Transcript Overview of recombinant technology

DNA damage & repair
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 analogous
General types of DNA damage and causes
Replication errors and their repair
The nature of mutations: Point mutation
1. Switch of one base for another:
(transition)
(transversion)
purine
pyrimidine
2. insertion or deletion of a nucleotide
Drastic changes in DNA
Deletion
Insertion
Rearrangement of chromosome
By insertion of a transposon, or aberrant actions of recombination
Process.
Some replication errors escape proofreading
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
MutL activate MutH
Embracing mismatch;
Inducing a kick in DNA;
Conformational change in
MutS itself
Nicking is followed by Helicase (UvrD) and one of exonucleases
(III)
DNA methylation to recognize the parental strain
Once activated,
MutH selectively nicks the
Unmethylated strand.
Directionality in mismatch repair
Mismatch repair system in Eukaryotics
E. coli
Eukaryotics
MutS
MutL
MSH
MLH or PMS
(MutS homolog)
Hereditary nonpolyposis colorectal cancer
(mutations in human homologes of Muts and MutL)
DNA damage
Radiation, chemical mutagens, and spontaneous damage
spontaneous damage due to hydrolysis and deamination
deamination
Base pair with A
depurination
DNA damage
spontaneous damage to generate natural base
deamination
Methylated Cs are hot spot for spontaneous mutation in vertebrate DNA
Base deamination leads to the
formation of a spontaneous point
mutation
Damaged by alkylation and oxidation
Alkylation at the oxygen of carbon atom 6 of G : O6-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.
DNA damage by UV
Thymine dimer
These linked bases are incapable of base-pairing and cause
DNA polymerase to stop.
Mutations caused by base analogs and intercalating agents
Base analogs
Thymine analog
Analogs mispair to cause mistakes during replication
Mutations caused by intercalating agents
Intercalating agents
flat molecules
Causing addition or deletion of bases during replication
Chemical carcinogens react with DNA and
the carcinogenic effect of a chemical
correlates with its mutagenicity
Aflatoxin can lead to a
modification of guanosine
(in tobacco smoke)
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.
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 REPAIR
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).
(from T.A.Brown.
Genetics a molecular
approach)
Direct reversal of DNA damage
photoreactivation
Capture energy from light
breaking
covalent bond
Dark repair or light independent
mechanisms
3 mechanisms:
1. Excision repair – removal of damaged
DNA strand followed by DNA synthseis
2. Recombinational repair - using other
duplexes for repair.
3. SOS error-prone ‘repair’ – tolerance of
DNA damage
Dark repair processes are defined
by mutations in key genes
uvrA, uvrB, uvrC, uvrD - excision repair
recA, recB, recC
- recombination,
recA, - SOS error-prone repair
polA (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 dimer
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.
Base excision repair
If a damaged base is not removed by base excision before DNA
replication: a fail-safe system
oxoG:A repair
Excision Repair in E.coli
5’
3’
5’
3’
TT
TT
5’
3’
made on both sides of
3’ dimer
5’
TT
5’
3’
3’ Damage recognised
5’ by UvrABC, nicks
Dimer removed by
3’ UvrD, a helicase
5’
Gap filled by DNA
3’ pol I and the nick
5’
sealed by DNA
ligase
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)
Base excision repair
NOT a major form
of repair of UVinduced DNA damage,
but an important form
of DNA repair
generally.
(from T.A.Brown. Genetics a
molecular approach)
Homologous
DNA recombination
RecA protein is
essential for
homologous
recombination
(from T.A.Brown.
Genetics a molecular
approach)
Summary
Both the dark repair mechanisms and photoreactivation 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 errorprone (SOS) repair
This error –prone repair mechanism is a last resort to
ensure survival