Richard Losick
October 8
DNA Replication
1. The Meselson-Stahl experiment
2. Chemistry of DNA synthesis
3. The replication fork
4. DNA polymerase and proofreading
5. Mutation and repair
6. Story time!
1
Goal: To understand the chemistry of DNA synthesis
and how DNA is replicated with high accuracy.
Objectives. You should be able to:
• prove that DNA replication is semi-conservative.
• diagram phosphodiester bond formation.
• explain the energetics of DNA synthesis.
• describe the replication fork.
• explain how DNA is replicated accurately.
The Meselson-Stahl experiment
“The most beautiful experiment in biology”
Frank Stahl (1958) and Matt Meselson (1968)
2
Two models for DNA replication: which is correct?
E. coli was grown in medium containing 15N (isotope
with mass of 15) for many generations.
Next, the bacteria were shifted to medium containing
14N (mass 14) for one generation.
Growing cells take up nitrogen as a nutrient from the medium
and incorporate it into protein and nucleic acids.
3
E. coli was grown in medium containing 15N (isotope
with mass of 15) for many generations.
Next, the bacteria were shifted to medium containing
14N (mass 14) for one generation.
DNA was extracted and subjected to ultracentrifugation
in cesium, which separates DNAs of light, heavy
and hybrid densities:
2. CHEMISTRY OF DNA SYNTHESIS
What are the substrates for DNA
synthesis?
4
DNA is synthesized from 2’-deoxy nucleoside
triphosphates (nucleotides) in which the
phosphates are labeled a, b and g.
What is the direction of DNA synthesis?
5
Nucleotides extend the 3’ end of the growing strand in a 5’ to
3’ direction.
Incoming dCTP is selected by pairing with the
complementary base (G) in the template strand.
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A phosphodiester bond is formed by nucleophilic
attack of the 3’ oxygen on the dCTP a-phosphate.
The leaving group for phosphodiester bond formation is
pyrophosphate.
PPi
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The leaving group for phosphodiester bond formation is
pyrophosphate.
PPi
DNA synthesis is an energetically coupled
reaction
Reaction one:
dXTP + (dXMP)n
(dXMP)n+1 + PPi
o
G rxn= moderately favorable
Reaction two:
PPi
2 Pi
o
G rxn = moderately favorable
Combined reaction:
dXTP + (dXMP)n
(dXMP)n +1 + 2Pi
o
G rxn < -7.3 kcal/mol
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The equilibrium constant (Keq)
Keq
[products]
[(XMP) n+1]  [Pi]2


[reactan ts] [XTP]  [(XMP )n ]
Gorxn = -RT ln Keq
Where R is universal gas constant and T is absolute temperature.

If Gorxn= -2.7 kcal/mol, then Keq ≈ 102 (worth remembering!)
Therefore if Gorxn = -7.3 kcal/mol, then Keq ≈ 105 which is strongly
in favor of the products.
Main points
1. DNA synthesis takes place in a 5’ to 3’ direction.
2. The 3’ oxygen of the growing chain attacks the a
phosphate of the substrate to create the
phosphodiester bond.
3. Bond formation results in release of pyrophosphate.
4. Phosphodiester bond formation is driven by
pyrophosphate hydrolysis.
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3. The Replication Fork
DNA synthesis takes place at a
moving “replication fork”.
5’ 3’
If strand growth is 5’ to 3’, then how
can BOTH strands be copied?
The fig leaf problem!
3’
5’
10
5’ 3’
Direction of
fork movement
?
3’
5’
5’
Removing the fig leaf from the replication fork
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5’ 3’
Direction of
fork movement
5’
3’
5’
The replication fork is asymmetric.
5’
DNA synthesis occurs continuously on the “leading” strand and
“discontinuously” on the “lagging” strand.
5’ 3’
Direction of
fork movement
5’
3’
5’
The replication fork is asymmetric.
5’
DNA synthesis occurs continuously on the “leading” strand and
“discontinuously” on the “lagging” strand.
In lagging strand synthesis, short stretches of DNA are synthesized,
which are stitched together by an enzyme.
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4. DNA polymerase
•
Catalyzes phosphodiester bond
formation
•
Fast
•
“Processive”
•
Proof reads its own work
DNA polymerase resembles a hand with the
catalytic center in the palm
growing strand
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14
15
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DNA polymerase is fast
DNA polymerase synthesizes DNA at rate of 800
nucleotides/second.
The chromosome of E. coli is 4.6 X106 base pairs.
DNA polymerase is fast
DNA polymerase synthesizes DNA at rate of 800
nucleotides/second.
The chromosome of E. coli is 4.6 X106 base pairs.
The chromosome is replicated from two forks, with an
overall rate is 1,600 nucleotides/second.
Ergo, it takes only ~40 min to replicate the
chromosome of E. coli.
If you are not impressed, consider this!
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Suppose the DNA duplex were one meter in diameter and
nucleotides the size of a textbook, then:
Replication would proceed at 600 km/hour.
The replication machine would be the size of a FedEx truck.
Replicating the genome would be a 40 minute, 400 km trip (like
driving to the Bronx to watch the Red Sox-Yankees game),
while delivering 1,000 textbooks per second!
DNA polymerase is processive
A sliding clamp tethers DNA polymerase to the DNA
Lecture 8
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DNA polymerase proof reads its own
work and corrects its mistakes!
The enzyme has a built-in proof-reading
nuclease that removes misincorporated
nucleotides.
The editing nuclease of DNA polymerase
If the last base was mispaired, it slips into the editing
pocket where it is removed by an exonuclease.
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Main points
DNA polymerase is:
Fast:~800 nucleotides/second
Processive: a sliding clamp keeps it
anchored to DNA
Accurate: an editing exonuclease removes
misincorporated nucleotides.
5. DNA repair
DNA must endure the slings and arrows of
outrageous fortune!
Not only is DNA subject to replication errors but
also to post-replication damage.
Cells have a squadron of “repair” proteins that
surveil the genome and mend damage.
20
Mutations arise from chemicals and radiation.
For example, ultraviolet radiation from the sun
damages pyrimidines.
Even water is mutagenic!
DNA is bathed in water at 55 molar.
Water causes hydrolytic damage.
21
An example of hydrolytic damage
uracil
cytosine
Hydrolytic attack converts cytosine to uracil via
deamination at carbon 4.
Uracil is mutagenic because it has the base pairing
specificity of thymine.
U
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Deamination leads to replacement of C:G with T:A
after two rounds of replication.
To prevent this, cells have a “DNA repair” enzyme that
detects uracil and replaces it with cytosine.
U
A
C
G
U
G
U
A
T
A
C
G
“If the DNA were copied badly, we would have
diseases such as cancer at a much higher
frequency, and we would not get a faithful copy of
our parental inheritance. Our species would not be
preserved, and we would not live long. If the DNA
were copied perfectly, there would be no room for
evolution, and the basis for creation of new species
with better environmental adaptation would have
vanished long ago. The DNA repair system allows a
happy medium.…”
Dan Koshland, former editor of Science magazine, 1994
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Story time!
Genome editing by CRISPR Cas9
Emmanuelle Charpentier
Jennifer Doudna
Ionizing radiation, such as X-rays, cause double-strand breaks in DNA
Ionizing radiation
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During mitosis identical chromosomes from DNA replication are segregated to different
daughter cells.
Cells repair breaks by “Non-Homologous End Joining” but
it is highly error prone
.
So the price for repairing a break, which if left unmended
would be catastrophic, is the introduction of mutations.
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CRISPR is an immune-like system that protects bacteria
against invading viruses.
It consists of:
• an endonuclease called Cas9 that creates doublestrand breaks in DNA
• an RNA complementary to the viral DNA that guides
the endonuclease to its target.
CRISPR Cas9 has been turned into a tool for
introducing mutations into cells of higher organisms
efficiently and at pre-determined sites.
Guided by RNA, the “Cas9” nuclease makes a double
strand break in DNA, which when repaired by the cell
leaves behind a mutation.
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