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DNA damage & repair DNA damage & repair

DNA damage & repair - PowerPoint Presentation

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DNA damage & repair - PPT Presentation

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 ID: 536175

repair dna light damage dna repair damage light base excision dimer replication form dark damaged pyrimidine survival mismatch error mutations spontaneous uvra

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Slide1

DNA damage & repairSlide2

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 analogousSlide3

General types of DNA damage and causesSlide4

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 repairSlide5

Drastic changes in DNA

Deletion

Insertion

Rearrangement of chromosome

By insertion of a transposon, or aberrant actions of recombination

Process. Slide6

Some replication errors escape proofreadingSlide7

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 in

MutS

itself

(III)

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

MutL activate MutHSlide8

DNA methylation to recognize the parental strain

Once activated,

MutH selectively nicks the

Unmethylated strand.Slide9

Directionality in mismatch repairSlide10

Mismatch repair system in Eukaryotics

MutS

MutL

E. coli

MSH

(MutS homolog)

Eukaryotics

MLH or PMS

Hereditary nonpolyposis colorectal cancer

(mutations in human homologes of Muts and MutL)Slide11

DNA damage

Radiation, chemical mutagens, and spontaneous damage

deamination

spontaneous damage due to hydrolysis and deamination

Base pair with A

depurinationSlide12

DNA damage

spontaneous damage to generate natural base

deamination

Methylated Cs are hot spot for spontaneous mutation in vertebrate DNASlide13

Base deamination leads to the formation of a spontaneous point mutationSlide14

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.Slide15

DNA damage by UV

Thymine dimer

These linked bases are incapable of base-pairing and cause

DNA polymerase to stop.Slide16

Mutations caused by base analogs and intercalating agents

Base analogs

Analogs mispair to cause mistakes during replication

Thymine analogSlide17

Mutations caused by intercalating agents

Intercalating agents

flat molecules

Causing addition or deletion of bases during replicationSlide18

Chemical carcinogens react with DNA and the carcinogenic effect of a chemical correlates with its mutagenicitySlide19

Aflatoxin can lead to a

modification of guanosine

(in tobacco smoke)Slide20

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.Slide21
Slide22

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 REPAIRSlide23

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).Slide24

(from T.A.Brown. Genetics a molecular approach)Slide25

Direct reversal of DNA damage

photoreactivation

breaking

covalent bond

Capture energy from lightSlide26

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 damageSlide27

Dark repair processes are

defined

by mutations in key genes

uvrA

,

uvrB

,

uvrC

,

uvrD - excision repair

recA, recB, recC - recombination,

recA, - SOS error-prone repairpolA

(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 dimerSlide28

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.Slide29

Base excision repair

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

oxoG:A repairSlide30

T

T

T

T

Damage recognised

by UvrABC, nicks

made on both sides of

dimer

T

T

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’Slide31
Slide32

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)Slide33

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)Slide34

Homologous

DNA recombination

RecA protein is essential for homologous recombination

(from T.A.Brown. Genetics a molecular approach)Slide35

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