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DNA damage & repair

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 - PowerPoint PPT Presentation

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Page 1: DNA damage & repair

DNA damage & repair

Page 2: 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 radiationIf uncorrected, such changes may interfere with the ability of the cell to functionDNA 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 cancerProkaryotic and eukaryotic DNA-repair systems are analogous

Page 3: DNA damage & repair

General types of DNA damage and causes

Page 4: DNA damage & repair

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 repair

Page 5: DNA damage & repair

Drastic changes in DNA

DeletionInsertionRearrangement of chromosome

By insertion of a transposon, or aberrant actions of recombinationProcess.

Page 6: DNA damage & repair

Some replication errors escape proofreading

Page 7: DNA damage & repair

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 inMutS itself

(III)

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

MutL activate MutH

Page 8: DNA damage & repair

DNA methylation to recognize the parental strain

Once activated, MutH selectively nicks the Unmethylated strand.

Page 9: DNA damage & repair

Directionality in mismatch repair

Page 10: DNA damage & repair

Mismatch repair system in Eukaryotics

MutS MutLE. coli

MSH(MutS homolog)

Eukaryotics MLH or PMS

Hereditary nonpolyposis colorectal cancer(mutations in human homologes of Muts and MutL)

Page 11: DNA damage & repair

DNA damageRadiation, chemical mutagens, and spontaneous damage

deamination

spontaneous damage due to hydrolysis and deamination

Base pair with A

depurination

Page 12: DNA damage & repair

DNA damage

spontaneous damage to generate natural base

deamination

Methylated Cs are hot spot for spontaneous mutation in vertebrate DNA

Page 13: DNA damage & repair

Base deamination leads to the formation of a spontaneous point

mutation

Page 14: 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.

Page 15: DNA damage & repair

DNA damage by UVThymine dimer

These linked bases are incapable of base-pairing and cause DNA polymerase to stop.

Page 16: DNA damage & repair

Mutations caused by base analogs and intercalating agents

Base analogs

Analogs mispair to cause mistakes during replication

Thymine analog

Page 17: DNA damage & repair

Mutations caused by intercalating agents

Intercalating agents

flat molecules

Causing addition or deletion of bases during replication

Page 18: DNA damage & repair

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

correlates with its mutagenicity

Page 19: DNA damage & repair

Aflatoxin can lead to amodification of guanosine

(in tobacco smoke)

Page 20: DNA damage & repair

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.

Page 21: DNA damage & repair
Page 22: DNA damage & repair

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

Page 23: DNA damage & repair

Photoreactivation repairThe enhanced survival of UV-irradiated bacteria following exposure visible light is now known to be due to PHOTOLYASE, an enzyme that is encoded byE. 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).

Page 24: DNA damage & repair

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

Page 25: DNA damage & repair

Direct reversal of DNA damagephotoreactivation

breaking covalent bond

Capture energy from light

Page 26: DNA damage & repair

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

Page 27: DNA damage & repair

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 lightuvrA-recA- mutants are totally defective at dark repair and are killed by the presence of just one pyrimidine dimer

Page 28: DNA damage & repair

Excision repairIn 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.

Page 29: DNA damage & repair

Base excision repairIf a damaged base is not removed by base excision before DNA replication: a fail-safe systemoxoG:A repair

Page 30: DNA damage & repair

TT

TT

Damage recognised by UvrABC, nicks made on both sides ofdimer

TT 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’

Page 31: DNA damage & repair
Page 32: DNA damage & repair

Excision repairThe 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)

Page 33: DNA damage & repair

Base excision repairNOT 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)

Page 34: DNA damage & repair

Homologous DNA recombination

RecA protein is essential for homologous recombination

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

Page 35: DNA damage & repair

SummaryBoth 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