114c lecture 8

Overview of DNA Replication and Repair

  • DNA Repair Mechanisms
    • Mismatch repair and nucleotide excision repair are two key systems in DNA damage management.
    • Nucleotide Excision Repair: Involves the UVRABC proteins to remove thymine dimers.
    • This is a backup system, as multiple types of damage can occur.
    • Ineffective repair processes can lead to mutations and significantly increase cancer risk. Significant global research focuses on identifying gene variants responsible for certain types of cancer.

Importance of p53

  • p53 Protein: Known as the "guardian of the genome".
    • It serves as a transcription factor regulating cellular responses to DNA damage.
    • Functions:
    • Activates genes that repair DNA when damage is minimal.
    • Initiates apoptosis (programmed cell death) when DNA damage is severe.
    • Approximately 50% of tumors have mutations leading to loss of p53 function, enabling uncontrollable cell growth.
    • The decision-making analogy: Repairing minor harm versus destroying a completely damaged cell (e.g., house analogy).

Cisplatin and Chemotherapy

  • Cisplatin: A platinum-based compound used in cancer treatment.
    • Discovered accidentally by Barnett Rosenberg during electrolysis experiments.
    • Mechanism of Action: Damages DNA further in cancer cells with already compromised repair systems, leading to cell death.
    • Notable side effects due to broad DNA damage potential affecting normal cells as well.

Ames Test

  • Overview: A rapid test designed to assess whether a compound acts as a carcinogen using non-pathogenic Salmonella typhimurium.
    • Employs bacteria that cannot synthesize histidine; requires histidine-supplemented media for growth.
    • Procedure:
    • Plate mutant cells on agar without histidine.
    • Introduce a filter paper with a compound to test.
    • If the compound causes mutations, revertants can revert to wild type, causing colony growth.
    • Additional liver homogenate may be used to test for metabolic activation of the compound.

DNA Recombination

  • Recombination Types:
    • Homologous Recombination: Requires nearly identical sequences, crucial during meiosis for genetic diversity.
    • Site-Specific Recombination: Involves particular sequences of DNA, often seen in viral integrations.
    • Transposition: Transposons (jumping genes) move within the genome without requiring sequence similarity.
  • Applications of Recombination:
    • Antibody diversity in immune cells (VDJ recombination).
    • Creation of genetically modified organisms via targeted gene insertion or knockout.
    • Importance in resolving issues during DNA replication fork stalling or double-strand breaks.

Mechanisms of Recombinant DNA Formation

  • Process:
    • Involves protein complexes (e.g. RuvABC, RecA) for strand invasion and Holliday junction formation.
    • Holliday junctions allow for genetic material exchange and repair of DNA breaks.
  • Protein Involvement:
    • RecA homolog (Rad51 in humans) binds to single-stranded DNA and facilitates search for homologous sequences in double-stranded DNA.
    • RuvAB machinery facilitates migration of Holliday junctions for further processing.
  • Example: RUV A and B in E.coli actively separate strands, enabling continuity in DNA repair.

Generating Antibody Diversity

  • Mechanism: VDJ recombination enables B cells to produce antibodies with unique specificity.
    • Random selection of variable segments creates functional antibody genes that recognize diverse antigens.
    • This process significantly enhances the immune system’s adaptability to various pathogens.