Accuracy of DNA Replication

🔎 Why Accuracy Matters

• DNA polymerases do not always select the correct nucleotide.

• Misincorporation causes base substitutions → can result in mutations.

• Errors like insertions or deletions can cause frameshifts.

• DNA polymerases have intrinsic fidelity due to:

  • Base pairing strength

  • Shape of base in polymerase active site

🔁 Proofreading: Error Correction During Synthesis

• Some polymerases have 3' → 5' exonuclease activity to remove errors.

• Key enzymes:

  • Prokaryotes: DNA Pol III

  • Eukaryotes: DNA Pol δ & Pol ε

• Mispaired bases → distort shape → polymerase switches to exonuclease site, removes error, then resumes synthesis.

🧠 Proofreading Process (Step-by-step)

1. Incorrect base is added.

2. Polymerase pauses; strand flips to exonuclease site.

3. Incorrect nucleotide is removed.

4. Strand flips back to polymerase site → synthesis resumes.

🧬 Mismatch Repair (MMR): Fixing What Proofreading Misses

• Some errors slip through proofreading.

• Results in bulges due to mismatched base pairs.

• MMR recognises & corrects these.

• Strand selection: The correct strand is identified via methylation (e.g. in prokaryotes).

📊 Overall Fidelity

• Combined proofreading + MMR = extremely accurate replication.

• Error rate ≈ 1 in 1 billion nucleotides.

🔧 DNA Repair Mechanisms

📉 DNA is Chemically Unstable

Daily DNA damage (per cell):

• ~18,000 purine bases lost

• ~200 cytosines deaminated to uracil

• ~50,000 single-strand breaks (SSBs)

• ~9 double-strand breaks (DSBs)

Caused by:

• UV radiation, smoking, chemicals, oxidative stress, etc.

✅ Most damage is temporary due to efficient repair systems.
❌ ~0.02% of lesions become permanent mutations.

⚠ Types of DNA Damage (Lesions)

• Base modifications (e.g. methylation)

• Photochemical lesions (e.g. UV-induced T-T dimers)

• Oxidative lesions: e.g. 8-oxoguanine mispairs with adenine

• Deaminated cytosine becomes uracil → mispairs with adenine

• Depurination → missing base leads to deletions

🧬 Lesions ≠ mutations unless they are not repaired before replication.

🛠 Common Themes in DNA Repair

1. Damage recognition (e.g. bulge, missing base, crosslink)

2. Removal of damaged region (endonuclease/helicase)

3. Gap filling (DNA polymerase)

4. Sealing (DNA ligase)

🔧 1. Base Excision Repair (BER)

• Fixes small, non-bulky lesions (e.g. deaminated cytosine → uracil)

• Process:

  1. DNA glycosylase removes wrong base

  2. Leaves AP site (apurinic/apyrimidinic)

  3. AP endonuclease cuts the sugar-phosphate backbone

  4. DNA polymerase fills the gap

  5. DNA ligase seals the nick

🔧 2. Nucleotide Excision Repair (NER)

• Repairs bulky lesions (e.g. T-T dimers from UV)

• Process:

  1. Damaged strand is recognised

  2. Segment removed by nuclease + helicase

  3. DNA polymerase synthesises new strand

  4. DNA ligase seals it

🧨 Repair of Double-Strand Breaks (DSBs)

❗ DSBs Are Dangerous

• Can result from radiation, replication stress, or chemical damage

• Can lead to genomic instability, cancer, or cell death

🔩 1. Non-Homologous End Joining (NHEJ)

• Quick but error-prone

• Occurs in prokaryotes and eukaryotes

• Process:

  1. DSB ends recognised by Ku protein

  2. Ends are “tidied up” (often losing bases)

  3. Ligated together

• Can cause:

  • Deletions

  • Chromosomal translocations → cancer (e.g. BCR-ABL1 in CML)

🔗 2. Homologous Recombination (HR)

• Accurate, template-based repair

• Uses sister chromatid or homologous chromosome

• Involves:

  • 5’ end resection

  • RecA (bacteria)/RAD51 (humans) coats ssDNA

  • Strand invasion, Holliday junction formation

  • Resolution by nucleases, sealing by ligase

• Requires BRCA1/BRCA2 (mutations → breast/ovarian cancer risk)

🔁 DNA Recombination

🧬 Why Recombine?

• Increases genetic diversity (e.g. during meiosis)

• Helps in:

  • DNA repair

  • Immunoglobulin gene rearrangement

  • Viral integration

📌 Types of Recombination

🧬 Homologous Recombination – Step-by-step

1. Initiation: Nick in DNA or DSB triggers process

2. Strand invasion: One strand invades homologous duplex

3. Strand displacement: Forms heteroduplex DNA

4. Holliday junction forms at crossover point

🧩 Holliday Model (1964)

• Explains strand exchange via a Holliday Junction (HJ)

• Can be resolved in 2 ways:

  • True crossover (recombinant chromosomes)

  • Non-crossover (gene conversion/repair only)

🧬 Recombination in Meiosis

• HR between homologous chromosomes in meiosis I

• Allows gene shuffling between maternal and paternal chromosomes

🧪 Recombination in DNA Repair

• HR is used to repair DSBs

• Involves:

  • Exonuclease resection

  • Protein-coated ssDNA

  • Invasion, copying, ligation

  • Resolution of HJ

⚠ Defects in DNA Repair = Disease

• Mutations in repair genes → genetic disorders and cancer risk

• Examples:

  • BRCA1/BRCA2 (HR repair)

  • MMR genes (Lynch syndrome)

  • NER genes (Xeroderma pigmentosum)

✅ Summary: Multi-Layered Defence Against Mutation