Unit 3: Structure of Nucleic Acids; DNA Packaging and Replication

I. Orientation: Nucleic Acids as Informational Polymers

Nucleic acids are unbranched polymers of nucleotides that store, transmit and express genetic information. Their properties (identified by Watson & Crick, 1953, using Franklin's X-ray data and Chargaff's rules) govern every later section.

• Nucleotide unit: a pentose sugar + phosphate + nitrogenous base; sugar is 2′-deoxyribose in DNA, ribose in RNA.
• Bases: purines (adenine, guanine) and pyrimidines (cytosine, thymine in DNA; uracil in RNA).
• Backbone: 3′→5′ phosphodiester bonds; chain has 5′-phosphate and 3′-OH ends giving directionality.
• Chargaff's rules: in DNA, A = T and G = C, so purines = pyrimidines.
• Complementarity: A–T (2 H-bonds), G–C (3 H-bonds); this drives base pairing, replication and hybridisation.

II. Structure of DNA — The Double Helix and Its States

Orientation: DNA is normally an antiparallel double helix whose conformation and topology respond to sequence and physical conditions.

A. Types and structure of DNA

The double helix exists in distinct conformations distinguished by handedness and geometry.

• B-DNA: physiological form; right-handed, ~10.5 bp/turn, ~3.4 Å rise per base, 2 nm diameter; distinct major and minor grooves that proteins read.
• A-DNA: right-handed, dehydrated form; ~11 bp/turn, shorter and wider; seen in DNA–RNA hybrids.
• Z-DNA: left-handed, zig-zag backbone; favoured by alternating purine–pyrimidine (e.g. GC repeats); ~12 bp/turn.
• Structural bonds: phosphodiester backbone + inter-strand hydrogen bonds + base-stacking van der Waals forces stabilise the helix.
• Antiparallel arrangement: one strand 5′→3′, the complement 3′→5′, essential for replication geometry.

B. Denaturation and re-naturation of DNA

Denaturation is the separation of strands by breaking hydrogen bonds; re-naturation is their reassociation.

• Denaturation trigger: heat, extreme pH, or urea/formamide disrupt H-bonds; covalent backbone is untouched.
• Melting temperature (Tm): temperature at which 50% of DNA is single-stranded, tracked by A₂₆₀ absorbance rise.
• Hyperchromic effect: unstacked bases absorb ~40% more UV at 260 nm than duplex DNA.
• GC dependence: higher G–C content (3 H-bonds) raises Tm; approximate relation:
  
  TEXT

  Tm ≈ 81.5 + 0.41(%GC) − (correction for salt/length)

• Re-naturation (annealing): slow cooling lets complementary strands re-pair; rate depends on sequence complexity (Cot analysis) — repetitive DNA reanneals fast.

C. Supercoiling of DNA

Supercoiling is the over- or under-winding of the helix, changing its topological state in a closed molecule.

• Linking number (Lk): total times strands cross; fixed for a covalently closed circle. Lk = Tw + Wr.
  • Twist (Tw): helical turns of one strand about the other.
  • Writhe (Wr): coiling of the axis upon itself.
• Negative supercoiling: underwinding (Lk < relaxed Lk₀); predominant in cells, aids strand separation for replication/transcription.
• Positive supercoiling: overwinding; accumulates ahead of moving polymerases.
• Topoisomerases: relieve strain — type I cuts one strand (ΔLk = ±1), type II (e.g. bacterial gyrase) cuts both and introduces negative supercoils using ATP.

III. Structure of RNA — Single-Stranded Functional Molecules

Orientation: RNA is generally single-stranded, uses ribose and uracil, and folds into functional secondary/tertiary structures.

A. Types and structures of RNA

RNA classes differ in role, length and folded architecture.

• mRNA (messenger): carries coding information; eukaryotic mRNA has a 5′ 7-methylguanosine cap, coding region and 3′ poly-A tail.
• tRNA (transfer): ~76 nt cloverleaf secondary structure folding into an L-shaped tertiary form; carries anticodon loop and 3′ CCA amino-acid attachment site.
• rRNA (ribosomal): structural/catalytic core of ribosomes (e.g. prokaryotic 16S, 23S, 5S); extensive intramolecular base pairing.
• Regulatory/small RNAs: snRNA (splicing), snoRNA (rRNA modification), miRNA/siRNA (gene silencing), and catalytic ribozymes.
• Secondary structure motifs: hairpins, stem-loops, bulges formed by intramolecular Watson–Crick and G–U wobble pairs.

IV. Hierarchical Packaging of DNA — Compacting the Genome

Orientation: ~2 m of human DNA is compacted into a nucleus (~6 µm) through successive folding levels built on histones.

A. Histone proteins

Histones are small, basic (arginine/lysine-rich) proteins that bind acidic DNA electrostatically.

• Core histones: H2A, H2B, H3, H4 — form the nucleosome octamer.
• Linker histone: H1 — binds linker DNA and stabilises higher-order folding.
• Histone-fold and tails: N-terminal tails project outward and carry modifications (acetylation, methylation) that regulate chromatin state.

B. The nucleosome assembly

The nucleosome is the fundamental repeating packaging unit.

• Core particle: ~147 bp of DNA wrapped ~1.65 left-handed turns around a histone octamer (two each of H2A, H2B, H3, H4).
• Beads-on-a-string: nucleosomes separated by ~20–80 bp linker DNA; ~10 nm fibre.
• 30 nm fibre: H1-mediated folding into solenoid/zig-zag arrangement.
• Higher order: looped domains anchored to a scaffold → condensed metaphase chromosome (~700 nm).
• Packing ratio: overall compaction of ~10,000-fold from naked DNA to metaphase chromosome.

V. Enzymes and Proteins in DNA Replication

Orientation: replication is semiconservative and requires a coordinated set of enzymes acting at the replication fork.

A. Enzymes and proteins in DNA replication

Each factor performs a defined step in unwinding, priming, synthesis and joining.

• Helicase: unwinds the duplex at the fork (bacterial DnaB), using ATP.
• Single-strand binding protein (SSB/RPA): coats separated strands, preventing reannealing.
• Topoisomerase/gyrase: relieves torsional strain ahead of the fork.
• Primase: synthesises short RNA primers providing a free 3′-OH.
• DNA polymerase: adds nucleotides 5′→3′; requires template and primer. Bacterial Pol III elongates; Pol I removes primers.
• Sliding clamp + clamp loader: (β-clamp/PCNA) tethers polymerase for processivity.
• DNA ligase: seals nicks between Okazaki fragments via phosphodiester bond.

VI. DNA Replication in Prokaryotes

Orientation: E. coli replicates its single circular chromosome bidirectionally from one origin.

• Origin: single oriC; DnaA binds AT-rich repeats to melt the duplex.
• Bidirectional forks: two forks move outward, forming a theta (θ) intermediate.
• Leading strand: synthesised continuously 5′→3′ toward the fork.
• Lagging strand: synthesised discontinuously as Okazaki fragments (~1000–2000 nt).
• Primer processing: Pol I excises RNA primers (5′→3′ exonuclease) and fills gaps; ligase seals.
• Termination: forks meet at ter sites bound by Tus protein; catenated daughters resolved by topoisomerase.

VII. DNA Replication in Eukaryotes

Orientation: larger linear chromosomes replicate from many origins during a defined S phase.

• Multiple origins: thousands of replicons fire in a temporally ordered programme.
• Licensing: pre-replication complex (ORC, Cdc6, Cdt1, MCM helicase) assembles in G1; activated once per cycle to prevent re-replication.
• Polymerases: Pol α (primase), Pol δ (lagging), Pol ε (leading); PCNA as sliding clamp.
• Chromatin coupling: nucleosomes disassemble ahead and reassemble behind the fork.
• End-replication problem: lagging-strand ends shorten; telomerase (RNA-templated reverse transcriptase) extends telomeric repeats.

VIII. Fidelity of DNA Replication

Orientation: cells achieve extremely low error rates through layered accuracy mechanisms.

A. Fidelity of DNA replication

Fidelity is the accuracy of nucleotide selection and correction.

• Base selection: correct Watson–Crick geometry is energetically favoured; error ~1 in 10⁵.
• 3′→5′ proofreading exonuclease: polymerase excises mismatched 3′ nucleotides, improving accuracy ~100-fold.
• Mismatch repair (MMR): post-replication system (MutS/MutL in bacteria) recognises mismatches and excises the newly synthesised strand.
• Combined error rate: ~1 mistake per 10⁹–10¹⁰ nucleotides after all mechanisms.
• Strand discrimination: in E. coli, transient hemimethylation (Dam methylation) marks the parent strand for MMR templating.

IX. Rolling Circle Replication

Orientation: an alternative mode used by plasmids, some phages (e.g. φX174) and for viral/rDNA amplification.

A. Rolling circle replication

A circular template generates long single-stranded concatemers.

• Initiation: an initiator protein nicks one strand at the origin, exposing a 3′-OH.
• Elongation: polymerase extends the 3′ end, displacing the old strand as the circle "rolls".
• Concatemer: continuous synthesis yields a multimeric single-stranded product with repeated genome copies.
• Resolution: the displaced strand is cleaved into unit lengths and circularised; the complementary strand is later synthesised.
• Uses: bacteriophage genome replication, bacterial conjugation transfer, and amplification of ribosomal DNA in some eukaryotes.