Lecture 15 Repair mechanism and repair

DNA Replication and Repair

Importance of DNA Replication

• Essential to prevent mutations that could lead to diseases such as cancer. Accurate DNA replication ensures genetic fidelity and proper cellular function.

Mismatch Repair (MMR)

• Occurs immediately after DNA replication.

• Distinct from proofreading during replication, focusing on correcting errors that escaped the replication machinery.

• Reduces the overall DNA replication error rate significantly, maintaining genomic stability.

• Key Players:

  • Exonucleases: Active in both 3' to 5' and 5' to 3' directions to remove mistakes made during DNA synthesis.

  • Model Organism: E. coli is primarily used to understand the underlying mechanisms of mismatch repair due to its well-characterized genetic system.

Mechanism of Mismatch Repair

• Methylation: Helps distinguish between original and newly synthesized strands.

  • The original strand remains methylated, while the newly synthesized strand is initially unmethylated, allowing the repair machinery to identify which strand contains the error.

• Components Involved:

  • MutS: Detects and binds to mismatches in the DNA.

  • MutL: Activates further repair processes and helps coordinate the repair.

  • MutH: Cuts the unmethylated strand to allow for the removal of the error.

  • Exonucleases: Remove incorrect bases, creating a gap that is then filled by DNA polymerase III, ensuring that accurate base pairing is restored.

Additional Repair Mechanisms

• Photoreactivation: This method specifically targets thymine dimers formed by UV light exposure.

  • Utilizes the enzyme photolyase, which gets activated by light, breaking the bonds of the dimer. This is a direct repair mechanism that restores the original DNA structure.

• Methyltransferase: Enzyme responsible for removing improper methyl groups from bases to restore normal base pairing.

  • Critical for correcting damage resulting from deamination, which can lead to base mispairing.

• Base Excision Repair:

  • Glycosylases: Identify and eliminate abnormal bases from DNA strands, effectively preventing potential mispairing.

  • Generates gaps which are subsequently filled by DNA polymerase I, which synthesizes the correct bases.

  • The mechanism may involve cutting back the DNA backbone to enable repair processes.

• Nucleotide Excision Repair:

  • Targets larger DNA lesions, including complex structures such as thymine dimers.

  • Involves a series of enzymes, notably the UVRABC complex, which recognizes and removes damaged DNA sections.

  • Following removal, DNA polymerase I fills in the gaps, and ligase repairs the DNA backbone to restore integrity.

• Translesion Repair: A specialized process that allows DNA replication to continue, even in the presence of damage in template strands.

  • Utilizes error-prone polymerases (e.g. DNA polymerases II, IV, and V) to add nucleotides in an imprecise manner.

  • This process significantly increases the risk of mutations due to its lack of proofreading, with a roughly 25% chance of correctness in base pairing.

• Recombination: Plays a critical role in repairing double-strand breaks, a severe form of DNA damage.

  • Types of Recombination:

    • Homologous Recombination: A sequence-specific repair mechanism using similar DNA sequences as templates to ensure correct repair.

    • Non-homologous Recombination: A more random process that may result in mutations due to improper alignment and joining of broken ends.

    • Site-specific Recombination: Commonly used in laboratory settings for precise genetic engineering applications.

  • RecA: A key recombinase involved in homologous recombination, facilitating strand invasion and exchange necessary for repair processes.

Summary of Repair Mechanism Functions

• Prevent mutations before replication: Through mechanisms such as directed and excision repair.

• Repair errors after they occur: Mismatch repair efficiently corrects replication errors.

• Handle significant damage: Processes like recombination and translesion repair manage severe DNA damage that would prevent replication from occurring.