DNA Mutation and Repair Mechanisms

DNA Mutations and Repair Mechanisms

I. Mechanisms of Mutation Accumulation

• Replication-Dependent Mutation: The number of mutations that accumulate in cells is directly proportional to the number of DNA replications undergone by those cells.

• Polymerase Errors: DNA polymerases, though highly accurate, are not $100\%$ error-free. They occasionally make mistakes during DNA synthesis.

• Error Rates (Laboratory Measurements):

  • Viruses: Approximately $1$ error per $10,000$ nucleotides synthesized.

  • Humans: Approximately $1$ error per $5 \times 10^{11}$ (five hundred billion) nucleotides synthesized.

• Reasons for Lower Observed Mutation Rates: The significantly lower observed mutation rates in living organisms compared to in vitro polymerase error rates are due to two primary factors:

  • Survival Bias: Mutations that quickly lead to cell death are rarely observed because the affected cells are eliminated.

  • DNA Repair Pathways: Cells possess sophisticated DNA repair mechanisms that actively correct mutations. This is crucial because the vast majority of potential mutations are deleterious, and minimizing mutation rates increases the chance of cell survival.

II. Mismatch Repair (MMR)

• Detection of Mismatches:

  • Mismatched base pairs (non-canonical pairings) perturb the regular, correct shape of the DNA double helix.

  • This structural alteration serves as a signal for proteins to recognize the mismatch.

• Challenge of Repair: In a mismatched base pair, it is not immediately obvious which base is incorrect and which is the correct, original base.

• Solution: Differentiating New vs. Template Strands:

  • In the context of DNA replication, the newly synthesized strand contains the error, while the template strand contains the correct nucleotide.

  • Cells distinguish the new strand from the template strand through methylation status.

  • GATC sequences in E. coli are transiently hemimethylated after replication, meaning the adenines in the template strand are methylated, but the new strand remains unmethylated for a brief period.

• MMR Protein Complex:

  • MutL and MutS: These proteins recognize the characteristic structural perturbation caused by a mismatch and bind as a complex.

  • ATP-Dependent Binding: The binding of MutL and MutS to the mismatch requires energy derived from ATP hydrolysis.

  • MutH Recruitment: The MutL-MutS complex recruits two units of another protein called MutH.

  • MutH Function: MutH acts as an endonuclease. It searches in both directions along the DNA for a nearby hemimethylated GATC site (where only the template strand is methylated).

  • Nick Formation: Once a hemimethylated GATC site is found, MutH nicks the unmethylated (new) strand, either $5'$ or $3'$ to the mismatch, depending on the orientation of the GATC site relative to the mismatch.

• Excision and Resynthesis:

  • An exonuclease (e.g., UvrD helicase and an appropriate exonuclease) removes the segment of the new strand between the nick and the mismatch (and beyond, creating an intermediate loop).

  • DNA Polymerase III: Being in the vicinity of the replication fork, DNA Polymerase III (used in replication) resynthesizes the excised segment, ensuring the incorporation of the correct nucleotide.

  • Single-Stranded Binding Proteins (SSBs): SSBs bind to the exposed single-stranded template DNA to prevent secondary structure formation and protect it from degradation.

  • DNA Ligase: Seals the remaining nick, completing the repair.

III. Huntington's Disease: An Example of Replication Error during Expansion

• Nature of the Disease: Huntington's disease is a dominant, neurodegenerative disorder. Only one copy of the aberrant gene is needed for symptoms to manifest.

• Affected Cells: It primarily targets medium spiny neurons in the striatum region of the brain, causing them to degenerate.

• Symptoms: Involuntary movements, unsteady gait, and in later stages, dementia.

• Genetic Cause: A mutation within the Huntington gene involving an expansion of a CAG trinucleotide repeat sequence.

• Mechanism of Mutation:

  • During DNA replication, the DNA polymerase can