🧬 Genomic Instability and DNA Repair in Mammalian Cells

1. Introduction & Agenda

Tinna’s lecture focuses on:

• What genomic instability is and why it matters
• How DNA damage occurs
• How DNA repair is measured
• How repair varies between cells, tissues, and with aging
• And finally, what regulates DNA repair capacity

⚠️ Genomic Instability

What it means:

Genomic instability = the increased tendency of an organism’s genome to acquire mutations or structural changes (e.g., deletions, rearrangements, telomere shortening, or epigenetic alterations). It threatens the integrity of genetic information, the “template for life”.

Why it matters:

Instability can:

• Inactivate essential proteins 🧩
• Activate oncogenes 🔥 → Leading to diseases like cancer, Huntington’s disease, myotonic dystrophy, and premature aging.

Causes:

DNA is constantly attacked by both external and internal factors:

• ☀️ UV light, 💀 ionizing radiation
• 🚬 Smoking, 🔬 metabolism
• 🧫 Mitochondrial ROS
• 🧪 Chemicals, pollution, chemotherapeutics

A single cell experiences ~70,000 lesions/day!

🔋 Mitochondria and DNA Damage

Mitochondria have their own small genome (~16 kb), crucial for metabolism and apoptosis. They’re also major sources of ROS, which damage both mtDNA and nuclear DNA. Superoxide dismutase (SOD) helps detoxify ROS — but not completely.

🧯 DNA Repair Mechanisms

Cells have multiple repair systems to protect their genome.

Four major pathways:

1. Base Excision Repair (BER) – fixes small base lesions (e.g., oxidation, alkylation).
2. Nucleotide Excision Repair (NER) – removes bulky distortions (like UV dimers).
3. Mismatch Repair (MMR) – corrects replication errors.
4. Homologous Recombination (HR) – repairs double-strand breaks accurately.

DNA Repair Disorders:

• Xeroderma pigmentosum (NER defect) → UV sensitivity
• Cockayne syndrome (NER/BER defect) → premature aging
• Bloom syndrome (HR defect)
• Hereditary colon cancer (MMR defect)

🧪 Measuring DNA Repair

Several experimental assays are used to study repair activity:

1. Incision assay – measures specific enzyme activity (e.g., glycosylases)
   • Tissue extracts + damaged DNA → backbone incision = repair.
2. Incorporation assay – measures total BER activity via labeled nucleotides.
3. Comet assay – detects DNA breaks in single cells (tail = damage).
4. Reporter assay – uses fluorescent or reporter genes to measure repair efficiency.

🧫 DNA Repair Variation and Aging

Between tissues:

Different organs show distinct mtBER capacities: 🧠 Brain < 💪 Muscle < ❤️ Heart < 🧬 Liver, etc. → Reflects metabolic activity and oxidative stress.

During aging:

Studies (Karahalil 2002, Imam 2006, Gredilla 2010–2012):

• Repair of oxidative lesions like 8-oxoG and uracil declines with age.
• Brain regions (hippocampus, cortex, cerebellum) show region-specific decline.
• Even within a single neuron, repair can differ across compartments.

In humans:

Centenarians’ blood cells show decreased APE1 activity, a key BER enzyme. Human brain microarray data reveal that after ~60 years, BER gene expression drops significantly.

🧠 Regulation of DNA Repair

DNA repair isn’t static — it’s dynamically regulated at multiple levels.

1. Transcriptional regulation

• BDNF (Brain-Derived Neurotrophic Factor) → activates TrkB receptor → triggers CREB phosphorylation.
• CREB binds promoters of BER genes (e.g., POLB, APE1, NEIL2) and enhances transcription.
• Verified via in silico prediction, ChIP assays, and electrophoretic mobility shift assays (EMSA).

Result: BDNF signaling → CREB activation → higher BER gene expression 🔝

2. Experimental validation

• In BDNF+/- mice, BER genes (POLB, APE1) are downregulated in hippocampus.
• In cultured neurons, BDNF treatment increases both pCREB and BER enzyme levels (POLB, NEIL2, APE1).
• Leads to higher total BER and APE1 activity (Lautrup et al., Aging Cell, 2023).

🧩 Summary: BDNF positively regulates brain DNA repair via the CREB pathway.

⚙️ Post-Translational Regulation

NEIL2 Phosphorylation (Myrup Holst et al., Antioxidants 2023)

• NEIL2 is a DNA glycosylase removing oxidized bases.
• Found to be phosphorylated in vivo (in SH-SY5Y cells).
• Predicted kinase: Protein Kinase C (PKC).
• In vitro, PKC phosphorylates NEIL2, which reduces its activity (↓ Kcat/KM).

🧩 So: PKC → phosphorylates NEIL2 → repair activity decreases.

🔗 Protein–Protein Interactions in DNA Repair

The CSB (Cockayne Syndrome B) protein interacts with and stimulates NEIL2 activity. CSB-deficient cells (from Cockayne patients) show reduced BER efficiency and premature aging. CSB also interacts with other BER proteins (OGG1, APE1, PARP1). → Emphasizing how DNA repair relies on multi-protein cooperation.

🧓 Consequences of Reduced DNA Repair in the Brain

Poor repair = accumulated mutations + oxidative stress → cognitive decline & neurodegeneration.

Human studies:

• Centenarian study (1915-West cohort, Sanchez-Roman et al., 2022): Higher APE1 expression and activity correlate with better MMSE scores 🧠✨ → Suggests that efficient BER helps preserve cognition.
• Alzheimer’s and MCI brains (Weissman et al., 2007): Reduced BER gap-filling activity in parietal lobes of affected individuals. → BER defects appear early in neurodegeneration.

🧩 Overall Summaries

Summary I

• DNA repair is essential for genome stability.
• Damage and repair vary by cell type, organ, and age.
• Both mitochondrial and nuclear genomes are at risk.

Summary II

• BER gene expression declines with age, especially in the brain.
• BDNF–CREB signaling enhances BER, maintaining brain resilience.
• Posttranslational regulation (PKC, CSB) fine-tunes enzyme activity.

🎓 Take-Home Messages

• Every cell faces constant DNA damage — stability = survival.
• Aging and oxidative stress impair repair systems.
• Neural DNA repair is tightly regulated by growth factors (BDNF) and phosphorylation (PKC).
• Defects in repair link directly to aging, cancer, and neurodegeneration.
• Supporting repair (e.g., via BDNF pathways) may promote brain health and longevity.