1 lecture 25: dna mutation, proofreading, and repair figure 16.7a, c (c) space-filling model c t a a...
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![Page 1: 1 Lecture 25: DNA mutation, proofreading, and repair Figure 16.7a, c (c) Space-filling model C T A A T C G GC A C G A T A T AT T A C T A 0.34 nm 3.4 nm](https://reader036.vdocument.in/reader036/viewer/2022062714/56649d3f5503460f94a17e94/html5/thumbnails/1.jpg)
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Lecture 25: DNA mutation, proofreading, and repair
Figure 16.7a, c (c) Space-filling model
C
T
A
A
T
CG
GC
A
C G
AT
AT
A T
TA
C
TA0.34 nm
3.4 nm
G
1 nm
G
T
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Lecture Outline 11/2/05
• Review DNA replication machine
• Fidelity of replication and proofreading
• Replicating the ends of chromosomes
• Mutation– Types of mutations– Repair mechanisms
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Figure 16.13
New strand Template strand5 end 3 end
Sugar A TBase
C
G
G
C
A
C
T
PP
P
OH
P P
5 end
Pyrophosphate
Phosphate
DNA synthesis goes 5’ to 3’
• DNA polymerases, add nucleotides to the 3 OH at the end of a growing strand
Nucleosidetriphosphate
OH
P
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4Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Model for the “replication machine,” or replisome
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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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6Figs. from http://www.mun.ca/biochem/courses/3107
DNA Polymerase III• A complex enzyme with many subunits• one part adds the nucleotides• another helps it slide along the template• another checks for mis-pairing
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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 incorrect bases
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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?
3’
5’
Leaves a gap when the RNA primer is removed
5’
3’
3’
5’
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10Figure 16.18
End of parentalDNA strands
Leading strandLagging strand
Last fragment Previous fragment
RNA primer
Lagging strand
Removal of primers andreplacement with DNAwhere a 3 end is available
Second roundof replication
New leading strand
New lagging strand 5
Further roundsof replication
Shorter and shorterdaughter molecules
5
3
5
3
5
3
5
3
3
Primer removed butcannot be replacedwith DNA becauseno 3 end available
for DNA polymerase
The ends of eukaryotic chromosomal DNA get shorter with each round of replication
If they get short enough, essential genes will eventually be deleted
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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.
Additions and deletions
Others are silent
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Mutations can be caused by:
• Chemical mutagens
• Ionizing radiation
• Slippage during DNA replication
• Spontaneous errors
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--C-----G---
--U-----A---
--U-----G--- --G---
--C---
--T-----A---
Deamination changes C to U
After replication, new strand has an A
Chemical changes in one of the nucleotide bases
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UV damage (e.g. pyrimidine dimers)
UV radiation can cause thymine dimers
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![Page 20: 1 Lecture 25: DNA mutation, proofreading, and repair Figure 16.7a, c (c) Space-filling model C T A A T C G GC A C G A T A T AT T A C T A 0.34 nm 3.4 nm](https://reader036.vdocument.in/reader036/viewer/2022062714/56649d3f5503460f94a17e94/html5/thumbnails/20.jpg)
20Figure 16.17
Nuclease
DNApolymerase
DNAligase
A thymine dimerdistorts the DNA molecule.1
A nuclease enzyme cutsthe damaged DNA strandat two points and thedamaged section isremoved.
2
Repair synthesis bya DNA polymerasefills in the missingnucleotides.
3
DNA ligase seals theFree end of the new DNATo the old DNA, making thestrand complete.
4
• In nucleotide excision repair– Enzymes cut out and replace damaged
stretches of DNA
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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)
4000-5000
Mis-match repair
(mutS)
760
Base excision repair
(mutY mutM)
8200
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Mismatch repairHere is a mis-paired base that must be
repaired:
GT
How does the mismatch repair system know which strand is the new one and which strand is the old one?
How is the mistake recognized?
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GT
MutS/L/H
GT GATC
CTAG
CH3
MutS/L/H
The old (template) DNA has methyl groups in certain places
Certain enzymes detect the deformed helix that results from the incorrect pairing
Cut the newly synthesized strand here
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GGATC G
CH3
CH3
GC
GATCCTAG
DNA pol I/IIIDNA Ligase
Re-synthesize DNA from the template using the normal DNA polymerases
Corrected base pair
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• Various similar mechanisms for other types of mutations