1. DNA damage & repair

2. 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

3. General types of DNA damage and causes

4. Replication errors and their repair
The nature of mutations: Point mutation
1. Switch of one base for another:
purine
pyrimidine
(transition)
(transversion)
2. insertion or deletion of a nucleotide

5. Rearrangement of chromosome
Drastic changes in DNA
Deletion
Insertion
Rearrangement of chromosome
By insertion of a transposon, or aberrant actions of recombination
Process.

6. Some replication errors escape proofreading

7. 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)

8. DNA methylation to recognize the parental strain
Once activated,
MutH selectively nicks the
Unmethylated strand.

9. Directionality in mismatch repair

10. Mismatch repair system in Eukaryotics
MutS
MutL
E. coli
MSH
(MutS homolog)
MLH or PMS
Eukaryotics
Hereditary nonpolyposis colorectal cancer
(mutations in human homologes of Muts and MutL)

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

12. DNA damage spontaneous damage to generate natural base
deamination
Methylated Cs are hot spot for spontaneous mutation in vertebrate DNA

13. Base deamination leads to the formation of a spontaneous point mutation

14. 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.

15. DNA damage by UV Thymine dimer
These linked bases are incapable of base-pairing and cause
DNA polymerase to stop.

16. Mutations caused by base analogs and intercalating agents
Thymine analog
Analogs mispair to cause mistakes during replication

17. Intercalating agents Mutations caused by intercalating agents
flat molecules
Causing addition or deletion of bases during replication

18. Chemical carcinogens react with DNA and the carcinogenic effect of a chemical correlates with its mutagenicity

19. modification of guanosine
Aflatoxin can lead to a
modification of guanosine
(in tobacco smoke)

20. 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.

22. 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

23. 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).

24. (from T.A.Brown. Genetics a molecular approach)

25. Direct reversal of DNA damage
photoreactivation
Capture energy from light
breaking
covalent bond

26. Dark repair or light independent 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 damage

27. 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

28. 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.

29. Base excision repair
If a damaged base is not removed by base excision before DNA replication: a fail-safe system
oxoG:A repair

30. Excision Repair in E.coli
Damage recognised
by UvrABC, nicks
made on both sides of
dimer 5’ 3’ 3’ 5’ T T 5’ 3’ 3’ 5’ T T
Dimer removed by UvrD, a helicase T 3’ 5’ 5’ 3’
Gap filled by DNA pol I and the nick sealed by DNA ligase 3’ 5’ 5’ 3’

31. 31

32. 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)

33. 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)

34. RecA protein is essential for homologous recombination
DNA recombination
RecA protein is essential for homologous recombination
(from T.A.Brown. Genetics a molecular approach)

35. 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