Chapter 11 DNA replication, repair
and recombination
DNA复制、修复及重组
Overview
„ Brief
introduction
„ DNA replication
„ DNA repair
„ DNA recombination
DNA replication is essential for life
Introduction
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Cells divide and make copies
DNA must be copied
„ DNA replication
Must be accurate
„ DNA repair
Evolutionary change
„ DNA recombination
Short on DNA structure
“It has not escaped our notice
that the specific basepairing we
have postulated immediately
suggests a possible copying
mechanism for the genetic
material”
- Watson and Crick, 1953
Basepairing:
Adenine (A) binds thymine (T)
Guanine (G) binds cytosine (C)
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Basepairing enables DNA replication
DNA replication
Each strand a template
for synthesizing a
complementary strand
DNA polymerase 聚合酶 at the heart of
replication
Bacterial DNA Polymerases
Synthesize DNA using a template
Add nucleotides to 3’ end
of growing DNA chain
Require:
- DNA template 模板
- The four nucleoside triphosphates
外切
三磷酸核苷
外切
- Primer 引物
Li et al. 1998
DNA Polymerase III
Semiconservative replication
- One new strand and one conserved strand
全保留
半保留
散布式
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Multiple Origins in Eukaryotes
Replication begins at replication origins
- Local areas where the stable DNA helix is opened up
复制原点
Helicase 解螺旋酶 breaks
hydrogen bonds
DNA polymerase
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Replication forks 复制叉
Due to chromosome size and replication fork migration rate,
eukaryotic chromosomes tend to have many origins for DNA
replication
The new strand is synthesized 5’→3’
- Replication moves in both directions (bidirectional)
Opposite directions
DNA polymerase at work
Leading and
Lagging Strands
Results in problem at replication fork
- Assymetrical with leading and lagging strand
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Lagging strand
后随链
Leading strand
前导链
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Discontinuous synthesis
discovered by Okazaki
Pulse-chase experiment
revealed that new DNA was
initially small pieces but
became very large with time
冈崎片断
DNA polymerase synthesize 5’→3’
Problem at lagging strand!
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DNA polymerase is self-correcting
- Through error-correcting activity called proofreading
校正阅读
- 3’- to 5’ exonuclease activity
More important replication components
- Topoisomerase to avoid tension build-up
DNA拓扑异构酶
DNA polymerase in work...
Topoisomerase
Proofreading detects mispaired
nucleotide and removes it (3’- to 5’)
Correct nucleotide added and
polymerase continues work...
Helicase
Events at the replication fork
More important replication components
- Summary
- Single-strand binding protein prevents base pair re-forming
- Sliding clamp keeps polymerase attached to DNA template
滑动夹
单链结合蛋白
Accuracy in replication important
DNA repair
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Changes in DNA sequence
often detrimental
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Sickle cell anemia as an
example
Permanent change –
mutation
At the same time
important for evolution!
Need for DNA repair
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DNA mismatch repair system
DNA replication is very accurate
- Corrects 99% of errors in the replication
Examples of error rates
US postal delivery of local first-class mail 13 late deliveries per 100 parcels
Airline luggage
1 lost bag per 200
Professional typist
1 mistake per 250 characters
Driving a car in the US
1 death per 104 people per year
DNA replication (no mismatch repair)
1 mistake per 107 nucleotides
DNA replication (with mismatch repair)
1 mistake per 109 nucleotides
Hostile 恶劣的 environment causes mutations
Sources of Induced Mutations
- Such as thermal collisions, radiation and chemicals
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Fungal toxins 真菌毒素
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Cosmic rays
UV light
Chemical pollutants
X rays
Chemicals
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Purines A and G lost
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C converted to U
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Hostile environment causes mutations
Aflatoxin (peanuts)
Industrial
Cigarette smoke
Basic mechanism of DNA repair
- Such as thermal collisions, radiation and chemicals
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DNA damage removed by different nucleases
核酸酶
2. Repair DNA polymerase fills the gap
聚合酶
3. DNA ligase seals the nick
连接酶
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How can evolution act on DNA sequences?
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DNA replication and repair is
VERY effective
Maintains genetic message over
millions of years
Humans very similar to
chimpanzees 黑猩猩 genetically
DNA recombination
~98% similar...genetically!
Genetic (DNA) recombination!
Homologous recombination fundamental
Mechanism of homologous recombination
- Homology here referring to sequence similarity
Regions with similar sequence
Site-specific recombination 位点特异性重组
- Between nonhomologous DNA sequences
- Shuffle mobile genetic elements such as transposons
- Very common ~45% of human genome
Viruses are fully mobile genetic elements
Mediates the movement of the transposon
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Sequences recognized by transposase
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Antibiotic resistance markers
Extremely small
Genes enclosed by a coat
Contain RNA or DNA
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Single-stranded
Double-stranded
Herpes simplex virus 1
单纯疱疹病毒1
Ebola virus
埃博拉病毒
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Retroviruses common in Eucaryotic cells
- DNA synthesized using RNA template (retro)
- HIV is a retrovirus
逆转录病毒
Reverse transcriptase
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