Download Mismatch repair

Chapter 10 DNA damage and repair
1. Defects in repair cause disease
2. Common types of DNA damage
3. DNA repair pathways
Direct repair
Base excision repair
Nucleotide excision repair
Mismatch repair
Recombination repair
SOS response
DNA Damage
• The vast majority of DNA damage affects
the primary structure of the double helix
• Occurs at a rate of 1,000 to 1,000,000
molecular lesions per cell per day, only
0.000165% of the human genome's
approximately 6 billion bases (3 billion base
pairs)
DNA repair defects cause disease
Damage from where?
•
•
•
•
Consequences of DNA replication errors
Chemical agents acting on the DNA
UV light imparting energy into DNA molecule
Spontaneous changes to the DNA
Common types of DNA damage -- 1
1. Depurination （脱嘌呤） : A, G
2. Deamination （脱胺作用） : C --> U, A -->
Hypoxanthine （次黄嘌呤）
DNA level view of the same two events as last slide
06_25_mutations.jpg
Common types of DNA damage -- 2
Pyrimidine dimers 嘧啶二聚体 (UV induced).
Common types of DNA damage -- 3
Two carcinogens that
mutate (the P53 gene)
by base alkylation 烷化
+ Mismatches (mistakes in
DNA synthesis)
Interstrand cross-links,
Double-strand DNA breaks
Total damage from all mechanisms: 104 - 106 lesions/day!
DNA repair
• Damaged DNA must be repaired
• If the damage is passed on to subsequent
generations, then we use the evolutionary
term - mutation. It must take place in the
germ cells - the gametes - eggs and sperm
• If damage is to somatic cells (all other cells of
the body bar germ cells) then just that one
individual is affected.
Why repair DNA?
• DNA pol does a great job, but not good enough
• Introduces errors in about 1 in 10E7 nucleotides
added, which it does not correct
• Other mechanisms exist (as we will see) to correct
many of the errors left by the replication system
• Most mistakes and damage corrected (99% leaving just a few - only 1 in 10E9 errors are left)
• Mutations are permanent changes left in the DNA
Diverse DNA repair systems
General mechanisms shared in eukaryotes
1. Direct repair, 直接修复e.g. pyrimidine dimers
2. Base excision repair 碱基切除修复
3. Nucleotide excision repair 核苷酸切除修复
4. Mismatch repair 错配修复
5. Recombination repair 重组修复
6. SOS response （SOS反应）
• Two thymines
connected together
by UV light.
• Photoreactivation
(bacteria, yeast,
some vertebrates not humans)
DNA photolyase
Diverse DNA repair systems
•General mechanisms shared in eukaryotes
1. Direct repair, 直接修复e.g. pyrimidine dimers
2. Base excision repair 碱基切除修复
3. Nucleotide excision repair 核苷酸切除修复
4. Mismatch repair 错配修复
5. Recombination repair 重组修复
6. SOS response （SOS反应）
Base excision repair
Damaged base
Base excision repair pathway (BER).
(a) A DNA glycosylase recognizes a
damaged base and cleaves between the
base and deoxyribose in the backbone.
(b) An AP endonuclease cleaves the
phosphodiester backbone near the AP
site.
(c) DNA polymerase I initiates repair
synthesis from the free 3’ OH at the
nick, removing a portion of the damaged
strand (with its 5’3’ exonuclease
activity) and replacing it with
undamaged DNA.
(d) The nick remaining after DNA
polymerase I has dissociated is sealed
by DNA ligase.
AP= apurinic (脱嘌呤 ) or
apyrimidinic(脱嘧啶 )
(a=without)
A DNA glycosylase initiates base excision repair
Damaged base
Examples of bases cleaved
by DNA glycosylases:
Uracil (deamination of C)
8-oxoG paired with C (oxidation of G)
Adenine across from 8-oxoG
(misincorporation)
Thymine across from G (5-meC
deamination)
Alkyl-adenine (3-meA, 7-meG,
hypoxanthine)
Diverse DNA repair systems
• Augment DNA polymerase proofreading
• Mostly characterized in bacteria
• General mechanisms shared in eukaryotes
1. Direct repair, e.g. pyrimidine dimers
2. Base excision repair
3. Nucleotide excision repair
4. Mismatch excision repair
5. Recombination repair
Nucleotide excision repair
UvrA recognizes
bulky lesions
UvrB and
UvrC make cuts
Structural distortion = signal
UvrD
(a) Two excinucleases (excision endonucleases) bind DNA at the site of bulky
lesion. (b) One cleaves the 5’ side and the other cleaves the 3’ side of the lesion,
and the DNA segment is removed by a helicase. (c) DNA polymerase fills in the
gap and (d) DNA ligase seals the nick.
Nucleotide excision repair -- eukaryotes
Mutations in any of at
least seven genes, XP-A
through XP-G, cause an
inherited sensitivity to
UV-induced skin cancer
called xeroderma
pigmentosum. The XP
proteins are among >30
required for nucleotide
excision repair.
Two pathways of increasing complexity
Base
Excision
repair
Nucleotide
Excision
repair
Diverse DNA repair systems
• Augment DNA polymerase proofreading
• Mostly characterized in bacteria
• General mechanisms shared in eukaryotes
1. Direct repair, e.g. pyrimidine dimers
2. Base excision repair
3. Nucleotide excision repair
4. Mismatch excision repair -- replication errors
5. Recombination repair
Mismatch repair
Which strand is new and which is the parent?
Mut S binds mismatch
Mut L links S to H
Mut H recognizes the parental strand
Mismatch repair
Which strand is new and which is the parent?
The mutation is in the new strand!
-CH3 marks the parental strand!
MutH - Binds 7-meGATC
MutS - Binds mismatch
MutL - links MutH and MutS
Mismatch repair -- Recognition
Which strand is new and which is the parent?
The mutation is in the new strand!
A-CH3 marks the parental strand!
MutS - Binds mismatch
MutL - links MutH and MutS
MutH - Binds GmeATC
DNA is threaded through the
MutS/MutL complex. The complex
moves simultaneously in both
directions along the DNA until it
encounters a MutH protein bound at
a hemimethylated GATC sequence.
MutH cleaves the unmethylated
strand on the 5’ side of the G in the
GATC sequence.
Mismatch repair -- Resolution
1. The combined action of DNA helicase II, SSB, and one of many
different exonucleases (only two are labeled) removes a segment of
the new strand between the MutH cleavage site and a point just
beyond the mismatch.
2. The resulting gap is filled in by DNA polymerase III, and the nick is
sealed by DNA ligase.
Dam methylation
The bacterial can discriminate between old and new replicated
strands.
Old strands are methylated at adenine in -GATC- sequences
by an enzyme called the Dam methylase.
So the mismatch on the newly replicated strand is
preferentially excised.
Mismatch repair systems are present in human cells and
defects lead to colon cancer.
Mismatch repair -- Hereditary Non-Polyposis Colon
Cancer (HNPCC) gene (Humans)
遗传性非息肉病性结肠癌
HNPCC results from mutations in genes involved in DNA
mismatch repair, including:
• several different MutS homologs
• Mut L homolog
• other proteins: perhaps they play the role of MutH, but
not by recognizing hemi-methylated DNA (no 6meA
GATC methylation in humans, no dam methylase)
Diverse DNA repair systems
• Augment DNA polymerase proofreading
• Mostly characterized in bacteria
• General mechanisms shared in eukaryotes
1. Direct repair, e.g. pyrimidine dimers
2. Base excision repair
3. Nucleotide excision repair
4. Mismatch excision repair -- replication errors
5. Recombination repair
Thymine
dimer
3’
5’
Recombinational Repair
3’
5’
The gap in the undamaged
parental strand is filled by DNA
pol I and ligase. The thymine
dimer can now be repaired by
excision repair
5’
3’
Thymine
dimer
3’
5’
Thymine
dimer
5’
5’
3’
3’
3’
5’
3’
5’
3’
5’
SOS response
• The SOS response is a global response to DNA
damage in which the cell cycle is arrested and DNA
repair and mutagenesis are induced.
• The SOS uses the RecA protein (Rad51 in eukaryotes).
• During normal growth, the SOS genes are negatively
regulated by LexA repressor protein dimers .
• Activation of the SOS genes occurs after DNA damage
by the accumulation of ssDNA regions generated at
replication forks, where DNA polymerase is blocked.
The SOS response
In response to extensive genetic damage there is a regulatory system
that co-ordinates the bacterial cell response. This results in the
increased expression of >30 genes, involved in DNA repair, these
include:
recA
- activator of SOS response, recombination
sfiA (sulA) - a cell division inhibitor (repair before
replication)
umuC, D
- an error prone bypass of thymine dimers
(loss of fidelity in DNA replication)
uvrA,B,C,D - excision repair
The SOS response is regulated by two key genes:
recA & lexA
Gene mutation
• Mutations are heritable permanent changes
in a genomic sequence.
• Mutation can be caused by
– spontaneous （自发的） errors in DNA replication
or meiotic recombination；
– radiation, viruses, transposons and mutagenic
chemicals;
– by the organism itself, by cellular processes
such as hypermutation.
Classification of mutation types
• By effect on structure
– Small-scale mutations
– Large-scale mutations in chromosomal structure
• By effect on function
–
–
–
–
Loss-of-function mutations
Gain-of-function mutations
Lethal mutations
A back mutation or reversion
• By effect on fitness
– A harmful mutation
– A beneficial mutation
• By impact on protein sequence
–
–
–
–
A frameshift mutation
A nonsense mutation
A Missense mutations
A neutral mutation
By impact on protein sequence
•
•
•
•
A frameshift mutation
A nonsense mutation
A Missense mutations
A neutral mutation
A frameshift mutation
• A frameshift mutation is a mutation caused
by insertion or deletion of a number of
nucleotides that is not evenly divisible by
three from a DNA sequence
Mutations: Insertion
A frame shift mutation
Normal gene
GGTCTCCTCACGCCA
↓
CCAGAGGAGUGCGGU
Codons
↓
Pro-Glu-Glu-Cys-Gly
Amino acids
Addition mutation
GGTGCTCCTCACGCCA
↓
CCACGAGGAGUGCGGU
↓
Pro-Arg-Gly-Val-Arg
Mutations: Deletions
A frame shift mutation
Normal gene
GGTCTCCTCACGCCA
↓
CCAGAGGAGUGCGGU
Codons
↓
Pro-Glu-Glu-Cys-Gly
Amino acids
Deletion mutation
GGTC/CCTCACGCCA
↓
CCAGGGAGUGCGGU
↓
Pro-Gly-Ser-Ala-Val
A nonsense mutation
• A nonsense mutation is a point mutation in a
sequence of DNA that results in a premature stop
codon, or a nonsense codon in the transcribed
mRNA
DNA:
5' - ATG ACT CAC CGA GCG CGA AGC TGA - 3'shut
3' - TAC TGA GTG GCT CGC GCT TCG ACT –5'
mRNA:
5' - AUG ACU CAC CGA GCG CGA AGC UGA - 3'
Protein:
Met Thr His Arg Ala Arg Ser Stop
DNA:
5' - ATG ACT CAC TGA GCG CGA AGC TGA - 3'shut
3' - TAC TGA GTG ACT CGC GCT TCG ACT –5'
mRNA:
5' - AUG ACU CAC TGA GCG CGA AGC UGA - 3'
Protein:
Met Thr His Stop
A Missense mutations
• A missense mutation is a point mutation in
which a single nucleotide is changed,
resulting in a codon that codes for a different
amino acid (mutations that change an amino
acid to a stop codon are considered
nonsense mutations, rather than missense
mutations).
A Missense mutations
Normal gene
GGTCTCCTCACGCCA
↓
CCAGAGGAGUGCGGU
Codons
↓
Pro-Glu-Glu-Cys-Gly
Amino acids
Substitution mutation
GGTCACCTCACGCCA
↓
CCAGUGGAGUGCGGU
↓
Pro-Arg-Glu-Cys-Gly
A neutral mutation
• A neutral mutation is a mutation that has no
effect on fitness.
• Many or even most mutations to noncoding
DNA are neutral.
Point mutation: a single base change
CATTCACCTGTACCA
GTAAGTGGACATGGT
transition (T-A to C-G)
CATCCACCTGTACCA
GTAGGTGGACATGGT
normal sequence
transversion (T-A to G-C)
CATGCACCTGTACCA
GTACGTGGACATGGT
base pair substitutions
转换 transition: pyrimidine to pyrimidine, purine to purine
颠换 transversion: pyrimidine to purine
Deletion/ insertion
CATTCACCTGTACCA
GTAAGTGGACATGGT
deletion
CATCACCTGTACCA
GTAGTGGACATGGT
normal sequence
insertion
CATGTCACCTGTACCA
GTACAGTGGACATGGT
deletions and insertions can involve one
or more base pairs
Inversion
CATTCACCTGTACCA
GTAAGTGGACATGGT
CATCTACCTGTACCA
GTAGATGGACATGGT
normal sequence
The consequence of mutation
• Have no effect
• Alter the product of a gene
• Prevent the gene from functioning properly or
completely
• About 70 percent of these mutations having
damaging effects
06_19_sickle_cell.jpg
Consider…
• Sunlight - sunbathing or daily exposure
– Impact of ozone depletion 臭氧层破坏
– Impact on different skin tones
• Environmental degradation 劣化
Evolution acts on mutations
• If we did not have mutation then we would all
be the same!
• Any changes in the environment would be
deleterious to all members of the population
equally
• But mutation does exist and it is supported by
comparison of related organisms…
请大家自学突变剂的作用