Dna damage repair
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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

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DNA damage & repair

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Dna damage repair

DNA damage & repair


Dna damage and repair and their role in carcinogenesis

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

General types of DNA damage and causes


Dna damage repair

(transition)

(transversion)

purine

pyrimidine

Replication errors and their repair

The nature of mutations: Point mutation

1. Switch of one base for another:

2. insertion or deletion of a nucleotide


Dna damage repair

Drastic changes in DNA

Deletion

Insertion

Rearrangement of chromosome

By insertion of a transposon, or aberrant actions of recombination

Process.


Dna damage repair

Some replication errors escape proofreading


Dna damage repair

Mismatch repair removes errors escape proofreading

2. The system must correct the mismatch accurately.

1. It must scan the genome.

Scan DNA

MutL activate MutH

Distortion in the backbone

Embracing mismatch;

Inducing a kick in DNA;

Conformational change in

MutS itself

Nicking is followed by Helicase (UvrD) and one of exonucleases

(III)


Dna damage repair

DNA methylation to recognize the parental strain

Once activated,

MutH selectively nicks the

Unmethylated strand.


Dna damage repair

Directionality in mismatch repair


Dna damage repair

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)


Dna damage repair

DNA damage

Radiation, chemical mutagens, and spontaneous damage

spontaneous damage due to hydrolysis and deamination

deamination

Base pair with A

depurination


Dna damage repair

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

Base deamination leads to the formation of a spontaneous point mutation


Dna damage repair

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 repair

DNA damage by UV

Thymine dimer

These linked bases are incapable of base-pairing and cause

DNA polymerase to stop.


Dna damage repair

Mutations caused by base analogs and intercalating agents

Base analogs

Thymine analog

Analogs mispair to cause mistakes during replication


Dna damage repair

Mutations caused by intercalating agents

Intercalating agents

flat molecules

Causing addition or deletion of bases during replication


Dna damage repair

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


Dna damage repair

Aflatoxin can lead to a

modification of guanosine

(in tobacco smoke)


Dna damage by uv light

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

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

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 pyrimidinedimers 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).


Dna damage repair

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


Dna damage repair

Direct reversal of DNA damage

photoreactivation

Capture energy from light

breaking

covalent bond


Dark repair or light independent mechanisms

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 damage


Dark repair processes are defined by mutations in key genes

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

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.


Dna damage repair

Base excision repair

If a damaged base is not removed by base excision before DNA replication: a fail-safe system

oxoG:A repair


Dna damage repair

T

T

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

3’

5’

5’

3’

Gap filled by DNA pol I and the nick sealed by DNA ligase

3’

5’

5’

3’


Excision repair1

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)


Dna damage repair

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)


Dna damage repair

Homologous

DNA recombination

RecA protein is essential for homologous recombination

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


Summary

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


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