1 0 TH W E E K

DNA damage, repair & Mutagenesis

DNA damage, repair & mutagenesis

Mutagenesis
Mutation: replication fidelity, mutagens, mutagenesis

DNA damage
DNA lesions: oxidative damage, alkylation, bulky adducts

DNA repair
Photoreaction, alkyltransferase, excision repair, mismatch repair, hereditary repair defects

DNA damage, repair & Mutagenesis

1 Mutagenesis
Mutation

Replication fidelity
Mutagens: chemical & physical Mutagenesis: direct & indirect

Mutation Replication Fidelity Mutagens Mutagenesis

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1-1 Mutation
Permanent, heritable alterations in the base sequence of DNA Reasons
1. Spontaneous errors in DNA replication or meiotic recombination 2. A consequence of the damaging effects of physical or chemical mutagens on DNA

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Point mutation (a single base change)

Transition : Purine or pyrimidine is replaced by the other AG T C Transversion: a purine is replaced by a pyrimidine or vice verse A T or C T A or G G T or C C A or G

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Effects of a point mutation

Phenotypic effects Silent mutation

Noncoding DNA Nonregulatory DNA 3rd position of a codon

Coding DNA altered

No

Missense mutation Yes or No

Coding DNA stop codon truncated protein

Nonsense mutation

Yes

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Insertions or deletions
The addition or loss of one or more bases in a DNA region

Frameshift mutations
The translation of a protein encoded gene is frameshifted , then changed the C-terminal side of the mutation is completely changed.

Examples of deletion mutations

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1-2 Replication fidelity

Important for preserve the genetic information from one generation to the next

Mutation relevant 1. Spontaneous errors in DNA replication is very rare, one error per 1010 base in E. coli.

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Molecular mechanisms for the replication fidelity

1. DNA polymerase: Watson-Crick base pairing
2. 3 5 proofreading exonuclease. 3. RNA priming: proofreading the 5 end of the lagging strand 4. Mismatch repair

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Proofreading by E. coli polymerase

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Mutagens
Mutation relevant Cause DNA damage that can be converted to mutations.

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Physical mutagens High-energy ionizing radiation: X-rays and g-rays

strand breaks and base/sugar destruction

Nonionizing radiation : UV light pyrimidine dimers Chemical mutagens

Base analogs: direct mutagenesis

Nitrous acid: deaminates C to produce U

Alkylating agents
Intercalating agents

Lesions-indirect mutagenesis

Base analogs: derivatives of the normal bases incorporated in DNA, altering base pairing properties. Nitrous acid: deaminates C to produce U, resulting in GC

AU

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Mutagenesis
The molecular process in which the mutation is generated.
Note: the great majority of lesions introduced by chemical and physical mutagens are repaired by one or more of the error-free DNA repair mechanisms before the lesions is encounter by a replication fork

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Direct mutagenesis The stable, unrepaired base with altered base pairing properties in the DNA is fixed to a mutation during DNA replication.

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OH

H
O

Br

AGCTTCCTA TCGAAGGAT

:G

1. Base analog incorporation

AGCTBCCTA TCGAAGGAT

enol form

2. 1st round of replication

H O

Br

AGCTTCCTA TCGAAGGAT

AGCTBCCTA TCGAGGGAT

:A

3. 2nd round of replication

AGCTBCCTA TCGAAGGAT AGCTCCCTA TCGAGGGAT

Keto form 5-BrU

ATGC transition

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Indirect mutagenesis The mutation is introduced as a result of an error-prone repair.

Translesion DNA synthesis to maintain the DNA integrity but not the sequence accuracy: when damage occurs immediately ahead of an advancing fork, which is unsuitable for recombination repair the daughter strand is synthesized regardless of the the base identity of the damaged sites of the parental DNA.

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E. coli translession replication: SOS

response: Higher levels of DNA damage
effectively inhibit DNA replication and trigger a stress response in the cell, involving a regulated increase (induction) in the levels of a number of proteins. This is called the SOS response. 1. Some of the induced proteins, such as the UvrA and UvrB proteins, have roles in normal DNA repair pathways. 2. A number of the induced proteins, however, are part of a specialized replication system that can REPLICATE PAST the DNA lesions that block back DNA polymerase III.

Proper base pairing is often impossible and not strictly required at the site of a lesion because of the SOS response proteins, this translesion replication is error-prone.
The resulting increase in mutagenesis does not contradict the general principle that replication accuracy is important (the resulting mutations actually kill many cells). This is the biological price that is paid, however, to overcome the general barrier to replication and permit at least a few mutant cells to survive.

DNA damage, repair and mutagenesis

DNA damage and repair

Mutagen
chemical reactivity of the bases DNA damage (lesions) Direct mutagenesis Extensive, right before Replication Fork (not repairable) Indirect mutagenesis

minor or moderate Error-free Repairing

Completely repaired

mutations