DNA Damage, Repair, and Mutation

15.1 Molecular consequences of point mutation

15.2 Molecular basis of spontaneous mutations

15.3 Molecular basis of induced mutations

15.4 DNA repair mechanisms

Molecular Consequences of Point Mutation

Definition: Point mutation refers to the alteration of a single (or few) base pair(s) of DNA.

Types of point mutations:

• Base Substitution: Replacement of one nucleotide with another, leading to potential changes in amino acid sequences.

  • Transition: Purine ↔ purine or pyrimidine ↔ pyrimidine, generally resulting in fewer drastic effects due to the similarity of the bases.
  • Transversion: Purine ↔ pyrimidine or vice versa, often having more significant effects as it introduces greater biochemical diversity.

• Insertions and Deletions (INDELs): Alteration involving the addition or loss of nucleotide(s), which can disrupt the coding sequence entirely and result in frameshift mutations.

Consequences of Point Mutations

Point mutations can occur within genes, leading to various types of amino acid alterations, significantly affecting protein function and activity:

• Silent (synonymous) mutation: No change in corresponding amino acid, often occurring in non-critical regions of the gene.

• Conservative missense (nonsynonymous) mutation: Change to a chemically similar amino acid, potentially maintaining similar protein function.

• Nonconservative missense (nonsynonymous) mutation: Change to a chemically dissimilar amino acid, which may alter protein folding and function significantly.

• Nonsense mutation: Change resulting in a premature stop codon (e.g., UAA, UAG, UGA), usually leading to truncated proteins, often nonfunctional.

• Frameshift mutation: Insertions or deletions affecting the reading frame, causing profound impacts on translation and often resulting in complete loss of function if occurring early in the coding sequence.

Effects of Mutations on Protein Function

The Ras protein plays a crucial role in cell signaling related to growth and cell death. The functionality of Ras is greatly influenced by mutations:

• G12V mutation: Converts Ras into a permanently ‘active’ form, promoting uncontrolled cell proliferation and the development of cancer; often used in cancers like pancreatic cancer.

Spontaneous Mutations

• Question: Do spontaneous mutations arise in response to environmental factors or randomly?

• Luria-Delbruck fluctuation test: Demonstrates that mutations can occur randomly, illustrated by variable mutation rates among different bacterial cultures, elucidating the stochastic nature of mutation appearance.

Mechanisms of Spontaneous Mutations

Potential Causes:

• Errors during DNA replication:

  • Tautomerization & Ionization: Result in nucleotide mispairing, contributing to base substitutions, leading to permanent mutations.
  • Replication slippage: Often involves repeated sequences, causing indel mutations and frameshifts, significantly impacting genetic integrity.

• Spontaneous lesions:

  • Depurination: Loss of purine base (e.g., adenine or guanine), forming an apurinic (AP) site, leading to replication errors.
  • Deamination: Loss of an amine group resulting in various mispairing, often leading to cytosine being converted to uracil.
  • Oxidative damage: Damage caused by reactive oxygen species (ROS), such as hydroxyl radicals, contributing to broad genomic instability.

Induced Mutations

Induced mutations arise via exposure to mutagens, leading to:

• Replacement (substitution) or alteration of bases.

• Potential mechanisms include:

  • Alkylating agents: Modify bases (e.g., EMS, MNNG), causing various types of base substitutions.
  • Base analogs: Similar in structure to normal bases (e.g., 2-aminopurine), which can be wrongly incorporated during replication.
  • Intercalating agents: Insert themselves between bases, causing indels and frameshifts, significantly disrupting gene sequences.
  • UV light: Leads to thymine dimers that halt DNA replication until repaired, often causing errors if not correctly excised.
  • Ionizing radiation: Causes extensive breakage and reactivity in the DNA, leading to chromosome aberrations and severe genomic instability.

Repair Mechanisms

Overview of DNA repair:

• Direct repair: Specific enzymes recognize and reverse damage through direct chemical reversal.
• Base excision repair: Targets small damages from alkylation, oxidation, and deamination, ensuring high fidelity of the genetic code.
• Nucleotide excision repair: Handles bulky damage and UV-induced mutations through two flavors:
• Global genome NER: Scans the entire genome for lesions.
• Transcription-coupled NER: Prioritizes repair of actively transcribed regions.
• Mismatch repair: Utilizes methylation status to identify and repair mismatches post-replication, filling in gaps caused by replication errors.
• Translesion DNA synthesis: Completes replication when stalled by lesions but retains errors, enabling the bypass of severe blocks.
• Double-strand break repair: Ensures that breaks in DNA are effectively repaired to maintain chromosomal integrity.
• Nonhomologous end joining: An error-prone method, it fixes double-strand breaks without a template, often leading to mutations.
• Homologous recombination: An error-free method using homologous double-stranded DNA as a template for repair; occurs mainly during DNA replication, employing two pathways for efficient repair.

Summary of Repair Mechanisms
Each repair mechanism plays a critical role in maintaining genomic stability, addressing various types of DNA damage, and preventing the propagation of mutations that could lead to diseases such as cancer.

Key Points
Understanding these concepts is crucial for grasping the complexities of genetic mutations and the intricate systems our cells have developed for