13.5 Molecular Genetics & Enzymes: Replication, Repair, Gene Expression, Molecular Techniques, Lysosomal Storage Diseases & Enzyme Kinetics

Why this area matters

The TOS lists:

• "gene expression (molecular basis of oncogenesis, techniques in molecular biology research, molecular and genetic-based therapeutic methods)";
• "enzymes: properties and function, substrate binding, kinetics, regulation, inhibition".

Molecular diagnostics such as GeneXpert for tuberculosis, RT-PCR for COVID-19, newborn screening and HbA1c are now routine in Philippine practice, so the principles behind them are tested.

Eukaryotic DNA Replication Machinery and Telomere Maintenance

DNA replication in human cells is semiconservative, bidirectional, and occurs during the S-phase of the cell cycle at multiple origins of replication (AT-rich consensus sequences).

Enzymatic Orchestration at the Replication Fork

• DNA Helicase: Unwinds the double helix at the replication fork by breaking hydrogen bonds between base pairs. Mutations in the BLM (RecQ helicase) gene cause Bloom Syndrome (dwarfism, telangiectatic erythema, chromosomal instability, and cancer predisposition).
• Single-Stranded DNA-Binding Proteins (RPA in eukaryotes): Bind exposed single strands to prevent reannealing and hairpins.
• Topoisomerases (Type I and II): Create transient single-strand (Type I) or double-strand (Type II) nicks to relieve negative and positive torsional supercoiling ahead of the advancing replication fork.
  • Pharmacological Targets: Fluoroquinolones (ciprofloxacin) inhibit prokaryotic topoisomerase II (DNA gyrase) and topoisomerase IV. Etoposide/teniposide inhibit eukaryotic Topoisomerase II. Irinotecan/topotecan inhibit eukaryotic Topoisomerase I.
• Primase: Specialized RNA polymerase that synthesizes short ($10\text{ bp}$) RNA primers required to provide free $3'\text{-OH}$ groups for DNA polymerases.
• Eukaryotic DNA Polymerases:
  • DNA Polymerase $\alpha$: Associates with primase to initiate synthesis by adding a short run of DNA onto the RNA primer.
  • DNA Polymerase $\delta$: Synthesizes the lagging strand (Okazaki fragments); possesses $3'\to 5'$ exonuclease proofreading activity.
  • DNA Polymerase $\epsilon$: Synthesizes the continuous leading strand in the $5'\to 3'$ direction; possesses $3'\to 5'$ exonuclease proofreading activity.
  • DNA Polymerase $\beta$: Exclusively involved in Base Excision Repair (BER).
  • DNA Polymerase $\gamma$: Replicates and repairs mitochondrial DNA.
• DNA Ligase: Catalyzes the formation of phosphodiester bonds to seal Okazaki fragment nicks, consuming ATP.

Telomerase Dynamics in Aging and Oncogenesis

Because DNA polymerases require an RNA primer and synthesize strictly $5'\to 3'$, the removal of the terminal lagging-strand primer leaves an unreplicated $3'$ gap (the "end-replication problem"). Telomerase is a specialized ribonucleoprotein complex acting as an RNA-dependent DNA polymerase (reverse transcriptase). It carries an internal RNA template ($3'\text{-CAAUCCCAAUC-}5'$) that directs the addition of tandem non-coding hexanucleotide repeats ($5'\text{-TTAGGG-}3'$) onto the $3'$ ends of eukaryotic chromosomes.

• Active in germ cells, hematopoietic stem cells, and basal skin keratinocytes; inactive in differentiated somatic cells, leading to progressive telomere shortening that triggers replicative senescence (the Hayflick limit).
• Reactivated in $>85-90\%$ of human malignancies, enabling limitless replicative immortality.

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DNA Repair Mechanisms and Human Genomic Instability Syndromes

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Eukaryotic Transcription and Post-Transcriptional Processing

Eukaryotic transcription is catalyzed by three distinct RNA polymerases, each dedicated to specific RNA subclasses:

• RNA Polymerase I: Synthesizes large ribosomal RNAs (28S, 18S, and 5.8S rRNA) in the nucleolus.
• RNA Polymerase II: Synthesizes messenger RNA (pre-mRNA), small nuclear RNAs (snRNAs), and microRNAs.
  • Toxicity: Ingestion of the Death Cap mushroom (Amanita phalloides) causes ingestion of $\alpha$-amanitin, a cyclic octapeptide that binds tightly to RNA Polymerase II, halting mRNA synthesis and triggering massive hepatic centrilobular necrosis and renal tubular failure.
• RNA Polymerase III: Synthesizes tRNA and 5S rRNA.
• Mnemonic: "RNA Pol I, II, III = Rampant, Massive, Tiny" (rRNA, mRNA, tRNA).

Eukaryotic Promoters and Pre-mRNA Processing

Transcription initiation requires the recognition of promoter consensus sequences: the TATA box (Hogness box, located $-25\text{ to }-30\text{ bp}$ upstream) and the CAAT box ($-75\text{ to }-80\text{ bp}$ upstream). Enhancers (binding activators) and Silencers (binding repressors) can reside upstream, downstream, or within introns, looping DNA to modulate transcription rates.

Nascent pre-mRNA undergoes three obligatory processing events inside the nucleus prior to nuclear export:

1. $5'$ Capping: Addition of 7-methylguanosine via an unusual $5'\text{-to-}5'$ triphosphate bridge; protects against exonuclease degradation and serves as the ribosomal binding signal for translation initiation.
2. $3'$ Polyadenylation: Cleavage at the downstream consensus sequence ($5'\text{-AAUAAA-}3'$), followed by the template-independent addition of approximately $200$ adenine residues by Poly-A Polymerase; protects against $3'$ degradation and mediates nuclear export.
3. Splicing of Introns: Precise excision of non-coding introns by the spliceosome, a dynamic complex of five small nuclear ribonucleoproteins (snRNPs: U1, U2, U4, U5, U6). Splicing occurs via two transesterification reactions: the branch-point adenosine ($2'\text{-OH}$) attacks the $5'$ splice donor site ($5'\text{-GU-}3'$), forming a looped lariat intermediate, followed by cleavage at the $3'$ splice acceptor site ($3'\text{-AG-}3'$). Autoantibodies to snRNP core proteins (anti-Smith / anti-Sm) are highly specific for Systemic Lupus Erythematosus (SLE); anti-U1 RNP antibodies characterize Mixed Connective Tissue Disease (MCTD).

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The Genetic Code, Translation, and Ribosomal Peptidyl Transfer

Characteristics of the Genetic Code

• Unambiguous: Each codon (triplet of nucleotides) specifies exactly one amino acid.
• Degenerate / Redundant: 61 sense codons specify only 20 amino acids (e.g., leucine is encoded by 6 different codons). The exceptions are Methionine (AUG) and Tryptophan (UGG), encoded by single codons. Redundancy is accommodated by Crick's Wobble Hypothesis, which permits non-Watson-Crick base pairing between the $3'$ base of the mRNA codon and the $5'$ base of the tRNA anticodon.
• Universal: Conserved across all known life (with minor exceptions in human mitochondrial DNA).
• Non-overlapping and Comma-less: Read continuously, three nucleotides at a time, from a fixed start codon.

Translation Phases

1. Initiation: Small ribosomal subunit ($40\text{S}$) binds the $5'$ cap and scans for the AUG start codon (coding for Methionine in eukaryotes; N-formylmethionine in prokaryotes). Assembly of the $60\text{S}$ subunit forms the active $80\text{S}$ ribosome, placing Met-tRNA in the P site (requires GTP).
2. Elongation: Incoming aminoacyl-tRNA binds the A site (facilitated by elongation factors and GTP). Peptidyl Transferase (a ribozyme within the $28\text{S}$ rRNA of the large subunit) forms a peptide bond between the P-site amino acid and the A-site amino acid. The ribosome translocates three nucleotides downstream, shifting the deacylated tRNA to the E site (exit) and the growing peptide chain to the P site.
3. Termination: When a stop codon (UAA, UAG, UGA; Mnemonic: "U Are Away, U Are Gone, U Go Away") enters the A site, eukaryotic release factors (eRFs) bind, cleaving the completed polypeptide chain from the tRNA.

Regulation of gene expression and the molecular basis of oncogenesis

Genes are switched on and off by:

• transcription factors at promoters and enhancers (for example steroid-hormone receptors);
• chromatin modification: histone acetylation opens chromatin, while DNA methylation at CpG islands silences genes, the basis of imprinting and X-inactivation;
• alternative splicing;
• microRNAs, which block translation or degrade mRNA.

In cancer, proto-oncogenes are activated by point mutation (KRAS), amplification (HER2) or translocation (BCR-ABL, MYC). Tumor suppressors (TP53, RB, APC) are lost by two hits. Epigenetic silencing, such as MLH1 promoter methylation, can also inactivate a gene.

Techniques in molecular biology

Molecular and gene-based therapy includes:

• recombinant proteins (insulin, erythropoietin, clotting factors);
• monoclonal antibodies (rituximab, trastuzumab);
• targeted kinase inhibitors (imatinib);
• antisense oligonucleotides;
• gene therapy with viral vectors (for ADA-SCID, spinal muscular atrophy);
• mRNA vaccines;
• emerging CRISPR-Cas9 gene editing (approved for sickle cell disease).

Enzymes: properties, kinetics and regulation

Enzymes are biological catalysts, mostly proteins (ribozymes are RNA). They lower the activation energy but do not change the equilibrium or the free energy of the reaction. Many need cofactors (metal ions such as Zn2+, Mg2+ and Cu2+) or coenzymes, which are usually vitamin-derived (TPP, FAD, NAD+, PLP, biotin).

Michaelis-Menten kinetics:

$$v = \frac{V_{max}[S]}{K_m + [S]}$$

• Km is the substrate concentration at which velocity is half of Vmax. A low Km means high affinity: hexokinase has a low Km and glucokinase a high Km.
• Vmax is proportional to the amount of enzyme.
• On a Lineweaver-Burk (double-reciprocal) plot, the y-intercept is 1/Vmax, the x-intercept is -1/Km and the slope is Km/Vmax.

Regulation of enzyme activity:

• Allosteric control gives sigmoidal kinetics, for example ATP and citrate inhibiting PFK-1.
• Covalent modification: phosphorylation by kinases, dephosphorylation by phosphatases (glycogen phosphorylase and synthase).
• Zymogen activation by proteolysis: trypsinogen, clotting factors.
• Induction or repression of enzyme synthesis: insulin induces glucokinase; heme represses ALA synthase.
• Compartmentalization, as with the urea cycle split between mitochondria and cytosol.
• Feedback inhibition by the end product.

Isoenzymes catalyze the same reaction but differ in structure and tissue distribution, for example LDH 1 to 5, CK-MM, CK-MB and CK-BB, and alkaline phosphatase from bone versus liver.

Clinical enzymology:

• ALT (more liver-specific) and AST (also heart and muscle; AST more than twice ALT suggests alcoholic liver disease);
• ALP and GGT (cholestasis);
• amylase and lipase (pancreatitis; lipase more specific);
• CK-MB (myocardium, now largely replaced by cardiac troponins, which are not enzymes);
• acid phosphatase (prostate, semen);
• G6PD (newborn screening).

Sphingolipidoses and Lysosomal Storage Disorders

Sphingolipidoses are autosomal recessive disorders (except X-linked Fabry disease) caused by deficiencies of specific lysosomal acid hydrolases, leading to progressive accumulation of undegraded sphingolipids within the reticuloendothelial system and central nervous system.

High-Yield Differential Diagnosis of Sphingolipidoses