41

MCB 250: Mutation and Mutagenesis

Lecture Information

  • Presenter: Dr. James M. Slauch

  • Department: Department of Microbiology

  • Vcast: 41

Point Mutations

Types of Point Mutations

  • Transition:

    • Definition: A point mutation where a purine is replaced by another purine, or a pyrimidine is replaced by another pyrimidine.

  • Transversion:

    • Definition: A point mutation where a purine is replaced by a pyrimidine, or a pyrimidine is replaced by a purine.

  • Commonality: Transition mutations are more common than transversion mutations.

Consequences for Proteins

Mutation Types

  • Silent Mutation:

    • Definition: Mutation that does not result in a change in amino acid sequence.

  • Missense Mutation:

    • Definition: Mutation that results in a different amino acid in the protein sequence.

  • Nonsense Mutation:

    • Definition: Mutation that results in a premature stop codon in the protein sequence.

Mutation Designation

  • Format: wild type amino acid – position in the protein – mutant amino acid.

  • Example: E214K

    • Explanation: The mutation resulted in Glu (glutamic acid) at amino acid position 214 changing to Lys (lysine).

Insertion/Deletion Mutations

Description

  • Wild Type Example:

    • The original sequence: THE CAT ATE THE FAT RAT.

  • Insertion Example:

    • Resulting sequence after insertion: THE CAT AAT ETH EFA TRA T.

  • Deletion Example:

    • Resulting sequence after deletion: THE CAT TET HEF ATR AT.

  • Frameshift Concept:

    • Definition: Insertion or deletion of a number of bases that is not divisible by 3 leads to a changes in the reading frame, causing a frameshift mutation.

Non-frameshift Mutations

  • Note: Insertion or deletion of 3 or multiples of 3 bases still causes a mutation but not a frameshift mutation.

Spontaneous Mutation

Causes of Spontaneous Mutation

  • Errors in DNA Replication:

    • Possible causes include incorporation errors due to tautomerization and misalignment due to strand slippage.

  • Chemical Instability of DNA:

    • Deamination of Cytosine (C)

    • Depurination (loss of purine bases)

  • Oxidative DNA Damage:

    • Occurs through:

    • Formation of modified bases

    • Creation of single and double-stranded breaks

Tautomerization

  • Tautomeric Forms:

    • Each base can exist in two tautomeric forms.

    • Predominant forms are amino and keto; rare tautomer presence during replication leads to misincorporation.

    • Figure Reference: Tautomerism (Fig 6-8)

Mispairing Example due to Tautomerization

  • Sequence Example:

    • A mispair can occur during replication resulting in one mutant and one wild-type strand.

    • Parent strands: 5' AGTCAATAG 3' and 3' TCAGTTATC 5'

    • After replication, the first generation shows a replication error: 3' TCAGCTATC 5' and 5' AGTCAATAG 3'.

    • Illustration of Mispairing and Replication Errors (Fig 12-2)

Strand Slippage

Trinucleotide Repeat Expansion

  • Occurs during DNA replication leading to expansions of repeats, linked to inherited diseases.

  • Normal DNA Replication Example:

    • Example sequence: 5' GTC GTC GTC GTC 3' and 3' CAG CAG CAG CAG CAG CAG 5' while experiencing slips can lead to mutated sequences after replication.

    • Illustration of Slippage Effects (Fig 12-4)

    • Understanding of the connection between trinucleotide repeat expansions and diseases is encouraged, but specific disease names are not required to be memorized.

Chemical and Oxidative Damage

Implications of Chemical Instability

  • Deamination of Cytosine:

    • Converts Cytosine to Uracil.

    • Thymine is used in DNA instead of Uracil due to this risk; a repair system exists to remove Uracil from DNA.

    • Figure Reference: Fig 12-8

  • Depurination:

    • Hydrolysis of the N-glycosidic bond of purines leads to an “abasic” site, where no base exists.

    • Every cell has approximately 1,000 abasic sites at any moment.

    • Figure Reference: Fig 12-9

Oxidative Damage Mechanism

  • Oxygen Usage:

    • Oxygen acts as a terminal electron acceptor producing water: ( O2 + 4H^+ \to 2H2O )

    • Side products (e.g., superoxide, hydroxyl radicals) from O2 metabolism can be highly toxic.

  • Effects of Oxygen Radicals:

    • Can cause double-stranded and single-stranded breaks; they can also damage the deoxyribose ring, stalling replication at lesions.

Example of Modified Bases from Oxidation

  • Example: 8-oxoG

    • Can mispair with either C or A, showing altered base pairing properties.

    • Illustration Reference: Fig 12-11

Induced Mutations - Mutagens

Sources of Induced Mutations

  • Ultraviolet Light:

    • Leads to the formation of thymine dimers, which block replication.

  • Chemical Modification:

    • Alkylating agents (e.g. mustard gas, vinyl chloride, cigarette smoke) modify DNA, and may cause mispairing or replication blockage.

  • Ionizing Radiation:

    • Sources: X-rays and radiation.

    • Can cause direct damage (modified bases, single and double strand breaks) or indirect damage through generating reactive oxygen species.

  • Intercalating Agents:

    • Compounds such as ethidium bromide can cause frameshift mutations by stabilizing slipped strands allowing for base additions or deletions.

Ultraviolet Light and Thymine Dimers

  • Description: Thymine dimers occur as intrastrand links between two adjacent thymines, physically blocking replication.

    • Illustration Reference: Figs from Watson et al. Molecular Biology of the Gene, © 2014, Pearson Education

Ionizing Radiation Effects

  • Can cause:

    • Direct Damage: Modified bases, single and double-strand breaks

    • Indirect Damage: Creates reactive oxygen species which can harm DNA.

Intercalating Compounds

  • Function: They stabilize slipped strands, causing the polymerase to add or delete bases, contributing to frameshift mutations.

Examples of Intercalating Mutagens

  • Not required to memorize composition, but recognition of types of compounds that act as intercalating mutagens is necessary.