Lecture 35: DNA Repair and Recombination

Learning Objectives:
- Understand types of DNA damage during and after replication.
- Familiarity with DNA repair pathways.
- Understanding the Ames test for identifying mutagenic compounds.

Genome Integrity:
- Essential for cellular function; mutations must be repaired to maintain integrity.
Types of Mutations:
- Substitutions: Changes to a single base.
- Eg: Sickle cell anemia (GAG to GUG causes Glu → Val mutation).
- Insertions: Addition of bases.
- Example: Tay Sachs mutation - four nucleotide insertion in Ashkenazi population.
- Deletions: Removal of bases.
- Example: Cystic fibrosis - removal of three nucleotides.
- Chromosomal Rearrangements:
- Example: Philadelphia chromosome.
- Regulatory Region Changes: Affect gene expression and regulation.

Replication Errors:
- Despite proofreading, errors can occur:
- Incorrect nucleotide incorporation.
- Insertion/deletion of bases or double-strand breaks due to polymerase stalling.
Insertions and Deletions:
- Cause: Interstrand slippage, especially in repeat-rich regions.
- Example: Huntington's disease (increase in CAG repeats leads to aggregated Poly Q protein).
Double-Strand Breaks:
- Causes: Collapse of replication fork, exposure to ionizing radiation.
Chemical Damage:
- Guanine Oxidation:
- Reactive oxygen species can damage DNA leading to 8-oxoguanine, resulting in GC to TA conversion if not corrected.
Guanine Alkylation:
- Damage from added hydrocarbons, e.g., aflatoxin converting to reactive epoxide leading to bulky mutagenic adducts, often resulting in GC to AT conversion.
- Example of historical significance involving chimney sweeps and cancer.
Adenine and Cytosine Deamination:
- Loss of amino groups alters base pairing, e.g., adenine to hypoxanthine (pairs with cytosine) which converts AT to GC.
- Cytosine deamination occurs at detectable rates, approximately 100 events/cell/day, resulting in uracil which can lead to CG to TA conversion if unrepaired.

Damage caused by ultraviolet (UV) radiation:
- Formation of pyrimidine dimers (cyclobutane rings).
- Blocks replication and transcription; high frequency of formation at elevated UV exposure.

**Ubiquity of Repair Mechanisms:
- Example: Deinococcus radiodurans can survive extreme radiation.
Basic Steps in Repair Pathways:
1. Recognition: Identifying the damaged base(s).
2. Removal: Cutting out the offending base(s).
3. Repair: Filling in the gaps with DNA polymerase and sealing with ligase.
- Key players in repair include:
- Proteins: Recognize differences from standard DNA structures;
  - Endonucleases: Cut one strand.
  - Exonucleases: Remove nucleotides.
  - Excinucleases: Cut strands at both sides of modified bases.
  - Glycosylases: Remove bases by cleaving the sugar-base bond.
  - DNA polymerases and ligases: Fill gaps and seal connections.

Mismatch Repair:
- Handles incorrectly paired bases:
- Recognition: MutS identifies mismatches.
- Removal: MutH nicks the strand near the mismatch; exonuclease removes the segment.
- Repair: DNA polymerase III restores the sequence and DNA ligase seals it.
Base Excision Repair:
- Handles abnormal bases (e.g., uracil, alkylated bases):
- Recognition: Glycosylases identify and remove the damaged bases.
- Removal: AP endonuclease cuts the strand near the AP site, followed by phosphodiesterase action.
- Repair: DNA polymerase I fills in the gap and ligase seals the strand.
Nucleotide Excision Repair:
- Recognizes structural distortions like pyrimidine dimers:
- Recognition: UvrABC recognizes distortions and binds.
- Removal: UvrB and UvrC perform the cutting.
- Repair: Similar to other pathways, polymerase I fills and ligase seals.
Direct Repair:
- Uses energy to repair without cutting DNA:
- Example: DNA photolyases use light energy to reverse dimerization of pyrimidines.

Each type of repair pathway handles different aspects of DNA damage, employing specialized enzymes for recognition and repair.

Causes:
- Replication fork collapse or ionizing radiation.
Repair Steps (Eukaryotes):
1. 5' exonuclease creates ssDNA.
2. Strand invasion forms a D-loop.
3. DNA synthesis occurs.
4. Second strand invasion results in a Holliday junction.
5. Cleavage of the junction leads to re-ligation.

Overview of the Ames Test:
- Rapid identification of potential mutagens.
- Involves a histidine-requiring Salmonella strain that has lost the ability due to a mutation.
- Measures survival rates in the presence of test compounds against controls.
- If unchanged, bacteria die; if mutated, bacteria survive.
- Uses liver extract to simulate metabolism; known carcinogens show >90% accuracy.

Genome Integrity:
- Types of DNA damage and repair strategies.
Understanding Repair Processes:
- Recognize important enzymes and the pathways involved in DNA repair.
- Significance of the Ames test and how to understand mutagenic damage.

Maintaining DNA integrity through effective repair mechanisms is essential for the prevention of mutations that can lead to diseases, including cancer.