Download DNA Replication and Repair

DNA Replication and Repair
n During
cell division nucleus is
divided equally by mitosis
Cytokinesis
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Cell is split into two cells, each daughter
cell has an exact copy of the parent’s
cells DNA
Each DNA strand acts as a template to
build the complementary strand
Hydrogen bonds between
complementary bases break and DNA
unzips
DNA Replicates Semiconservatively
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Each DNA molecule is composed of
one parent strand and one newly
synthesized strand
Compare other possible models of DNA
replication with the semi-conservative
model on the next slide
Meselson & Stahl
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Experiment with E. coli (bacteria)
Grew E. coli in nutrient medium rich in
15N
Bacteria reproduced 17 generations
Same bacteria is transferred to nutrient
medium containing 14N
M & S devised a new centrifugation
method to measure DNA densities
before and after switch to light N
Experiment
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Samples of DNA were centrifuged in a
solution of cesium chloride, for many
hours at high speeds
Movement of DNA stops once the DNA
reaches the same density in the
gradient as its own
M & S hypothesized that DNA strand of
14N and one of 15N, therefore
intermediate density
Results Confirmed Hypothesis
DNA Repliction is SemiConservative
Process of DNA Replication
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First: Separating the DNA Strands
Begins when proteins bond at a specific
site on DNA
DNA helicase unwinds the double helix
by breaking the hydrogen bonds
between bases
Strands have a natural tendency to
anneal - pairing of bases
Single Stranded Binding Proteins
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SSB’s bind to the exposed DNA single
strands and block H bonding
DNA gyrase: enzyme that relieves any
tension brought about by the unwinding
of DNA in bacteria
DNA cannot be fully unwound because
of its large size
Replication Fork
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The replication fork is the junction
where the DNA strands are still joined.
When two replication forks are quite
near each other a replication bubble
forms
See figure 4 page 220
Eukaryotes
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In eukaryotes there are hundreds of
replication forks, eventually bubbles
become continuous and two new
double-stranded daughter molecules
are completely formed
Building the Complementary
Strands
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DNA Polymerase III enzyme
responsible for synthesizing
complementary strands of DNA during
DNA replication in prokaryotes
Adds complementary nucleotides in the
5’ to 3’ direction using RNA primers as
starting points
DNA Polymerase III
RNA Primer
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DNA polymerase synthesizes DNA only
in the 5’ to 3’ direction
It adds free deoxyribonucleoside
triphosphates to a 3’ end
RNA Primer is a sequence of 10 to 60
RNA bases which is annealed to the
template because DNA polymerase III
cannot initiate a complementary strand
by itself
Primase
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Primase is the enzyme that builds RNA
primers
RNA primer is removed easily once
DNA polymerase III starts elongation
Deoxyribonucleoside triphosphates are
added, the energy obtained from
breaking the bond between the P’s adds
a complementary nucleotide to the
elongating strand
Direction
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DNA is always synthesized in the 5’ to
3’ direction
Therefore only one strand can be built
continuously
Leading strand - 5’ to 3’ direction - is
built towards replication fork
Lagging Strand
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The lagging strand is the strand that is
synthesized in short fragments - known
as Okazaki fragments
DNA polymerase I is an enzyme that
removes RNA primers and replaces
them with the appropriate
deoxyribonucleosides
DNA ligase - joins the Okazaki
fragments
See figure 7 page 221
Quality Control
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Both DNA polymerase III and DNA
polymerase I act as quality control
checkers (search for bumps)
If error is detected either can act as an
exonuclease
This enzyme backtracks and excises
the incorrect nucleotide
Review
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How does the DNA strand unzip?
Hydrogen bonds between
complementary bases break
What does it mean when we say DNA
replicates semi-conservatively?
New DNA is composed of one parent
strand and one new strand
Review cont’d
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How did Meselson & Stahl demonstrate
this semi-conservative form of
replication?
They grew bacteria on different isotopes
of N and centrifuged DNA
Review Con’t
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What is the name of the enzyme that
unwinds the double helix?
DNA helicase
What keeps the individual strands from
annealing to each other?
SSB’s single-stranded binding proteins
bind to the exposed DNA strands
Review Cont’d
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What is the name of the enzyme that
relieves any tension brought about by
the unwinding of DNA?
DNA gyrase
What is a replication fork?
The junction where the DNA strands are
still joined.
Review Cont’d
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What is the name of the enzyme
responsible for synthesizing strands of
DNA during DNA replication?
DNA polymerase III
In what direction does this enzyme add
nucleotides?
It adds nucleotides in the 5’ to 3’
direction.
Review Cont’d
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What is a RNA primer?
A sequence of 10 to 60 RNA bases that
is annealed to the template.
What happens to RNA primer once
DNA polymerase III starts elongation?
RNA primer is removed easily.
Review Cont’d
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What is the difference between the
leading strand and lagging strand?
Leading strand is built continuously, the
lagging strand is built in short
fragments.
What are the short fragments in the
lagging strand called?
Okazaki fragments
Review Cont’d
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What enzyme joins the Okazaki
fragments together?
DNA ligase
How does the cell ensure that it has
made DNA with the nucleotides in the
correct sequence.
DNA polymerase III and DNA
polymerase I backtrack and search for
bumps
Experimental Methods