Download 25_replication2

Lecture 25: DNA mutation,
proofreading, and repair
G
C
A
T
T
A
1 nm
C
G
C
3.4 nm
G
A
T
G
C
T
A
T
A
A
T
T
A
G
A
Figure 16.7a, c
C
0.34 nm
T
(c) Space-filling model
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Lecture Outline 11/2/05
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Review DNA replication machine
Fidelity of replication and proofreading
Replicating the ends of chromosomes
Mutation
– Types of mutations
– Repair mechanisms
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DNA synthesis goes 5’ to 3’
• DNA
New strand Template strand
polymerases,
3 end
5 end
add nucleotides
to the 3 OH at
Sugar
A
T
Base
the end of a
Phosphate
growing strand
C
G
G
C
OH
A
C
OH
5 end
Figure 16.13
Nucleoside
triphosphate
P
P
Pyrophosphate
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Model for the “replication machine,” or replisome
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Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Replication overview
• Look at animations on your textbook CD
• Look again at the animation from DNAi
– http://www.dnai.org
– (go to the section on copying the code)
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DNA Polymerase III
• A complex enzyme with many subunits
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one part adds the nucleotides
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another helps it slide along the template
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another checks for mis-pairing
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Figs. from http://www.mun.ca/biochem/courses/3107
Proofreading
• Even though bases preferentially pair G-C and A-T,
the initial error rate is about 1 in 10,000.
• Many polymerases have “proofreading” ability. They
can excise an mis-paired base and try again.
• This reduces the error rate to about 1 in a million.
One polymerase subunit adds nucleotides
Another “edits” out incorrect7 bases
Fidelity of replication
Replication step
error rate
5′→3′ polymerization
1 × 105
3′→5′ proofreading
1 × 102
Strand-directed mismatch repair 1 × 102
Total error rate
1 × 109
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What happens to the lagging strand
at the very end of the chromosome?
Leaves a gap when the RNA
primer is removed
3’
5’
5’
3’
3’
5’
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The ends of eukaryotic chromosomal DNA get
shorter with each round of replication
5
Leading strand
Lagging strand
End of parental
DNA strands
3
Last fragment
Previous fragment
RNA primer
Lagging strand
5
3
Primer removed but
cannot be replaced
with DNA because
no 3 end available
for DNA polymerase
Removal of primers and
replacement with DNA
where a 3 end is available
5
3
If they get short enough,
essential genes will
eventually be deleted
Second round
of replication
5
New leading strand 3
New lagging strand 5
3
Further rounds
of replication
Figure 16.18
Shorter and shorter
daughter molecules
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Telomerase
Carries its own RNA
template
Extends the old
(template) strand
Normal synthesis of
new DNA
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What happens to the lagging strand
that the end of the chromosome?
• Telomeres contain hundreds of simple tandem repeats.
• In humans, the repeat sequence is TTAGGG
TTAGGG TTAGGG TTAGGG TTAGGG TTAGGG . . . . . . .
Lots of junk, so if the ends get slightly shorter, no essential genes are lost
• Cell lines with active telomerase live longer than those without
telomerase.
– That may be important in allowing cancer cells to continue to divide.
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Mutations and repair
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Various kinds of mutations:
Purine -> Purine or Pymimidine -> Pyrimidine: common
Purine -> Pymimidine: rare
Some mutations change the code to a new amino acid
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Types of base pair substitutions and mutations.
Others are silent
Additions and deletions
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Mutations can be caused by:
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Chemical mutagens
Ionizing radiation
Slippage during DNA replication
Spontaneous errors
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Chemical changes in one of the
nucleotide bases
After replication, new strand has an A
Deamination changes C to U
--C----G---
--U----G---
--U----A---
--T----A---
--G----C---
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UV damage (e.g. pyrimidine dimers)
UV radiation can
cause thymine
dimers
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• In nucleotide excision repair
– Enzymes cut out and replace damaged
stretches of DNA
1 A thymine dimer
distorts the DNA molecule.
2 A nuclease enzyme cuts
the damaged DNA strand
at two points and the
damaged section is
removed.
Nuclease
DNA
polymerase
3 Repair synthesis by
a DNA polymerase
fills in the missing
nucleotides.
DNA
ligase
Figure 16.17
4 DNA ligase seals the
Free end of the new DNA
To the old DNA, making the
strand complete.
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Certain bacterial mutations
cause increased mutation rates
Defect in:
Rifr mutants per 108 cells
Wild-type (mut+ )
5-10
Pol III proofreading
(mutD)
Mis-match repair
(mutS)
Base excision repair
(mutY mutM)
4000-5000
760
8200
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Mismatch repair
Here is a mis-paired base that must be
repaired:
G
T
How is the mistake recognized?
How does the mismatch repair system
know which strand is the new one and
which strand is the old one?
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MutS/L/H
Certain enzymes detect the
deformed helix that results from the
incorrect pairing
G
T
The old (template) DNA
has methyl groups in
certain places
MutS/L/H
G
T
CH3
GATC
CTAG
Cut the newly
synthesized strand
here
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G
CH3
GATC
G
DNA pol I/III
DNA Ligase
Re-synthesize DNA from the
template using the normal DNA
polymerases
CH3
G
C
GATC
CTAG
Corrected base pair
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• Various similar
mechanisms for
other types of
mutations
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