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 MutS MutL
Eukaryotics
MSH
(MutS homolog)
MLH or PMS
Hereditary nonpolyposis colorectal cancer
(mutations in human homologes of Muts and MutL)

Radiation, chemical mutagens, and spontaneous damage spontaneous damage due to hydrolysis and deamination deamination
Base pair with A depurination

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 : 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.

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.

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

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

defined
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

5’
3’
5’
3’
5’
3’
5’
3’
Excision Repair in E.coli
TT
TT
3’
5’
Damage recognised by UvrABC, nicks
3’
5’ made on both sides of dimer
TT
3’
5’
Dimer removed by
UvrD, a helicase
3’
5’
Gap filled by DNA pol I and the nick sealed by DNA ligase

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