Mutations, Mutagenesis and DNA Repair – Core Vocabulary

Key Learning Outcomes

• Be able to define, classify, and illustrate all major types of DNA mutations.
• Explain how mutations arise (spontaneously vs. induced) and calculate mutation rate \left(\text{mutations}/\text{bp}/\text{replication}\right).
• Discuss the evolutionary benefits of mutation vs. the short-term need for genomic fidelity.
• Relate mutations to population diversity, inherited metabolic disease, cancer, and pathogen variation.
• Describe and diagram DNA repair pathways (specific & general) and recognise their clinical significance.

The Human Genome & Genetic Material Recap

• Humans carry 23 homologous chromosome pairs (total 46).
• Linear double-stranded DNA ≈ (3 \times 10^{9}) nucleotide pairs.
• Arranged into \sim20{,}000{-}25{,}000 protein-coding genes (unit of heredity).
• Inter-individual sequence identity ≈ 99.9\%; remaining 0.1\% = genetic variation / mutation.
• Links & resources: Human Genome Project, NCBI Chapter 1, NIGMS “Studying Genes”.

Mutations: Definitions & Positional Categories

• Mutation = any heritable change in DNA sequence.
• Positional importance
‣ Exonic (coding) → directly affect amino-acid sequence.
– Synonymous (silent) ≈ no aa change (redundancy: e.g. \text{GGT}, \text{GGA}, \text{GGC}, \text{GGG} all → Gly).
– Non-synonymous → aa change (missense, nonsense).
‣ Splice-site (exon/intron border) → exon skipping or intron retention.
‣ Promoter / regulatory → alters transcription rate.
‣ UTR, enhancers, non-coding RNAs → post-transcriptional control.
• Germline vs. Somatic
‣ Germline: in egg/sperm → heritable.
‣ Somatic: acquired in body cells; important in cancer, mosaicism.
• Evolutionary perspective: “survival of the fittest” balance – variation vs. stability.

Genetic Variation & Polymorphisms

• Polymorphism = DNA variant with ≥1\% allele frequency.
• Most frequent form = SNP (single-nucleotide polymorphism); can also be indels, VNTRs, CNVs.
• Usually neutral but may modulate disease risk, drug response, quantitative traits (eye colour: OCA2, HERC2 locus examples; brown ↔ blue alleles).
• Combination of multiple SNPs + environment ⇒ complex diseases (arthritis, cancer stratification).
• Forensics: SNP & STR patterns form DNA fingerprints.

Mutation Rate

• Expressed as mutations per cell division / per gamete / per replication round.
• Empirical estimate in fast-growing bacteria: \approx1 \text{ bp}/10^{9} \text{ bp} replicated.
• DNA polymerase proofreading + repair keep errors low; failure → mutations.

Detailed Types of Mutations

Point Mutations (single-base)

Transitions: purine↔purine (A↔G) or pyrimidine↔pyrimidine (C↔T).
Transversions: purine↔pyrimidine swaps (A/G↔C/T).
Consequences:
a. Same-sense (silent)
b. Missense – biochemical impact predicted by Grantham distance (aa similarity based on composition, polarity, volume); e.g. Leu↔Ile (conservative) vs. Cys↔Trp (drastic).
c. Nonsense – converts sense codon → stop (premature termination).

Small Indels

• +1 insertion or −1 deletion shifts reading frame ⇒ frameshift, alters every downstream codon, usually non-functional protein.

Multisite / Large-scale

• Deletions (most common) – may remove 3 bp (one aa) or kilobases (exons); no back-mutation.
• Insertions – often via transposons (“jumping genes”) that encode transposase; random genomic integration, major force in evolution.

Mutations & Human Disease

UK Newborn Blood-Spot Screening (9 disorders)

• SCD, CF, CHT, PKU, MCADD, MSUD, IVA, GA1, HCU.
• Heel-prick; early detection saves lives.

Phenylketonuria (PKU)

• Autosomal recessive; defective PAH gene on 12q.
• >50 distinct mutations (splice-site G→A first discovered).
• Pathophysiology: phenylalanine & phenylpyruvate neurotoxic → mental retardation, hypopigmentation, eczema, “musty” odour.
• Therapy: strict low-Phe diet, supplements; drug Sapropterin (Kuvan) ↑ Phe tolerance.

Sickle-Cell Anaemia

• β-globin missense transversion (A→T) at codon 6 ⇒ Glu→Val.
• Autosomal recessive; HbS polymerizes → sickled RBCs, pain crises, infection risk.
• Heterozygote advantage = malarial resistance.

Thalassaemia

• Diverse nonsense & other mutations reduce/abolish globin synthesis; Mediterranean prevalence.

Cancer Examples

• BRCA1/2 tumour-suppressors repair DSBs; inherited LoF allele raises breast cancer risk to \sim60\% by age 60 (vs 2\% population).
– BRCA1 closely linked to triple-negative phenotype; BRCA2 links to prostate.
– Prophylaxis: mastectomy, tamoxifen, intensified screening.
• Melanoma: UV-induced DNA damage; BRAF-targeted drugs (zebrafish models).

Mutations in Pathogens & Antimicrobial Resistance

SARS-CoV-2 Variants (2020)

• B.1.1.7 (UK): 17 mutations, key N501Y in spike ↑ACE2 affinity (≈+40\% transmissibility).
• B.1.351 (SA): N501Y + E484K (immune escape) + K417N.

Influenza

• Error-prone RNA polymerase → annual antigenic drift; guides seasonal vaccine design.

Antibiotic Resistance

• Incomplete antibiotic courses select for resistance mutations (e.g., MRSA).

Mutagenesis – Causes of Mutation

Spontaneous

• Replication errors despite proofreading \left(\text{~}1/10^{10}\right).
• Tautomeric shifts: keto↔enol (G, T, U) & amino↔imino (A, C) mispairing.

Induced – Chemical

Base Analogues
– 5-Br-uracil (5-BU): keto pairs A (silent); enol pairs G ⇒ A→G transition.
– 2-Aminopurine (2-AP): amino pairs T; imino pairs C ⇒ T→C.
Deaminating / Alkylating Agents
– HNO₂ deaminates C→U, 5-mC→T; hotspots at CpG.
– MNNG, mustard gas add CH₃/ethyl to bases → helix distortion, faulty repair → transitions & transversions.
Frame-shift Intercalators
– Acridines (cigarette smoke, also chemo drugs) slip between base pairs → indels at repeat runs ⇒ frameshifts.

Induced – Physical

• UV-C/UV-B (λ≈260 nm): adjacent T–T dimers.
• Ionising radiation
‣ Alpha (radon) – high energy/low penetration; lung cancer.
‣ Beta (^{32}\text{P} ingestion) – bone deposition.
‣ Gamma/X-ray/cosmic – deep penetration, DSBs & chromosomal breaks.

DNA Repair Mechanisms

Specific (Error-Free)

• Photolyase: light-dependent cleavage of T–T dimers in skin.
• DNA Glycosylases → Base Excision Repair (BER): recognise & remove abnormal bases → AP site → AP endonuclease → DNA pol → ligase.
• DNA Methyltransferase (Ada/OGT): transfers aberrant CH₃/ethyl from O⁶-G/T to enzyme cysteine.

General (Nucleotide Excision / Mismatch Repair)

Damage Recognition & Endonuclease Cleavage
– AP endonuclease (BER) cuts 3′ to AP.
– uvrA/B/C/D complex excises UV dimers with 5′ & 3′ nicks.
– mutH/L/S cleaves mismatches at GATC.
Gap-Filling by DNA polymerase (nick translation if single-strand).
Ligation seals nick via DNA ligase.

Ancillary & Emergency Systems

• Proofreading: DNA pol 3′→5′ exonuclease.
• Post-replication recombination: RecA swaps damaged strand with undamaged template.
• SOS response (LexA repressor relieved) – error-prone polymerases bypass lesions → survival with mutations.
• Genetic Suppression
‣ Intragenic: second frameshift restores frame.
‣ Intergenic: mutant tRNA anticodon reads stop codon → “pseudowildtype”.

Inherent DNA Advantages

• Double-stranded redundancy → one strand templates repair.
• Use of T not U: any deaminated C→U is flagged as “foreign”.
• Single-stranded genomes (e.g., many RNA viruses) lack this backup → higher mutation rates.

Ethical, Philosophical & Practical Implications

• Newborn screening illustrates preventive medicine vs. privacy/storage of genetic data.
• Carcinogenic mutagens (UV, tobacco, radiation) raise public-health legislation & occupational safety (PPE, sun-cream campaigns).
• Genome editing (e.g., CRISPR) now leverages controlled mutagenesis for therapy; ethical debate on germline editing.
• Antimicrobial stewardship combats mutation-driven resistance.
• Evolvability vs. stability: essential tension underlying life’s diversity and disease.

Suggested Further Study & Multimedia

• Animations: AP Endonuclease Repair, UVA repair (YouTube links).
• Articles: Science 324:29 (mutation rate), The Scientist guide to SARS-CoV-2 variants, Scientific American mutations review.