DNA damage and repair and their role in carcinogenesis A DNA sequence can be changed by copying errors introduced by DNA polymerase during replication and by environmental agents such as chemical mutagens or radiation ID: 536175
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Slide1
DNA damage & repairSlide2
DNA damage and repair and their role in carcinogenesis
A DNA sequence can be changed by copying errors introduced by DNA polymerase during replication and by environmental agents such as chemical mutagens or radiation
If uncorrected, such changes may interfere with the ability of the cell to function
DNA damage can be repaired by several mechanisms
All carcinogens cause changes in the DNA sequence and thus DNA damage and repair are important aspects in the development of cancer
Prokaryotic and eukaryotic DNA-repair systems are analogousSlide3
General types of DNA damage and causesSlide4
1. Switch of one base for another:
purine
pyrimidine
(transition)
(transversion)
2. insertion or deletion of a nucleotide
The nature of mutations: Point mutation
Replication errors and their repairSlide5
Drastic changes in DNA
Deletion
Insertion
Rearrangement of chromosome
By insertion of a transposon, or aberrant actions of recombination
Process. Slide6
Some replication errors escape proofreadingSlide7
Mismatch repair removes errors escape proofreading
1. It must scan the genome.
2. The system must correct the mismatch accurately.
Scan DNA
Distortion in the backbone
Embracing mismatch;
Inducing a kick in DNA;
Conformational change in
MutS
itself
(III)
Nicking is followed by Helicase (UvrD) and one of exonucleases
MutL activate MutHSlide8
DNA methylation to recognize the parental strain
Once activated,
MutH selectively nicks the
Unmethylated strand.Slide9
Directionality in mismatch repairSlide10
Mismatch repair system in Eukaryotics
MutS
MutL
E. coli
MSH
(MutS homolog)
Eukaryotics
MLH or PMS
Hereditary nonpolyposis colorectal cancer
(mutations in human homologes of Muts and MutL)Slide11
DNA damage
Radiation, chemical mutagens, and spontaneous damage
deamination
spontaneous damage due to hydrolysis and deamination
Base pair with A
depurinationSlide12
DNA damage
spontaneous damage to generate natural base
deamination
Methylated Cs are hot spot for spontaneous mutation in vertebrate DNASlide13
Base deamination leads to the formation of a spontaneous point mutationSlide14
Damaged by alkylation and oxidation
Alkylation at the oxygen of carbon atom 6 of G : O
6
-metylguanine,
often mispairs with T.
Oxidation of G generates oxoG, it can mispair with A and C. a G:C to T:A transversion is one of the most common mutation in human cancers.Slide15
DNA damage by UV
Thymine dimer
These linked bases are incapable of base-pairing and cause
DNA polymerase to stop.Slide16
Mutations caused by base analogs and intercalating agents
Base analogs
Analogs mispair to cause mistakes during replication
Thymine analogSlide17
Mutations caused by intercalating agents
Intercalating agents
flat molecules
Causing addition or deletion of bases during replicationSlide18
Chemical carcinogens react with DNA and the carcinogenic effect of a chemical correlates with its mutagenicitySlide19
Aflatoxin can lead to a
modification of guanosine
(in tobacco smoke)Slide20
DNA damage by UV light
The killing spectrum of UV light coincides with the peak absorbance of DNA for UV light, suggesting that DNA is the key macromolecule that is damaged.
UV light causes dimerization of 2 adjacent pyrimidine (thymines).
There are 2 forms of the dimer
a, cyclobutane dimer (most lethal form)
b, 6-4 photoproduct (most mutagenic form)
Both DNA lesions are bulky and distort the double helix
The thymine dimers block transcription and replication, and are lethal unless repaired.Slide21Slide22
UV survival curves
The UV survival curve for both mutant and wild-type indicates that there are repair systems to deal with UV –damaged induced DNA.
2 key observations
:
UV-irradiated bacteria if exposed to visible light showed an increased survival relative to those not exposed to visible light –
PHOTOREACTIVATION
UV-irradiated bacteria if held in non-nutrient buffer for several hours in the dark, also showed enhanced survival relative to controls which had not –
LIQUID HOLDING RECOVERY or DARK REPAIRSlide23
Photoreactivation
repair
The enhanced survival of UV-irradiated bacteria following exposure visible light is now known to be due to PHOTOLYASE, an enzyme that is encoded by
E. coli
genes
phrA
and
phrB
.
This enzyme binds to
pyrimidine dimers
and uses energy from visible light (370 nm) to split the dimers apart.
Phr-
mutants were defective at photoreactivation.
Similar enzymes are found in other bacteria, plants and eukaryotes (but not present in man).Slide24
(from T.A.Brown. Genetics a molecular approach)Slide25
Direct reversal of DNA damage
photoreactivation
breaking
covalent bond
Capture energy from lightSlide26
Dark repair or light independent mechanisms
3 mechanisms
:
Excision repair – removal of damaged DNA strand followed by DNA
synthseis
Recombinational
repair - using other duplexes for repair.
SOS error-prone ‘repair’ – tolerance of DNA damageSlide27
Dark repair processes are
defined
by mutations in key genes
uvrA
,
uvrB
,
uvrC
,
uvrD - excision repair
recA, recB, recC - recombination,
recA, - SOS error-prone repairpolA
(DNA pol I)All are very sensitive to UV light
uvrA-
recA
-
mutants are totally defective
at dark repair and are killed by the presence of
just one pyrimidine dimerSlide28
Excision repair
In this form of repair the gene products of the E. coli
uvrA
,
uvrB
and
uvrC
genes form an enzyme complex that physically cuts out (excises the damged strand containing the pyrimidine dimers.
An incision is made 8 nucleotides (nt) away for the pyrimidine dimer on the 5’ side and 4 or 5 nt on the 3’ side.. The damaged strand is removed by
uvrD
, a helicase and then repaired by DNA pol I and DNA ligase.
Is error-free.Slide29
Base excision repair
If a damaged base is not removed by base excision before DNA replication: a fail-safe system
oxoG:A repairSlide30
T
T
T
T
Damage recognised
by UvrABC, nicks
made on both sides of
dimer
T
T
Dimer removed by UvrD, a helicase
Gap filled by DNA pol I and the nick sealed by DNA ligase
Excision Repair in
E.coli
5’
3’
3’
5’
5’
3’
5’
3’
5’
3’
3’
5’
3’
5’
3’
5’Slide31Slide32
Excision repair
The UvrABC complex is referred to as an exinuclease.
UvrAB proteins identify the bulky dimer lesion, UvrA protein then leaves, and UvrC protein then binds to UvrB protein and introduces the nicks on either side of the dimer.
In man there is a similar process carried out by 2 related enzyme complexes: global excision repair and transcription coupled repair.
Several human syndromes deficient in excision repair, Xeroderma pigmentosum, Cockayne Syndrome, and are characterised by extreme sensitivity to UV light (& skin cancers)Slide33
Base excision repair
NOT a major form
of repair of UV-induced DNA damage, but an important form of DNA repair generally.
(from T.A.Brown. Genetics a molecular approach)Slide34
Homologous
DNA recombination
RecA protein is essential for homologous recombination
(from T.A.Brown. Genetics a molecular approach)Slide35
Summary
Both the dark repair mechanisms and photo-reactivation are very accurate and can deal with low levels of DNA damage.
However, extensive damage levels to elevated levels of excision and recombinational repair, and also the activation of another repair system which is error-prone (SOS) repair
This error –prone repair mechanism is a last resort to ensure survival