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Mechanisms of Mutation – Comprehensive Study Notes

Introduction & Lecture Overview

  • Course: PATH5151 – Molecular Pathology

  • Lecturer: Dr Clayton Fragall, School of Biomedical Sciences, UWA

  • Core theme: “Mechanisms of Mutation” – how genetic changes arise, are repaired, inherited and lead to disease

Stated Learning Outcomes

  • Appreciate the role and molecular mechanics of homologous recombination (HR)

  • Describe origins and inheritance patterns of different classes of genetic change

  • Recall the principal DNA-repair pathways

  • Explain multiple molecular routes by which mutations/polymorphisms translate into human disease

Human Genetic Variation – Conceptual Framework

  • Definition: Any alteration in the structure or linear sequence of the human genome

  • Levels of occurrence

    • Intra-individual (cell-to-cell mosaicism)

    • Inter-individual within a population

    • Inter-population among different geographic / ancestral groups

  • Mechanistic contributors

    • Meiotic recombination (allelic & non-allelic HR)

    • DNA replication infidelity & imperfect repair

    • Population-level forces:

    • Random genetic drift

    • Natural/sexual selection (adaptive advantage)

    • Migration, founder effects, bottlenecks

  • Reference genome serves as comparative baseline for cataloguing variation

Scales & Categories of Variation

  • Structural variants (>10^{3} bp)

    • Copy-number: deletions, duplications

    • Positional: insertions, translocations

    • Orientational: inversions

  • Sequence-level (<10^{3} bp)

    • Single-base substitutions

    • Small indels/duplications

    • Variable/repetitive elements (micro-/mini-satellites, STRs, VNTRs, SINEs/LINEs)

Cellular Context – Mitosis, Meiosis & Chromosome Biology

  • Human diploid complement: 46 chromosomes = 22 autosomal homologous pairs + XX/XY

  • Chromosome structure terms

    • Sister chromatids (post-S-phase identical copies)

    • Centromere (kinetochore attachment site)

  • Cell-cycle phases

    • G0, G1, S (DNA synthesis), G_2 = interphase

    • M (mitosis) + cytokinesis

  • Mitosis stages: prophase → metaphase → anaphase → telophase

    • Outcome: two genetically identical diploid daughter cells

  • Meiosis

    • Meiosis I (reductional): homologous chromosomes segregate (2N → N, 4C → 2C)

    • Meiosis II (equational): sister chromatids segregate (N, 2C → N, C)

    • Gametogenesis examples

    • Spermatogenesis: spermatogonium → primary/secondary spermatocyte → spermatid → spermatozoon

    • Oogenesis: oogonium → primary oocyte (arrest diplotene I) → secondary oocyte (arrest metaphase II) → ovum + polar bodies upon fertilisation

Homologous Recombination (Crossing-Over)

  • Exclusively meiotic; occurs during prophase I in tetrad (bivalent) configuration

  • Requires stretches of near-identical sequence; enzyme-mediated strand invasion & exchange

  • Produces novel allelic combinations and contributes to genetic diversity

  • Independent assortment + crossing-over exponentially increases gamete variety

Sources of Sequence Variation

  1. HR (allelic & non-allelic)

  2. Retrotransposition (LINE-1, Alu, SVA mobilisation)

  3. Spontaneous chemical change (tautomerism, hydrolysis, oxidation)

  4. Environmental damage

    • Ionising radiation (X-, γ-rays, α/β-particles, neutrons)

    • UV light (254–260 nm)

    • Chemical mutagens (alkylators, intercalators, base analogues, deaminators)

    • Biological agents (oncogenic viruses)

  5. Replication/repair errors

    • Proof-reading escape (\approx 10^{-4}! –!10^{-5} per base prior to repair)

    • Replication slippage at microsatellites

    • Fork stalling & template switching

Intrinsic Chemical Instability of DNA

  • DNA constantly assaulted by hydrolysis, oxidation, non-enzymatic methylation

  • Many lesions distort base-pairing geometry or sugar-phosphate integrity

  • Endogenous repair systems balance but do not fully eliminate mutagenesis

Tautomeric Shifts

  • DNA bases exist in stable keto (T,G) or amino (A,C) forms

  • Rare enol (T,G) or imino (A,C) tautomers mis-pair (e.g., T-G, A-C)

  • If replication occurs during tautomer residence, mis-pair becomes permanent substitution in daughter strand (transition)

Exogenous & Endogenous Mutagen Classes

Deaminating Agents

  • Spontaneous: hydrolytic deamination, esp. 5-methyl-C → T in CpG islands

    • Explains depletion of CpG to \approx 0.8\% (expected 4 %)

  • Induced: nitrous acid (HNO_2) converts

    • A → hypoxanthine (pairs like G) ⇒ A:T→G:C transition

    • C → uracil; 5mC → T

  • Hydroxylamine specifically targets cytosine → G:C→A:T transitions

Alkylating Agents

  • Donate \mathrm{C{n}H{2n+1}} groups; alter base pairing, provoke transitions/transversions, frameshifts, chromosomal breaks

  • Repair via direct reversal (e.g. O^{6}-alkylguanine-DNA methyltransferase) can itself be error-prone when overwhelmed

Depurination

  • Spontaneous hydrolysis of N-glycosidic bond removes A/G (abasic site)

  • Replicative bypass may insert any base (often A) or skip ⇒ single-bp deletion

Intercalating Agents

  • Flat, polycyclic, +ve molecules (proflavin, ethidium bromide) wedge between stacked bases

  • Induce single-bp indels; loss of agent post-replication causes reciprocal deletion

Base Analogues

  • 2-aminopurine (A-analogue) pairs with C

  • 5-bromouracil (T-analogue) pairs with G when ionised

UV Radiation

  • Generates intra-strand pyrimidine dimers (⇢ thymine dimer) causing helix kinks

  • Blocks replication; triggers nucleotide excision repair (NER)

  • Cytosine hydration leads to C→T substitutions

Ionising Radiation

  • Induces double-strand breaks (DSBs), single-strand breaks, abasic sites

  • High-linear energy transfer (LET) particles (α, neutrons) cluster damage

DNA Replication Fidelity & Post-Replicative Repair

  • Raw polymerase error rate \approx 10^{-4}–10^{-5}; after repair \approx 10^{-9} per nucleotide

  • Two lesion classes

    1. Mis-paired bases

    2. Structural damage (ss/ds breaks, bulky adducts, dimers)

Canonical Repair Pathways

  1. Direct Repair

    • Ligase seals “nicks” (requires 5’-P & 3’-OH)

    • Enzymatic reversal (e.g. O^{6}-meG MTase)

  2. Base-Excision Repair (BER)

    • DNA glycosylase excises altered base → AP site

    • AP endonuclease cleaves backbone

    • DNA pol + ligase fill/seal

  3. Nucleotide-Excision Repair (NER)

    • Detects helix distortions (e.g. UV dimers)

    • Excinuclease removes \sim24–32 nt

    • Gap filled by DNA pol, ligated

  4. Mismatch Repair (MMR)

    • Recognises post-replicative mis-pairs & insertion/deletion loops

    • Nicks new strand, exonuclease removes \le 10^{3} bp

    • Resynthesis + ligation

  5. Double-Strand Break Repair

    • Homologous Recombination (HR)

      • Uses sister chromatid template, high fidelity

      • Infrequent; mutations in HR genes (BRCA1/2, RAD51) ⇒ genomic instability syndromes

    • Non-Homologous End-Joining (NHEJ)

      • Direct ligation of broken ends; iterative resection/addition possible

      • Error-prone around junction, but can be precise

Replication Slippage (Microsatellite Instability)

  • Polymerase disengages/slips on short repeats ⇒ loop-out

  • If loop escapes MMR, indel results; expansions more common than contractions

  • Mechanism underlies triplet-repeat disorders (e.g., Huntington disease, Fragile X)

Limitations & Pathological Implications

  • Not all lesions repaired before replication

  • Repair attempts can fail or introduce new errors

  • Repair-gene mutations (e.g., MSH2, MLH1 in Lynch syndrome) drive disease

Taxonomy of Mutations

By Origin

  • Spontaneous: arise without exogenous agent

  • Induced: require mutagen exposure

By Cell Type

  • Germ-line: present in egg/sperm; therefore in every somatic cell

  • Somatic: post-zygotic; mosaic distribution

By Molecular Change

  • Substitution

    • Transition: purine↔purine or pyrimidine↔pyrimidine

    • Transversion: purine↔pyrimidine

  • Insertion / Deletion (indel)

  • Large structural rearrangements

By Effect on Translation

  • Synonymous (silent)

  • Missense (nonsynonymous)

  • Nonsense (premature STOP)

  • Frameshift (reading-frame shift)

  • Splice-site / splice-regulatory

By Functional Consequence

  • Loss-of-function (null/knockout)

  • Hypomorphic (reduced)

  • Hypermorphic (increased)

  • Gain-of-function (novel/ectopic)

  • Conditional (temperature-sensitive, etc.)

Context-Dependent Impact of Variants

  • “Junk” / intergenic DNA usually neutral but may harbour regulatory elements

  • Promoter variants affect transcription initiation

  • 5’/3’-UTR variants influence mRNA stability, localisation, translation efficiency

  • Coding-sequence variants alter amino-acid content or create stop codons

  • Intronic or exonic splice-site variants disrupt canonical or auxiliary splicing signals

  • Even nominally “silent” substitutions can modify exonic splicing enhancers/silencers

Mutation → Disease Pathways (Selected Illustrations)

  • Mutations in coding region altering protein structure/function

    • Haemoglobinopathies

    • Hb S (E6V in β-globin)

    • Hb Hammersmith, Hb Kempsey, Hb Kansas

    • Achondroplasia (FGFR3 G380R; gain-of-function)

  • Mutations affecting RNA stability/splicing

    • β-thalassaemia splice mutations → reduced β-globin

    • α-thalassaemia deletions

  • Mutations impacting gene dosage/regulation

    • Monosomies/Trisomies (e.g., Down syndrome – chr21 trisomy)

    • Charcot-Marie-Tooth 1A (PMP22 duplication)

    • Hereditary Persistence of Fetal Haemoglobin (HPFH) – promoter changes

    • Oncogene activation (MYC, RAS) via enhancer hijacking, amplification, or translocation

  • Tumour suppressor loss (p53, RB1) typically via loss-of-function

Laboratory & Diagnostic Relevance

  • Cytogenetics leverages cell-cycle staging to visualise chromosomes (metaphase spreads)

  • Understanding gametogenesis explains inheritance patterns (autosomal vs sex-linked, uniparental disomy)

  • Knowledge of repair & mutagenesis underpins assays for genotoxicity, MSI testing, HRD scoring

  • Principles of chromosomal rearrangement biology guide design/interpretation of FISH, karyotype, and sequencing data

Ethical, Philosophical & Practical Considerations

  • Germ-line editing (CRISPR) raises debate over intentional introduction/repair of mutations

  • Environmental mutagen exposure has public-health ramifications (UV sunlight, industrial chemicals, radiation)

  • Balancing somatic gene therapy vs oncogenic risk when manipulating repair pathways