���DNA mutation and repair����

What is a mutation?

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• Substitution, deletion, or insertion of a base pair.

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• Chromosomal deletion, insertion, or rearrangement.

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Somatic mutations - occur in somatic cells and only affect the individual in which the mutation arises.

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Germ-line mutations - alter gametes and passed to the next generation.

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Mutations are quantified in two ways:

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1. Mutation rate = probability of a particular type of mutation per unit time (or generation).

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• Mutation frequency = number of times a particular mutation occurs in a population of cells or individuals.

Two types of point mutations:

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1. Base pair substitutions.

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• Transitions
• Convert a purine-pyrimidine to the other purine-pyrimidine.
• 4 types of transitions; A ↔ G and T ↔ C
• Most transitions results in synonymous substitution because of the degeneracy of the genetic code.

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• Transversions

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• Convert a purine-pyrimidine to a pyrimidine-purine.
• 8 types of transversions; A ↔ T, G ↔ C, A ↔ C, & G ↔ T
• Transversions are more likely to result in nonsynonomous substitution.

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2. Base pair deletions and insertions

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Missense mutation :

Base pair substitution results in substitution of a different amino acid.

Nonsense mutation:

Base pair substitution results in a stop codon (and shorter polypeptide).

Neutral mutation:

Base pair substitution results in substitution of an amino acid with similar chemical properties (protein function is not altered).

Silent mutation:

Base pair substitution results in the same amino acid.

Frameshift mutations:

Deletions or insertions (not divisible by 3) result in translation of incorrect amino acids, stops codons (shorter polypeptides), or read-through of stop codons (longer polypeptides).

Types of base pair substitutions and mutations

Types of base pair substitutions and mutations

Effect of a nonsense mutation on translation

Reverse mutations and suppressor mutations:

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Forward mutation

Mutation changes wild type (ancestral) to mutant (derived).

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Reverse mutation (back mutation)

Mutation changes mutant (derived) to wild type (ancestral).

• Reversion to the wild type amino acid restores function.
• Reversion to another amino acid partly or fully restores function.

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Suppressor mutation

Occur at sites different from the original mutation and mask or compensate for the initial mutation without reversing it.

• Intragenic suppressors occur on the same codon;

e.g., nearby addition restores a deletion

• Intergenic suppressors occur on a different gene.

Intergenic suppressor genes:

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• Many function in mRNA translation.
• Each suppressor gene works on only one type of nonsense, missense, of frameshift mutation.
• Suppressor genes often encode tRNAs, which possess anti-codons that recognize stop codons and insert an amino acid.
• Three classes of tRNA nonsense suppressors, one for each stop codon (UAG, UAA, UGA).
• tRNA suppressor genes coexist with wild type tRNAs.
• tRNA suppressors compete with release factors, which are important for proper amino acid chain termination.
• Small number of read-through polypeptides are produced; tandem stop codons (UAGUAG) are required to result in correct translation termination.

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tRNA suppressor gene mechanism for nonsense mutation

Mutation caused by mismatch wobble base pairing

Addition and deletion by DNA looping-out.

Spontaneous mutations:

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Depurination

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Common; A or G are removed and replaced with a random base.

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Deamination

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Amino group is removed from a base (C → U); if not replaced U pairs with A in next round of replication (CG → TA).

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Prokaryote DNA contains small amounts of 5^MC; deamination of 5^MC produces T (CG → TA).

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Regions with high levels of 5^MC are mutation hot spots.

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Deamination

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Induced mutations

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Radiation (e.g., X-rays, UV)

Ionizing radiation breaks covalent bonds including those in DNA and is the leading cause of chromosome mutations.

Ionizing radiation has a cumulative effect and kills cells at high doses.

UV (254-260 nm) causes purines and pyrimidines to form abnormal dimer bonds and bulges in the DNA strands.

Thymine dimers induced by UV light.

Induced mutations:

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Chemical mutagens

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Base analogs

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• Similar to normal bases, incorporated into DNA during replication.

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• Some cause mis-pairing (e.g., 5-bromouracil).

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• Not all are mutagenic.

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Mutagenic efffects of 5-bromouracil

Mutagenic effects of 5-bromouracil

Induced mutations:

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Chemical mutagens

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Base modifying agents, act at any stage of the cell cycle:

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• Deaminating agents

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• Hydroxylating agents

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• Alkylating agents

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Base-modifying agents

Base-modifying agents

Induced mutations:

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Chemical mutagens

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Intercalating agents:

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• Thin, plate-like hydrophobic molecules insert themselves between adjacent base-pairs,

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• Mutagenic intercalating agents cause insertions during DNA replication.

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• Loss of intercalating agent can result in deletion.

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• Examples: Proflavin, Ethidium bromide

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Detecting environmental mutations:

Ames Test (after Bruce Ames)

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Ames Test is an inexpensive method used to screen possible carcinogens and mutagens.

• Histidine auxotroph Salmonella typhimurium (requires Histidine to grow) are mixed with rat liver enzymes and plated on media lacking histidine.

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• Liver enzymes are required to detect mutagens that are converted to carcinogenic forms by the liver (e.g., procarcinogens).

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• Test chemical is then added to medium.

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• Control plates show only a small of revertants (bacteria cells growing without histidine).

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• Plates innoculated with mutagens or procarcinogens show a larger of revertants.

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• Auxotroph will not grow without Histidine unless a mutation has occurred.

Ames test

DNA repair mechanisms:

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Types of mechanisms

• DNA polymerase proofreading - 3’-5’ exonuclease activity corrects errors during the process of replication.
• Photoreactivation (also called light repair) - photolyase enzyme is activated by UV light (320-370 nm) and splits abnormal base dimers apart.
• Demethylating DNA repair enzymes - repair DNAs damaged by alkylation.
• Nucleotide excision repair (NER) - Damaged regions of DNA unwind and are removed by specialized proteins; new DNA is synthesized by DNA polymerase.
• Methyl-directed mismatch repair - removes mismatched base regions not corrected by DNA polymerase proofreading. Sites targeted for repair are indicated in E. coli by the addition of a methyl (CH₃) group at a GATC sequence.

Nucleotide excision repair (NER) of pyrimidine dimmer and other damage-induced distortions of DNA

Mechanism of mismatch correction repair

Thank you…..