CH12 - DNA Mutation and Repair

Concept of Mutation

• Mutation = any heritable change in DNA sequence; propagated through cellular divisions.
  • Germ-line cells ➔ mutation can be passed to offspring.
  • Somatic cells ➔ transmitted only to daughter somatic cells.

Classification by Functional Consequence

• Loss-of-Function (LoF)
  • Product absent or present but inactive (e.g., kinase domain destroyed).
  • Usually recessive because wild-type allele supplies function.
• Gain-of-Function (GoF)
  • Product gains new or enhanced activity (e.g., kinase now phosphorylates extra targets).
  • Often dominant or codominant; both normal and new activities may be seen.

Molecular Types of Mutation

1. Point (Base-Substitution) Mutations

• Single nucleotide replaced by another.
• Two chemical classes:
  • Transition: purine↔purine (A↔G) or pyrimidine↔pyrimidine (C↔T).
  • Transversion: purine↔pyrimidine.
  • Transitions more frequent (ring-size conserved ➔ DNA pol swaps easier).
• Coding outcomes (examples shown for $\text{UCA}$ codon):
  • Missense: new codon encodes different aa (Ser ➔ other aa).
  • Nonsense: new stop codon (UCA➔UAA) ➔ premature truncation.
  • Silent: altered codon still encodes same aa (degeneracy of code).

2. Insertions & Deletions (Indels)

• Addition/removal of ≥1 nt.
• Frameshift if not multiple of 3 ➔ alters downstream reading frame; often introduces premature stop codon.
  • Analogy: THE DOG ATE ➔ insert "O" ➔ THO EDO GAT… (nonsense).
• In-frame ±3 nt (whole codon) less severe but may affect function.

3. Expanding Nucleotide (Triplet) Repeats

• Replication slippage enlarges short tandem repeats (usually 3-bp, e.g., $\text{CAG}$).
• More repeats ➔ higher slippage probability ➔ further expansion (positive feedback).
• >30 human diseases ("triplet-expansion" / "polyglutamine" disorders).
  • Ex: Huntington’s: normal $11!\text{–}!35$ repeats, disease >$35$ ➔ mid-life cortical degeneration.
• Diagnosis: PCR across repeat + agarose electrophoresis; amplicon size correlates with copy number (e.g., 20, 30, 44, 55 repeats).

4. Chromosomal-Scale Mutations

• Arise via aberrant recombination; large DNA segments moved.
  • Deletion: loss of segment (gene loss).
  • Duplication: extra copy (gene dosage imbalance).
  • Inversion: segment flipped; breakpoints may disrupt genes or alter regulation.
  • Translocation: segment transferred to non-homologous chromosome.
    • Reciprocal (exchange both ways) or non-reciprocal.
    • Genetic material conserved but breakpoints/fused genes problematic.
    • Classic: t(9;22) Philadelphia chromosome – BCR–ABL kinase fusion ➔ unregulated cell division ➔ chronic myelogenous leukemia.

DNA-Damaging Processes Leading to Mutation

Hydrolytic Damage (Water)

• Deamination (loss of –NH₂):
  • C ➔ U (most common) → pairs with A ⇒ $\text{CG} \to \text{TA}$ transition.
  • 5-methyl-C ➔ T ⇒ same transition (hotspot in methylated CpG sites).
  • A ➔ hypoxanthine (pairs with C) ⇒ $\text{AT} \to \text{GC}$.
  • G ➔ xanthine (pairs C, usually non-mutagenic).
• Depurination (hydrolysis of N-glycosidic bond) more frequent for purines ➔ abasic (AP) site; DNA pol may insert wrong base or SSBs.

Chemical Deaminators

• Food preservatives (sodium nitrite, bisulfites) form nitrous acid (HNO₂) in stomach.
  • Enhance C & A deamination; same transitions as above.

Oxidative Damage

• Reactive Oxygen Species (ROS): $\text{H}<em>2\text{O}</em>2$, $\text{O}_2^{-}!$, $\cdot OH$.
• G ➔ 8-oxoguanine (8-oxoG) pairs with A ⇒ GC➔TA transversion (most common mutation in human cancers).
• ROS can also produce abasic sites or oxidize deoxyribose.

Alkylation

• Alkylating agents add –alkyl groups.
  • Benzo[a]pyrene (from smoke/grilling): bulky adduct on purines ➔ mispairing.
  • Nitrogen mustard gas: crosslinks guanines (inter- / intra-strand) ➔ block replication.
  • Therapeutic cisplatin: Pt-mediated crosslinks; exploits faster division of cancer cells, triggers apoptosis.

Ultraviolet (UV) Radiation

• $200{-}400\,\text{nm}$; creates adjacent pyrimidine dimers:
  • Cyclobutane thymine dimer.
  • 6-4 photoproduct.
• Dimers stall replicative polymerase; lethal if unrepaired.

Ionizing Radiation (X-rays, γ-rays)

• Higher energy/shorter λ than UV.
• Direct DNA ionization ➔ SSBs/DSBs; indirect via radiolysis of water ➔ ROS.

Mutagen Detection & Biomedical Relevance

• Ames Test (Salmonella typhimurium His⁻):
  • Plates lacking histidine + disc containing test compound.
  • Reversion (His⁺) colony number ↑ indicates mutagenicity/carcinogenic potential.
• Rationale for cancer therapy: DNA-damaging agents kill rapidly dividing tumor cells (e.g., cisplatin).

DNA Repair Pathways

1. Mismatch Repair (MMR)

• Corrects replication errors (mispaired bases, small loops).
• In E. coli:
  1. MutS–MutL recognize helix distortion.
  2. DNA looped until nearest $\text{GATC}$ site.
  3. MutH nicks unmethylated (new) strand.
  4. Helicase + Exonuclease (DNA pol I 5′→3′) remove patch past mismatch.
  5. DNA pol III resynthesizes; ligase seals nick.
• Strand discrimination via hemi-methylated \text{G^mATC} sites.

2. Direct Repair

• Photoreactivation: Photolyase + visible light energy breaks cyclobutane dimer → restores Ts.
• O⁶-methylguanine-DNA methyltransferase (MGMT): transfers methyl from O⁶-Me-G to its own Cys → self-inactivation then degradation (single-use “suicide” enzyme).

3. Base Excision Repair (BER)

• Fixes single damaged bases or AP sites.
  1. DNA glycosylase recognizes specific lesion, cleaves N-glycosidic bond → AP site.
  2. AP endonuclease nicks phosphodiester backbone.
  3. DNA pol I (5′→3′ exonuclease & polymerase) removes sugar phosphate, inserts correct nt.
  4. DNA ligase seals.
• Handles deaminated bases, oxidized bases, single-strand breaks.

4. Nucleotide Excision Repair (NER)

• Removes bulky lesions (pyrimidine dimers, benzo[a]pyrene adducts).
  1. UvrA–UvrB scan for distortion; UvrA leaves.
  2. UvrB unwinds region; UvrC cuts ≈5 nt 3′ side & ≈8 nt 5′ side (13 nt total).
  3. UvrD helicase releases fragment.
  4. DNA pol I fills; ligase seals.
• In humans, NER defects → Xeroderma pigmentosum (XP): extreme UV sensitivity, >$10^3$-fold skin-cancer risk.

5. Translesion Synthesis (TLS)

• When replication fork meets unrepaired lesion.
  • Specialized low-fidelity polymerases (no 3′→5′ proofreading, wider active site) replace Pol III, insert “some” base opposite lesion.
  • After bypass, normal polymerase resumes.
  • Saves cell but introduces point mutations.

Quantitative & Miscellaneous Data

• Spontaneous DNA pol III error rate: $10^{-6}!\text{–}!10^{-8}$ per base.
• Huntington’s disease threshold: >$35$ $\text{CAG}$ repeats.
• UV wavelength range: $200{-}400\,\text{nm}$.

Ethical & Clinical Considerations

• Food additives producing nitrous acid raise mutagen exposure; regulatory limits exist.
• XP patients require strict UV avoidance; underscores importance of NER.
• Chemotherapeutic alkylators exploit differential repair capacity; side-effects stem from damage to normal proliferative tissues (hair, gut, marrow).
• Ames test remains standard screen in drug & chemical safety pipelines.

Integrative Connections

• Mutation types connect to earlier genetics (dominance/recessivity, codon table, recombination).
• Repair pathways illustrate enzyme mechanisms (glycosidase, endonuclease, ligase) introduced in replication chapter.
• Real-world relevance: carcinogenesis, hereditary diseases, molecular diagnostics (PCR sizing), targeted therapies (BCR-ABL kinase inhibitors complement understanding of translocation).