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13
DNA and Its Role in Heredity
13 DNA and Its Role in Heredity
13.1 What Is the Evidence that the
Gene Is DNA?
13.2 What Is the Structure of DNA?
13.3 How Is DNA Replicated?
13.4 How Are Errors in DNA Repaired?
13.5 How Does the Polymerase Chain
Reaction Amplify DNA?
13 DNA and Its Role in Heredity
Lance Armstrong’s cancer was stopped
by a drug called cisplatin that forms
linkages between DNA strands and
prevents replication.
Without DNA replication cells can’t
divide, and undergo programmed cell
death.
Opening Question:
How does cisplatin work?
13.1 What Is the Evidence that the Gene Is DNA?
By the 1920s it was known that
chromosomes consisted of DNA and
proteins.
A new dye that stained DNA provided
evidence that DNA is the genetic
material.
• It was in the right place
• It varied among species
• It was present in the right amounts
13.1 What Is the Evidence that the Gene Is DNA?
Experimental evidence came from work
on two strains of Streptococcus
pneumoniae.
A substance from cells of one strain
(even when dead) could produce a
heritable change in the other strain.
Figure 13.1 Genetic Transformation
13.1 What Is the Evidence that the Gene Is DNA?
To identify this substance, Oswald
Avery treated samples to destroy
different molecules.
If DNA was destroyed, the transforming
activity was lost.
There was no loss of activity with
destruction of proteins or RNA.
Figure 13.2 Genetic Transformation by DNA (Part 1)
Figure 13.2 Genetic Transformation by DNA (Part 2)
13.1 What Is the Evidence that the Gene Is DNA?
Hershey-Chase experiment: used
bacteriophage T2 virus to determine
whether DNA, or protein, is the
genetic material.
Part of the virus enters E. coli cells and
converts the cell into a virus
replication machine.
Figure 13.3 Bacteriophage T2: Reproduction Cycle
13.1 What Is the Evidence that the Gene Is DNA?
Bacteriophage were grown with either
35S to label the proteins, or with 32P to
label the DNA.
After infection, bacterial cells and viral
remains were separated—the bacteria
cells were labeled with 32P, indicating
that DNA had entered the cells.
Figure 13.4 The Hershey–Chase Experiment (Part 1)
Figure 13.4 The Hershey–Chase Experiment (Part 2)
13.1 What Is the Evidence that the Gene Is DNA?
Eukaryotic cells can also be
transformed (transfection).
A genetic marker (a gene that confers
an observable phenotype, such as
antibiotic resistance) is used to
demonstrate transfection.
Any cell can be transfected, even an
egg cell, resulting in a transgenic
organism.
Figure 13.5 Transfection in Eukaryotic Cells
13.2 What Is the Structure of DNA?
The structure of DNA was determined
using many lines of evidence.
One crucial piece came from X-ray
diffraction.
A purified substance can be made to
form crystals. When X-rays are
passed through it, position of atoms is
inferred from the pattern of diffraction.
Figure 13.6 X-Ray Crystallography Helped Reveal the Structure of DNA
13.2 What Is the Structure of DNA?
Rosalind Franklin prepared
crystallographs from DNA samples.
Her images suggested a doublestranded helix with 10 nucleotides in
each full turn.
The diameter of 2 nm suggested that
the sugar-phosphate backbone of
each strand must be on the outside.
13.2 What Is the Structure of DNA?
Chemical composition:
Biochemists knew that DNA is a
polymer of nucleotides.
Each nucleotide consists of
deoxyribose, a phosphate group, and
a nitrogen-containing base.
13.2 What Is the Structure of DNA?
The four different nucleotides differed
only in the bases:
• Purines: adenine (A), guanine (G)
• Pyrimidines: cytosine (C), thymine
(T)
13.2 What Is the Structure of DNA?
Erwin Chargaff noticed that in all DNA, the
amount of purines = the amount of
pyrimidines.
Chargaff’s rule
13.2 What Is the Structure of DNA?
Francis Crick and James Watson used
model building, plus the physical and
chemical evidence to solve the
structure of DNA.
They published their results in 1953.
Figure 13.7 DNA Is a Double Helix (Part 1)
13.2 What Is the Structure of DNA?
The X-ray diffraction data indicated that
the bases are on the inside and the
sugar-phosphate groups on the
outside of each strand,
and that the chains run in opposite
directions—antiparallel.
13.2 What Is the Structure of DNA?
Antiparallel chains:
13.2 What Is the Structure of DNA?
To satisfy Chargaff’s rule, the model
paired a purine on one strand with a
pyrimidine on the opposite strand,
resulting in uniform width.
Figure 13.7 DNA Is a Double Helix (Part 2)
13.2 What Is the Structure of DNA?
Four key features of DNA structure:
• It is a double-stranded helix
• It is right-handed
• It is antiparallel
• The outer edges of the bases are
exposed in major and minor
grooves
13.2 What Is the Structure of DNA?
The two chains are held together by:
1. Hydrogen bonding between bases –
complementary base pairing:
One purine (A or G) with one
pyrimidine (T or C)
In-Text Art, Ch. 13, p. 266 (1)
13.2 What Is the Structure of DNA?
2. Van der Waals forces between
adjacent bases on the same strand.
When the base rings come near one
another, they tend to stack like poker
chips.
13.2 What Is the Structure of DNA?
Antiparallel strands: direction of strand
is determined by the sugar–phosphate
bonds.
13.2 What Is the Structure of DNA?
Phosphate groups connect to the 3′ C
of one sugar, and the 5′ C of the next
sugar.
Results in one chain with a free 5′
phosphate group—the 5′ end;
The other chain has is a free 3′
hydroxyl group—the 3′ end.
Figure 4.5 DNA Replication and Transcription
13.2 What Is the Structure of DNA?
The backbones of the two DNA strands
are closer together on one side of the
double helix (forming the minor
groove) than on the other (forming the
major groove).
There are four possible configurations
of the base pairs in the grooves.
Figure 13.8 Base pairs in DNA Can Interact with Other Molecules
13.2 What Is the Structure of DNA?
The outer edges of the base pairs are
exposed and accessible for additional
hydrogen bonding.
The surfaces of the A-T and C-G base
pairs are chemically distinct.
Binding of proteins to specific base pair
sequences is the key to protein-DNA
interactions, which are necessary for
the replication and expression of
DNA.
13.2 What Is the Structure of DNA?
The double-helix structure is essential
to DNA function:
• Stores genetic information: with
millions of nucleotides, the base
sequences store a huge amount of
information
• Susceptible to mutations: alterations
in base sequences
13.2 What Is the Structure of DNA?
• Precisely replicated in cell division
by complementary base pairing
• Genetic information is expressed
as the phenotype—nucleotide
sequence determines sequence of
amino acids in proteins
13.3 How Is DNA Replicated?
The mechanism of DNA replication was
confirmed by replicating DNA in a test
tube.
Ingredients needed:
• Deoxyribonucleoside triphosphates
dATP, dCTP, dGTP, and dTTP
(dNTPs, the monomers of DNA)
13.3 How Is DNA Replicated?
• DNA molecules to serve as
templates for the sequence of
nucleotides
• DNA polymerase enzyme
• Salts and a pH buffer
These experiments confirmed that DNA
contains the information needed for
its own replication.
13.3 How Is DNA Replicated?
Three possible replication patterns:
• Semiconservative: Each parent
strand is a template; new molecules
have one old and one new strand
• Conservative: Original molecule
serves as a template only
• Dispersive: Fragments of DNA are
templates, old and new parts are
assembled into new molecules
Figure 13.9 Three Models for DNA Replication
13.3 How Is DNA Replicated?
Meselson and Stahl showed that
semiconservative replication was
the correct model:
E. coli cultures were grown with 15N (a
heavy, stable isotope that makes DNA
more dense), then transferred to a
medium with 14N.
DNA densities could only be explained
by the semiconservative model.
Figure 13.10 The Meselson–Stahl Experiment (Part 1)
Figure 13.10 The Meselson–Stahl Experiment (Part 2)
Working with Data 13.1: The Meselson–Stahl Experiment
In the Meselson–Stahl experiment,
DNA with 14N was separated from
DNA with 15N using an ultracentrifuge
to create a density gradient of cesium
chloride.
Working with Data 13.1, Figure A
Working with Data 13.1: The Meselson–Stahl Experiment
DNA bands from successive
generations of E. coli after
centrifugation.
Plots show quantitative analysis of the
bands, where height indicates amount
of DNA.
Working with Data 13.1, Figure B
Working with Data 13.1: The Meselson–Stahl Experiment
Question 1:
Use the heights of the peaks to
estimate the percent of total DNA that
was heavy, intermediate, and light at
each generational stage.
Create a table summarizing these
calculations and discuss whether they
support the authors’ conclusions.
Working with Data 13.1: The Meselson–Stahl Experiment
Question 2:
What would the data look like if the
bacteria had been allowed to divide
for three more generations?
Working with Data 13.1: The Meselson–Stahl Experiment
Question 3:
If Meselson and Stahl had done their
experiment starting with light DNA and
then added 15N for succeeding
generations, what would the bands
look like?
Draw them alongside the actual data,
above.
Working with Data 13.1: The Meselson–Stahl Experiment
Question 4:
What would the data look like if
conservative replication were the
correct model?
What would the data look like if
dispersive replication were correct?
Draw these alongside the actual data
above.
13.3 How Is DNA Replicated?
Two steps in DNA replication:
• Double helix is unwound, making
two template strands
• New nucleotides form
complementary base pairs with
template DNA and are linked by
phosphodiester bonds
13.3 How Is DNA Replicated?
Nucleotides are added to the new
strand at the 3′ end.
Formation of the phosphodiester
linkage is a condensation reaction.
Bonds linking the phosphate groups of
the triphosphate nucleosides are
broken, releasing energy that drives
the reaction.
Figure 13.11 Each New DNA Strand Grows from Its 5 End to Its 3 End (Part 1)
Figure 13.11 Each New DNA Strand Grows from Its 5 End to Its 3 End (Part 2)
13.3 How Is DNA Replicated?
DNA replication starts when a large
protein complex (pre-replication
complex) binds to a region called
origin of replication (ori).
In E. coli, DNA is unwound and
replication proceeds in both
directions, forming two replication
forks.
13.3 How Is DNA Replicated?
Eukaryote chromosomes are much
longer, and have multiple origins of
replication.
Otherwise, it would take weeks to
replicate chromosomes, which have
up to a billion base pairs.
Figure 13.12 The Origin of DNA Replication
13.3 How Is DNA Replicated?
DNA polymerase requires a primer, a
short starter strand—usually RNA.
The primer is complementary to the
DNA template and is synthesized by
an enzyme called a primase.
DNA polymerase then adds nucleotides
to the 3′ end until that section is
complete, and the primer is degraded.
Figure 13.13 DNA Forms with a Primer
13.3 How Is DNA Replicated?
DNA polymerases are larger than their
substrates, the dNTPs, and template
DNA.
The enzyme is shaped like an open
right hand—the “palm” brings the
active site and the substrates into
contact.
The “fingers” recognize the nucleotide
bases.
Figure 13.14 DNA Polymerase Binds to the Template Strand
13.3 How Is DNA Replicated?
Other proteins have roles in replication:
• DNA helicase uses energy from
ATP hydrolysis to unwind the DNA
• Single-strand binding proteins
keep the strands from getting back
together
Figure 13.15 Many Proteins Collaborate in the Replication Complex
13.3 How Is DNA Replicated?
At the replication fork DNA opens up
like a zipper in one direction.
The leading strand grows at its 3′ end
as the fork opens.
In the lagging strand the exposed 3′
end gets farther from the fork, and an
unreplicated gap forms.
Figure 13.16 The Two New Strands Form in Different Ways
13.3 How Is DNA Replicated?
Synthesis of the lagging strand occurs
in small, discontinuous stretches
called Okazaki fragments.
Each fragment requires its own primer,
synthesized by the primase.
DNA polymerase III adds nucleotides to
the 3′ end, until reaching the primer of
the previous fragment.
13.3 How Is DNA Replicated?
DNA polymerase I then replaces the
primer with DNA.
The final phosphodiester linkage
between fragments is catalyzed by
DNA ligase.
Figure 13.17 The Lagging Strand Story (Part 1)
Figure 13.17 The Lagging Strand Story (Part 2)
13.3 How Is DNA Replicated?
The enzymes of replication work very
fast and accurately.
In E. coli, new DNA is made at a rate in
excess of 1,000 base pairs per
second, with errors in fewer than one
base in a million.
13.3 How Is DNA Replicated?
DNA polymerases work fast because:
• They are processive: they catalyze
many linkages each time they bind
to DNA, rather than just one.
• The polymerase-DNA complex is
stabilized by a sliding DNA clamp,
a protein that keeps the enzyme and
DNA in close contact.
Figure 13.18 A Sliding DNA Clamp Increases the Efficiency of DNA Polymerization
13.3 How Is DNA Replicated?
In eukaryotes, the replication complex
remains stationary while the DNA
moves.
It goes into the complex as one doublestranded molecule, and emerges as
two double-stranded molecules.
13.3 How Is DNA Replicated?
Eukaryote chromosomes have
repetitive sequences at the ends
called telomeres.
In humans the sequence is TTAGGG,
repeated about 2,500 times.
It prevents the DNA repair system from
seeing the chromosome end as a
break.
13.3 How Is DNA Replicated?
On lagging strands, when the terminal
Okazaki primer is removed, no DNA
can be synthesized to replace it (no 3′
end).
The short piece of DNA is removed,
and the chromosome becomes
shorter with each replication.
After many divisions, genes may be
lost and the cell dies.
Figure 13.19 Telomeres and Telomerase
13.3 How Is DNA Replicated?
Continuously dividing cells, such as
bone marrow stem cells, have
telomerase, which catalyzes addition
of lost telomeres.
Telomerase is expressed in most
cancer cells, and is important in their
ability to keep dividing. It is a target for
anti-cancer drugs.
13.4 How Are Errors in DNA Repaired?
DNA polymerases initially make many
mistakes, and DNA can be damaged
by chemicals, UV radiation, and other
threats.
Cells have three repair mechanisms:
• Proofreading
• Mismatch repair
• Excision repair
13.4 How Are Errors in DNA Repaired?
DNA proofreading:
As DNA polymerase adds a nucleotide
to a growing strand, it can recognize
mismatched pairs.
If bases are paired incorrectly, the
nucleotide is removed.
Figure 13.20 DNA Repair Mechanisms (A)
13.4 How Are Errors in DNA Repaired?
Mismatch repair:
The newly replicated DNA is scanned
for mistakes by other proteins, and
mismatches can be corrected.
If mismatch repair fails, the DNA is
altered.
Figure 13.20 DNA Repair Mechanisms (B)
13.4 How Are Errors in DNA Repaired?
Excision repair:
Enzymes constantly scan DNA for
damaged bases—they are excised
and DNA polymerase I adds the
correct ones.
Lack of excision repair mechanisms
can lead to skin cancers.
Figure 13.20 DNA Repair Mechanisms (C)
13.5 How Does the Polymerase Chain Reaction Amplify DNA?
Principles of DNA replication were used
to develop the polymerase chain
reaction (PCR) technique.
An automated process makes multiple
copies of short DNA sequences for
genetic manipulation and research
(DNA amplification).
13.5 How Does the Polymerase Chain Reaction Amplify DNA?
A PCR mixture contains:
• A sample of double-stranded DNA
(the template)
• Two artificially synthesized primers
• The four dNTPs
• DNA polymerase that can tolerate
high temperatures
• Salts and buffer to maintain neutral
pH
13.5 How Does the Polymerase Chain Reaction Amplify DNA?
PCR amplification is a cyclical process:
• DNA strands are separated
(denatured) by heating
• Reaction is cooled to allow primers
to bind (anneal) to template strands
• Reaction is warmed to temperature
for DNA polymerase to catalyze new
strands
• The sequence is repeated many
times
Figure 13.21 The Polymerase Chain Reaction
13.5 How Does the Polymerase Chain Reaction Amplify DNA?
Base sequences at the 3ʹ ends of the
DNA strands must be known, so that
primers can be made.
The specificity of the primers is a key to
the power of PCR.
13.5 How Does the Polymerase Chain Reaction Amplify DNA?
An initial problem with PCR: the
temperature needed to denature the
DNA destroyed most DNA
polymerases.
A DNA polymerase that does not
denature at high temperatures (90°C)
was taken from a hot springs
bacterium, Thermus aquaticus.
13.5 How Does the Polymerase Chain Reaction Amplify DNA?
PCR has had a huge impact on genetic
research.
Applications range from identification of
individual persons, to detection of
diseases.
13 Answer to Opening Question
Cisplatin has a platinum atom bonded to
two chlorines and two amino groups.
The chlorines can be displaced easily by
nitrogens from guanine bases to form
strong covalent bonds.
This results in cross-linking the DNA
strands, so that replication can’t occur.
The cross-linking can’t be repaired by the
cell’s normal repair mechanisms.
Figure 13.22 Cisplatin: A Small but Lethal Molecule