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Mutation and Repair

Objective

At the end of this lecture, student will be able to

• Explain mutation and repair mechanisms

DNA Mutation and Repair

DNA Damage

Mechanisms for maintaining genetic stability associated with DNA replication in E. Coli

Mechanism frequency Cumulative error

Base pairing ~10-1 – 10-2

DNA polymerase actions (including base selection, 3′->5’proofreading) ~10-5 -10-6

Accessory proteins (e.g. SSBP) ~10-7

Post-replication mismatch correction ~10-10

Spontaneous alterations:

(a) Mismatches: Occurs during DNA synthesis (i.e. replication, repair, or recombination)

(b) Tautomeric shifts

• Nucleotides spontaneously undergo a transient rearrangement of bonding, e.g. a shift from NH2 (amino form) to NH (imino form) or C=O (keto) to C- OH (enol).

• Therefore, if any base in a template strand exists in its rare tautomeric form during DNA replication, misincorporation in the daughter strand can result.

Base Pairing of Imino A-C

(c) Deamination

• Three of the four bases normally present in DNA (cytosine, adenine, and guanine) contain amino group (NH2).

• The loss of the amino group (deamination) can occur spontaneously and result in the conversion of the affected bases to uracil, hypoxanthine, and xanthine, respectively.

(d) Loss of bases

• Depurination & depyrimidination: The loss of purines or pyrimidines from DNA usually occurs at acidic pH;

• This will results in breaking the 3’ phosphodiester bond called b- elimination.

Induced Mutations

(a) Physical agents that damage DNA:

– Ionizing radiation: OH, O2, H2O2, damage base and sugar residues.

– UV radiation: Cyclobutane pyrimidine dimers, Thymidine dimers (T-T) dimer

(b) Chemical agents that damage DNA:

• Alkylating agents: Alkylating agents are electrophilic compounds with affinity for nucleophilic centers in organic macromolecules.

• These include a wide variety of chemicals, many of which are proven or suspected carcinogens (such as nitrous acid, hydroxylamine, and ethylmethane sulfonate, EMS), Adding alkyl group to hydrogen-bonding oxygen of G or T, resulting in G-T mispairing

G-C —> G*T —>A-T

T-A —>T*-G —> CG

(c) Base-analogue Agents

• A base analogue is a substance other than a standard nucleic acid base that can be incorporated into a DNA molecule by the normal process of polymerization.

• Such a substance must be able to pair with the base on the complementary strand being copies, or the 3′->5′ editing function will remove it.

• For example, 5-bromouracil is an analogue of thymine and might cause an A-T to G-C transition mutation.

Base Analogue

(d) Intercalating agents:

• Substances whose dimensions are roughly the same as those of a purine-pyrimidine pair

• In aqueous solutions, these substances form stacked arrays

• Able to stack with a base-pair by insertion between two base-pairs. This may result in frameshift mutation.

Model of intercalating agent-induced mutagenesis

Type of Mutations

I. Point mutation:

A. Base substitution

Change in DNA

Transition: One purine replaced by a different purine; or one pyrimidine replaced by a different pyrimidine

A à G T à C

Transversion: A purine replaced by a pyrimidine or vice versa

A à T C à G

Change in protein

1. Silent mutation: altered codon codes for the same a.a.

2. Neutral mutation: altered codon codes for functional similar a.a.

GAG (Glu) —>GAA (Glu)

3. Missense mutation: altered codon codes for different dissimilar a.a.

GAG—>GAC (Asp)

4. Nonsense mutation: altered codon becsomes a stop codon

GAG —> AAG (Lys)

GAG —> UAG (stop)

B. Frameshift mutation: addition or deletion of one base-pair result in a shift of reading frame and alter amino acid sequence

Metabolite Mutagens

Chemicals that are metabolized to electrophilic reagents: Aflatoxins, benzo[a]pyrene

• A mutagen is a physical or chemical agent that causes mutations to occurs.

• Mutagenesis is the process of producing a mutation.

• Mutant refers to an organism or a gene that is different from the normal or wild type.

Reversion and the Ames test:

Mutants may have second mutation and become wild type again.

Reversion was used as a means of detecting mutagens and carcinogens- the Ames test

DNA Repair Mechanisms

(1) Repair by direct reversal: The simplest mechanism. e.g. UV induced T-T dimer is recognized by photolyase and is cleaved into intact thymine (light dependent). This is called photoactivation

(2) Excision Repair: The most ubiquitous repair mechanism, which can deal with a large variety of structural defects in DNA.

(3) Recombinational repair (Postreplicational repair): Occurs before excision repair has happened or when excision repair cannot fix the problem

(4) The SOS response: The SOS response system is only active in response to some signal such as a blocked of replication fork. In E. Coli, recA and lexA govern the expression of a number of other genes involved in DNA repair. This is an error-prone DNA repair mechanism and result in higher than normal mutagenesis.