lecture31

DNA Repair and Recombination

Overview of DNA Damage

  • Types of DNA Damage:

    • UV light exposure

    • Ionizing radiation

    • Chemical exposure

    • Replication errors

  • Cell Cycle Checkpoint Activation: Helps determine if the cell should repair damage or proceed to cell division.

DNA Repair Mechanisms

  • Direct Reversal: Repairs DNA damage directly without breaking the DNA backbone.

  • Base Excision Repair (BER): Cleans up non-helix-distorting base lesions.

  • Nucleotide Excision Repair (NER): Removes bulky DNA adducts such as UV-induced damage.

  • Mismatch Repair (MMR): Corrects erroneous insertion, deletion, and misincorporation of bases.

  • Double Strand Break Repair (DSBR): Restores integrity of DNA when both strands are broken.

  • Apoptosis: Programmed cell death if damage is irreparable.

  • Transcriptional Program Activation: Upregulation of genes involved in repair processes.

Repair of UV-Induced Damage

Pyrimidine Dimer Repair by DNA Photolyase

  • Mechanism of Action:

    • UV irradiation causes dimerization of adjacent thymine bases, forming a cyclobutane ring.

    • Disruption of normal base pairing occurs.

    • Photolyase Binding: Uses visible light energy to break the cyclobutane ring, restoring thymine to original form.

Chemical Mutagens and DNA Base Interaction

Effects of Chemical Mutagens

  • Chemical Mutagens: Overview

    • React chemically with bases altering DNA base structure.

    • Examples include alkylating agents like Dimethylsulfate.

  • Interactions with Bases:

    • Can cause mispairing or cross-linking of bases (e.g., methylation, formation of O6-methylguanine).

Methylation and its Consequences

Methylation of Guanine

  • Impact of Methylation on DNA:

    • O6-methylguanine can mispair with thymine during replication, leading to mutations.

  • Direct Repair Mechanism: Methyltransferase removes methyl groups from O6-methylguanine to revert back to guanine.

Base Excision Repair (BER)

Excision Repair Process

  • Steps:

    1. Damage recognition by DNA glycosylase.

    2. Base excision and sugar-phosphate backbone cleavage by AP endonuclease.

    3. DNA polymerase fills the gap with correct nucleotide.

    4. DNA ligase seals the nick.

Nucleotide Excision Repair (NER)

Mechanism of NER

  • Process in E. coli:

    • Excinuclease identifies and excises damaged regions, and DNA polymerase synthesizes new DNA in the gap.

Mismatch Repair (MMR)

Mechanism of Mismatch Repair

  • Key Enzymes:

    • Dam methylase distinguishes between the old and new strands in hemimethylated DNA.

    • MutS, MutL, and MutH proteins facilitate recognition and repair of mismatches.

DNA Recombination during Replication

Homologous Recombination

  • Occurs during Meiosis:

    • Involves alignment and exchange of genetic material between homologous chromosomes.

  • Holliday Model of Recombination: Depicts the formation and resolution processes of Holliday junctions during genetic recombination.

RecBCD and RecA Functions

  • RecBCD Complex: Initiates the homologous recombination process by generating single-stranded DNA.

  • Role of RecA: Facilitates strand invasion and pairing during recombination.

Resolution of Holliday Junctions

  • Ruv Complex Functionality: RuvA, RuvB, and RuvC work together to resolve Holliday junctions, ensuring genetic diversity during cell division.

CRISPR-Cas9 System

Overview

  • Functionality: Allows for site-specific cleavage of DNA, enabling precise gene editing.

  • Applications: Gene knockout, insertion and deletion mutations for research and therapeutic purposes.

Immunoglobulin Gene Rearrangement

Mechanism Overview

  • RAG1/RAG2 Recombination Complex: Cleaves DNA at recombination signal sequences allowing the recombination of V and J segments resulting in antibody diversity.