🌟 Genomic Instability & DNA Damage: Fun + Detailed Summary

🧬 1. What Is Genomic Instability?

Genomic instability = an increased tendency of the genome to acquire mutations or changes in gene dosage.

Key forms:

• Point mutations
• Repeat expansions or deletions (e.g., in Huntington’s disease)
• Chromosomal rearrangements (within or between chromosomes)
• Telomere shortening (happens with every cell division)
• Epigenetic alterations (mentioned but not explored)

Why it's dangerous:

• DNA → RNA → proteins Any mistake in DNA can mess up gene expression, protein levels, or protein function.
• Mutations in promoters → ↓ gene expression
• Mutations activating proto-oncogenes → cancer
• Structural changes → dosage imbalances

Associated diseases:

• Cancer (mutations activate oncogenes or disable tumor suppressors)
• Huntington’s disease (repeat expansion)
• Xeroderma pigmentosum (XP) (defective DNA repair → severe UV sensitivity)
• Myotonic dystrophy (repeat expansion)

🌞 2. Why Does Genomic Instability Occur?

Because DNA is constantly under attack.

Exogenous (environmental) sources:

• UV light → pyrimidine dimers
• Ionizing radiation → single- and double-strand breaks
• Cigarette smoke → bulky DNA adducts
• Combustion products / air pollution
• Chemotherapy chemicals (designed to damage DNA to kill cancer cells)

Endogenous sources:

• Reactive oxygen species (ROS) from mitochondria ROS attack bases, sugars, and the DNA backbone.
• Replication errors
• Spontaneous chemical reactions (e.g., depurination, deamination)

Every cell experiences ~70,000 DNA lesions per day (!). Repair systems must keep up.

🔋 3. Mitochondria & DNA Damage

Mitochondria:

• ~1000 per cell
• 4–10 copies of a 16 kb circular genome inside each mitochondrion
• Perform oxidative phosphorylation → ATP production BUT leak ROS → mitochondrial DNA is highly exposed to damage

ROS originate mainly from:

• Complex I and III of the electron transport chain Superoxide → hydrogen peroxide (via SOD enzymes)

Mitochondria are also involved in:

• Apoptosis
• Necrotic signaling
• Lipid and nitrogen metabolism

🛠 4. Types of DNA Damage

Types covered:

Replication-associated errors

• Misincorporation of bases → mismatches If not corrected → permanent mutation after the next replication round
• Replication slippage in repetitive sequences → insertions/deletions
• Misalignment in meiosis → unequal crossover → duplications/deletions

Chemical/spontaneous damage

• Depurination (loss of base)
• Oxidized bases
• Single-strand breaks (SSBs)
• Double-strand breaks (DSBs)
• Bulky adducts
• Intrastrand/Interstrand crosslinks

🧯 5. DNA Repair Pathways

Each type of damage → different repair pathway.

Main pathways mentioned:

1. Nucleotide Excision Repair (NER) Repairs:
   • UV damage
   • Bulky adducts Associated disease: Xeroderma pigmentosum Note: NER does NOT function in mitochondria.
2. Base Excision Repair (BER) ⭐ Main focus Repairs:
   • Oxidized bases
   • A-basic sites
   • Small non-bulky lesions
   • Single-strand breaks
   
   Steps of BER:
   1. Glycosylase → removes damaged base
   2. AP endonuclease → cuts backbone
   3. PNKP → cleans & prepares DNA ends
   4. Polymerase → inserts new nucleotide
   5. Ligase → seals the strand
3. Double-Strand Break Repair / Homologous Recombination Fixes double-strand breaks. Defective in Bloom syndrome.
4. Mismatch Repair (MMR) Fixes replication misincorporations. Defective in some colon cancers.
5. Other notes
   • Some enzymes are shared across pathways
   • Many age-related syndromes arise from repair deficiencies Examples:
     • Cockayne syndrome (NER + BER defects)
     • XP (NER defect)
     • Bloom syndrome (HR defect)

🔬 6. How Do We Measure DNA Repair Capacity?

A. Enzyme activity assays with extracts

Goal: Detect activity of specific BER enzymes.

1. AP endonuclease assay

• Use THF-containing DNA (mimics an abasic site)
• Label DNA end with radioactive isotope or fluorophore
• Incubate with cell extract
• Run gel
• Shorter DNA band = AP endonuclease cut = activity present

2. Glycosylase assay

• DNA contains an oxidized base (e.g., 5-hydroxy-uracil)
• Glycosylase removes base → AP site → cut appears
• More cut DNA = higher glycosylase activity

3. Full BER completion assay

• No label on DNA initially
• Add radiolabeled nucleotide (e.g., dCTP)
• If BER proceeds:
  • Base removed
  • Nucleotide inserted
  • Ligase seals nick
• Gel shows:
  • Small band = insertion but no ligation
  • Full-length band = complete repair

B. The Comet Assay (Single-Cell Gel Electrophoresis)

Visualizes DNA strand breaks in whole cells.

Procedure:

1. Embed cells in agarose
2. Lyse cells → DNA relaxes
3. Electrophorese DNA
4. Damaged DNA migrates toward the anode → creates a “tail”

Interpretation:

• Large comet tail = many strand breaks
• Round head only = low damage

Useful for:

• Comparing tissues
• Comparing individuals
• Time-course experiments after inducing damage

C. Plasmid-based Functional Repair Assay

Goal: Measure repair in live cells.

Method:

• Transfect cells with damaged EGFP plasmid
• Only cells that repair the lesion can express GFP
• Analyze % GFP-positive cells via flow cytometry → higher % = higher repair capacity

Allows:

• Large-scale comparative studies
• Tailoring lesion type to specific repair pathways

🎉 Quick Wrap-Up

This lecture section explains:

• What genomic instability is
• Why it happens (UV, ROS, replication errors, chemicals)
• How DNA repair pathways counteract it
• The biology of mitochondria in creating ROS
• Diseases caused by repair failures
• Experimental methods for measuring repair capacity (enzyme-level assays, comet assay, plasmid GFP assay)