Understanding DNA Mutations

What is a Mutation?

• Any change in the DNA sequence of an organism is a mutation
• DNA is made of a long sequence of smaller units strung together called A, T, G, and C
• Some parts of DNA control gene activity
• Some parts of DNA have no function
• Some parts of DNA have a function we don't yet understand
• Organisms have DNA repair mechanisms to remove mutations

Types of Mutations

• Point Mutations
  • Transitions
    • One purine replaced by a different purine
    • One pyrimidine replaced by a different pyrimidine
  • Transversions
    • A purine replaced by a pyrimidine or vice versa
• Frameshift Mutations
  • Insertions
    • Addition of one or more base pairs
  • Deletions
    • Deletion of one or more base pairs

Common Defects in DNA

• Incorrect base in one strand cannot hydrogen bond with corresponding base in the opposite strand
  • Normal base tautomerizes (i.e., isomerizes in such a way that is capable of an alternative form of hydrogen bonding)
  • A base substitution occurs during subsequent DNA replication
• Missing bases
  • Depurination: N-glycosylic bond joining puine base to deoxy-ribose is spontaneously broken without breaking DNA backbone
• Altered bases
  • Alkylating agents add methyl or ethyl groups to existing bases
• Addition or deletion of one or more bases
  • May occur spontaneously or be induced by chemical mutagens or biological agents
• Single-strand breaks
  • Phosphodiester bond is broken due to exposure to chemical agents or ionizing radiation
• Double-strand breaks
  • Phosphodiester bonds on opposite DNA strands are broken due to exposure to high doses of chemical agents or ionizing radiation
• Cross-linking of complementary DNA strands
  • Certain antibiotics or reagents form covalent bonds between two bases on complementary DNA strands

DNA Damage

• Chemical Modification
  • Thymine
  • Adenine
  • Cytosine
  • Guanine
• Depurination
  • AP site
• Photodamage thymine dimer
• Deamination
  • Cytosine
  • Uracil
• Chemical Modification by O₂ free radicals
  • Cytosine
  • Uracil

Types of Bases

• Thymine
  • Common: Keto form
  • Rare: Enol form
• Cytosine
  • Common: Amino form
  • Rare: Imino form
• Adenine
  • Common: Amino form
  • Rare: Imino form
• Guanine
  • Common: Keto form
  • Rare: Enol form

Causes of Mutations

• Spontaneous mutations:
  • Tautomeric shifts
    • A tautomeric shift immediately prior to DNA replication causes a mismatch that could be repaired. If repair fails, a mutation may occur.
  • Depurination
    • Loss of adenine or guanine bases
    • Can lead to the deletion of a base pair if not repaired.
• Induced mutations:
  • Alkylation
    • Alkylating agents add alkyl groups to the nucleotide bases, interfering with base pairing.
    • Examples: nitrogen mustard, EMS, and MMS.
  • Intercalating agents
    • Flat molecules that insert themselves between the base pairs of DNA, distorting the helix and leading to frameshift mutations.
    • Examples: acridine orange, ethidium bromide, and proflavin.
• Deamination
  • Removal of an amino group from the cytosine base
  • Other bases aren't readily deaminated, such as uracil
  • DNA repair enzymes recognize uracil as an inappropriate base and remove it
  • If repair fails, a C-G to A-T mutation will occur.
• Hydroxylamine
  • Converts cytosine into hydroxylaminocytosine
  • Leads to CG:TA transitions
  • Only affects cytosine, so it won't generate TA:CG transitions.

Induced Mutations

• Alkylating agents:
  • Add alkyl groups (like methyl or ethyl) to the purine or pyrimidine of the nucleotide.
  • Mustard gas adds alkyl groups to Guanine.
• Intercalating agents:
  • Wedge themselves between the base pairs of DNA
  • Cause single nucleotide insertions and deletions
• Agents that promote oxidative damage to DNA:
  • Damage DNA by producing reactive oxygen species (ROS) that can cause mutations.

Mutagens

• Physical mutagens
  • Ionizing radiations
    • Alpha, beta, fast neutrons, thermal neutrons, X-rays, gamma rays, UV rays
  • Non-ionizing radiations
• Chemical mutagens
  • Alkylating agents
    • Mustard gas, nitrogen mustard, EMS, MMS, EES
    • 5-Bromouracil, 2-amino purine, acriflavin, proflavin, acridine orange
  • Deamination agents
    • Nitrous acid (HNO2), hydroxylamine, sodium azide

Mutagenesis, Biological Mutagens, and Metals

• Physical Mutagens
  • X-rays
  • Gamma rays
  • Alpha rays
  • Ultraviolet radiations
  • Radioactive decay
  • Cosmic rays
• Chemical Mutagens
  • Reactive oxygen species (ROS)
  • Deaminating agents such as nitrous acid
  • Alkylating agents such as nitrosamines and ethylnitrosourea
  • Aromatic amines and amides
  • Alkaloids from plants
  • Bromine, benzene, and sodium azide
• Biological Mutagens
  • Transposons
  • Viruses
  • Bacteria
  • Prions
• Metals
  • Arsenic
  • Chromium
  • Cadmium
  • Nickel

Examples of Point Mutations

• Base Substitution Mutations
  • Transitions
    • A→T,  G→C
  • Transversions
    • A→T, A→T, T→A
• Silent mutation
  • Altered codon codes for the same amino acid
  • GAGGAA,  Glu→Glu
• Neutral mutation
  • Altered codon codes for a different but functionally similar amino acid
  • GAGGAU,  Glu→Asp
• Missense mutation
  • Altered codon codes for a different, dissimilar amino acid
  • GAGAAG,  Glu→Lys
• Nonsense mutation
  • New codon is a termination codon
  • GAGUAG,  Glu→Stop
• Frameshift Mutations
  • Result in a shift in the reading frame of the resulting mRNA molecule, leading to a nonfunctional protein
  • Wild type base sequence: ATG ACC AGG TC
  • Base addition: ATG ACA CAG GTC
  • Base deletion: ATG ACA GGT C

Frameshift Mutations

• Caused by insertions or deletions of a number of nucleotides in a DNA sequence that is not divisible by three.
• Can change the reading frame of a gene, resulting in a different amino acid sequence or a premature stop codon.