3
Chapter 5: DNA
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Figure 5-2
Chemical structure of a nucleic acid.
Figure 1-16 Double-stranded
DNA.
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•Each DNA base is hydrogen
bonded to a base on the
opposite strand forming a base
pair.
•A bonds with T and G bonds
with C forming complementary
strands.
Figure 5-3 Mechanism of basecatalyzed RNA hydrolysis.
Base induced deprotonation
of 2’-OH allows nucleophilic
attack on the adjacent
phosphate group.
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Results in a cyclic
intermediate that can be
hydrolyzed into either a 2’
product or 3’ product.
Because DNA is resistant to
this type of hydrolysis (lack of
2’-OH group) is likely reason
to be carrier of genetic info.
DNA is the transforming principle
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Diplococcus pneumoniae is a pneumococcus bacterium that
causes pneumonia.
Virulent strains a gelatinous polysaccharide coating that
contains binding sites through which it infects cells.
Mutant pneumococci that lack this coating are not virulent
(nonpathogenic).
Virulent and nonpathogenic pneumococci are known as S
(smooth) and R (rough) respectively.
Experiment: In 1928, Frederick Griffith injected mice with a
mixture of live R (nonpathogenic) and heat-killed S (virulent)
pneumococci that resulted in the death of most mice.
Dead mice contained live S pneumococci.
The progeny were also S.
Transformation could take place outside the cell by mixing R
cells with cell-free extract from R cells.
What is the transforming principle?
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Figure 5-4
Pneumococci.
DNA is the transforming principle
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1944: Avery, MacLeod, and McCarty reported that the
transforming principle was DNA.
Results: chemical had all the physical and chemical properties
of DNA, no detectable proteins, unaffected by enzymes that
hydrolyze proteins and RNA, totally inactivated by enzymes that
catalyze the hydrolysis of DNA-therefore, DNA must be the
carrier of genetic info.
Eukaryotes can also be transformed by DNA.
Gene for growth hormone (polypeptide) injected into the nuclei
of fertilized mouse eggs.
Fertilized mouse eggs implanted into foster mothers resulting in
supermice.
Genetically altered known as transgenic.
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Figure 5-5 Transgenic mice.
DNA is the genetic carrier for
many bacteriophages
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Phage acts like a hypodermic needle full of the
transforming principle that is injected into the
bacterial host cell.
Tested in 1952 by Hershey and Chase.
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Figure 5-6
Bacteriophages attached to the surface of
a bacterium.
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Figure 5-7
Diagram of T2 bacteriophage injecting its
DNA into an E. coli cell.
Figure 5-8 The
Hershey-Chase
experiment.
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•Used bacteriophage T2
grown on E. coli medium
containing 32P and 35S
radioisotopes.
Radiolabeled the capsid
(no P) with 35S and labeled
the DNA (no S) with 32P.
Keto
Enol
Keto-enol tautomerism
Equilibrium between ketone or aldehyde and enol forms.
Keto and enol forms are considered tautomers of one
another.
Carbonyl (C=O) is in rapid equilibrium with enol tautomer
that has a double-bonded (C=C) adjacjent to a hydroxyl
(OH) group.
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Figure 5-9
Some possible tautomeric conversions for
bases.
Figure 5-10 X-ray diffraction photograph of a vertically
oriented Na+ DNA fiber in the B conformation taken by
Rosalind Franklin.
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•Central X-shaped pattern of
spots indicative of helix.
•Heavy black arcs on top and
bottom correspond to a
distance of 3.4 Å
•DNA structure repeats every
3.4 Å.
Double helical DNA
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Structure determined by Watson and Crick-suggested the molecular
mechanism of heredity.
Tied together several studies:
Chargaff’s rules. A = T and G = C importance was shown.
Correct tautomeric forms of the bases.
Information that DNA is a helical molecule by X-ray diffraction done by
Rosalind Franklin. This photograph allowed Watson and Crick to deduce
that DNA was helical and the planar aromatic bases form a stack of parallel
rings parallel to the fiber axis.
Fibers of DNA assume the B confirmation of DNA (B-DNA).
Based on X-ray diffraction patterns.
Double helical DNA
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Consists of 2 polynucleotide strands that wind
around a common axis with a right-handed
twist
20 Å diameter double helix.
The strands are antiparallel.
Wrapped around each other and cannot be
separated without unwinding the helix.
Bases occupy the core and sugar-phosphate
chains are on the outside.
Planes of bases are perpendicular to the helix
axis.
Each bases is hydrogen bonded to opposite
strand to form a planar base pair
(complementary base pair).
Double helical DNA
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Ideal B-DNA helix has 10 base pairs (bp) per
turn.
Helical twist of 36° per bp.
Aromatic bases have van der Waals
thickness of 3.4 Å.
Helix has a pitch (rise per turn) of 34 Å.
Figure 5-12 Watson-Crick base pairs.
Adenine pairs with thymine.
Guanine pairs with cytosine.
These base pairs are
interchangeable in the double helix
without altering the positions of the
sugar phosphate backbone.
The top edge of each base pair is
structurally distinct from the bottom
edge.
The deoxyribose residues are
asymmetric.
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Minor groove exposes the edge
from the C1’ atom (open toward
bottom)
Major groove exposes the
opposite edge of each bp.
The Watson and Crick double helix model for DNA
Forces in DNA Double Helix
• DNA Double helix is stabilized by two types of
forces:
1. H-bonds between complementary bases on opposite
strands:
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2 H-bonds in A-T pair
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3 H-bonds in G-C pair
2. Van der Waals forces and hydrophobic interactions
between “stacked bases”
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Aromatic bases have p-electrons that interact via
attractive Van der Waals forces.
Structure of DNA determines
heredity
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Watson-Crick bp structure will allow any sequence on one
polynucleotide strand as long as the opposite strand has
complementary sequence.
Each polynucleotide strand can act as the template for its
complementary strand.
In order to replicate, the parental strands must separate so that a
complementary daughter strand can be synthesized on each parent
strand.
Results in duplex (double-stranded) DNA consisting of one
polynucleotide parental strand from the parental molecule and
another from the newly synthesized daughter strand.
This is called semi-conservative replication.
Shown by the Meselson-Stahl experiment in 1958.
Increased density of DNA by labeling with 15N and monitored the
overall DNA density as a function of growth using equilibrium density
gradient centrifugation.
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Denaturation and renaturation
• Duplex DNA can be heated above a certain
temperature to separate the complementary strands
into a random coil conformation.
• Denaturation is followed by a change in the physical
properties of DNA.
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Figure 5-14 Schematic representation of the strand
separation in duplex DNA resulting from its heat
denaturation.
Denaturation is cooperative
• DNA can be monitored by UV absorbance.
• When DNA denatures, UV abs is due to aromatic
bases and increases compared to the double
stranded DNA
• Results from disruptions of electronic interactions
among nearby bases.
• This is called the hyperchromic effect.
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Figure 5-15 UV absorbance spectra of native and
heat-denatured E. coli DNA.
Denaturation is cooperative
• The hyperchromic effect takes place over a narrow
temperature range.
• Indicates that collapse of one part of the DNA duplex will
destabilize the rest of the structure (cooperative
process).
• Melting curves are used to demonstrate the stability of
the DNA double helix and determine the melting
temperature (Tm) which is the midpoint of a melting
curve.
• Tm is dependent on the
– solvent
– concentrations and types of ions
– pH
– Mole fraction of GC base pairs
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Figure 5-16 Example of a DNA melting curve.
Denaturation is cooperative
• The hyperchromic effect takes place over a narrow
temperature range.
• Indicates that collapse of one part of the DNA duplex will
destabilize the rest of the structure (cooperative
process).
• Melting curves are used to demonstrate the stability of
the DNA double helix and determine the melting
temperature (Tm) which is the midpoint of a melting
curve.
• Tm is dependent on the
– solvent
– concentrations and types of ions
– pH
– Mole fraction of GC base pairs
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Figure 5-17 Variation of the melting temperatures, Tm,
of various DNAs with their G + C content.
Denatured DNA can be renatured
• If a solution of DNA is rapidly cooled below the Tm, the
resulting DNA is only partially base paired.
• However, if the temperature is maintained at 25 ºC
belowe the Tm, the base paired regions will rearrange
until DNA completely renatures.
• These are called annealing conditions and are
important for hybridization of complementary strands of
DNA or RNA-DNA hybrid double helices.
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Figure 5-18 Partially renatured DNA.
Size of DNA molecules
• DNA molecules are very large.
• Mass can be determined by
– ultracentrifugation
– length measurements by electron microscopy
– Autoradiography-technique in which the position of a
radioactive substance in a sample is recorded by exposure
to film.
• Contour lengths - end to end lengths of stretched out
native molecules of DNA.
• Genome - complement of genetic information
• kb - kilobase pair = 1000 bp
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Figure 5-19 Electron micrograph of a T2
bacteriophage and its DNA.
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Figure 5-20 Autoradiograph
of Drosophila melanogaster
DNA.
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Table 5-2
Sizes of Some DNA Molecules.
Size of DNA molecules
• DNA is highly susceptible to mechanical damage outside
of the cell.
• Shearing forces generated by ordinary lab techniques
can result in shearing of the DNA into small pieces.