DNA Study Guide

📖 Core Concepts DNA – double‑helix polymer of two antiparallel polynucleotide strands; each nucleotide = deoxyribose + phosphate + A, T, C, or G. Antiparallel orientation – one strand runs 5′→3′, the complementary strand runs 3′→5′. Watson‑Crick base pairing – A pairs with T (2 H‑bonds), G pairs with C (3 H‑bonds); a purine always pairs with a pyrimidine. B‑DNA – right‑handed helix dominant in cells; 10.4 bp per turn, 3.4 Å rise per base pair, 20 Å diameter. Replication – semiconservative; DNA polymerase adds nucleotides 5′→3′ using the template strand; leading strand continuous, lagging strand discontinuous (Okazaki fragments). Transcription – RNA polymerase II makes an RNA copy, substituting U for T; promoter recognition (major groove) → open complex → elongation → termination. Translation – mRNA codons (triplets) specify amino acids; three stop codons (UAA, UAG, UGA). DNA repair – base excision (small lesions), nucleotide excision (bulky lesions), homologous recombination (DSBs), non‑homologous end joining (error‑prone DSB repair). Chromatin – DNA wraps 147 bp around histone octamers → nucleosomes → higher‑order fibers; histone tail modifications (acetyl, methyl, etc.) regulate accessibility. Epigenetics – 5‑methylcytosine (CpG) → transcriptional repression; DNA methyltransferases use S‑adenosyl‑L‑methionine. Supercoiling – negative supercoiling = under‑wound (favours strand separation); topoisomerases relieve or introduce supercoils. --- 📌 Must Remember Chargaff rules: %A = %T, %G = %C. Helical geometry: 1 turn ≈ 34 Å, 10.4 bp/turn, radius ≈ 10 Å. Hydrogen bonds: A–T = 2, G–C = 3 → GC‑rich regions melt at higher temperature. Semiconservative replication (Meselson–Stahl). Leading vs. lagging synthesis – continuous vs. Okazaki fragments. RNA polymerase substitutes U for T; no thymine in RNA. Stop codons: UAA, UAG, UGA. DNA damage types: UV thymine dimers, oxidative 8‑oxoguanine, intercalators distort helix. Repair choice rule: Small base lesions → Base Excision Repair (BER); bulky lesions → Nucleotide Excision Repair (NER). Topoisomerase I = single‑strand break; Topo II = double‑strand break. --- 🔄 Key Processes DNA Replication (simplified) Origin binding & helicase loading – unwinds DNA. Primase synthesizes short RNA primers (5′‑RNA‑3′). DNA polymerase III (prokaryote) / DNA polymerase δ/ε (eukaryote) extends primers 5′→3′. Leading strand: continuous synthesis toward fork. Lagging strand: discontinuous synthesis → Okazaki fragments. DNA ligase seals nicks; DNA polymerase I replaces RNA primers with DNA (prokaryotes). Transcription Initiation RNA polymerase II + sigma (in bacteria) binds promoter (major groove). DNA melting → open complex. RNA synthesis begins at +1 site, proceeds 5′→3′. Base Excision Repair DNA glycosylase removes damaged base → abasic site. AP endonuclease cuts backbone 5′ to abasic site. DNA polymerase β fills gap; DNA ligase seals. Homologous Recombination (DSB repair) DSB → 5′→3′ resection → 3′ ssDNA overhangs. Rad51 mediates strand invasion into homologous duplex. DNA synthesis using sister chromatid as template. Holliday junction resolution → crossover or non‑crossover. --- 🔍 Key Comparisons A‑T vs. G‑C: 2 H‑bonds vs. 3 H‑bonds → GC ↑ melting temperature. B‑DNA vs. A‑DNA vs. Z‑DNA: B: right‑handed, wide shallow major groove, physiological conditions. A: right‑handed, deep narrow major groove, dehydration. Z: left‑handed, zig‑zag backbone, alternating purine‑pyrimidine repeats. Leading vs. Lagging strand synthesis: continuous vs. discontinuous (Okazaki fragments). Homologous recombination vs. Non‑homologous end joining: high‑fidelity template use vs. direct ligation (error‑prone). Base excision vs. Nucleotide excision repair: single‑base vs. bulky lesion removal. --- ⚠️ Common Misunderstandings DNA polymerase synthesizes 3′→5′ – false; all DNA polymerases add nucleotides only 5′→3′. RNA contains thymine – incorrect; RNA contains uracil instead of thymine. All DNA in cells is B‑form – neglects A‑DNA, Z‑DNA, and Hoogsteen pairing under stress or ligand binding. Supercoiling is always “negative” – cells also generate positive supercoils ahead of replication forks. Every DNA damage is fixed by BER – bulky lesions require NER or other pathways. --- 🧠 Mental Models / Intuition DNA as a twisted ladder: rungs = base pairs (purine‑pyrimidine), rails = sugar‑phosphate backbone. Replication fork = zipper: helicase opens the zipper, leading strand follows the pull, lagging strand works in short “teeth” (Okazaki). Chromatin as “beads on a string”: nucleosome = bead (DNA wrapped 2 turns), spacing determines accessibility. Repair pathways as “toolboxes”: small cuts → precise (BER); big dents → larger tools (NER, HR, NHEJ). --- 🚩 Exceptions & Edge Cases Hoogsteen base pairs: rotated purine; occur under superhelical stress or ligand binding. Positive supercoiling: generated ahead of transcription/replication; resolved by topoisomerase II. Non‑canonical bases: 5‑methylcytosine, N⁶‑methyladenine – epigenetic marks, not part of the canonical A‑T‑G‑C set. Antisense transcription: antisense strand can produce regulatory RNAs that modulate sense mRNA stability. --- 📍 When to Use Which Choose repair pathway: Small oxidized base → BER. UV‑induced thymine dimer → NER. DSB with sister chromatid available (S/G2) → Homologous recombination. DSB in G1 → Non‑homologous end joining. Select DNA polymerase: High‑fidelity cloning → proofreading polymerase (e.g., Pfu). Rapid PCR with tolerable error → Taq polymerase (no proofreading). Identify DNA conformation: Dehydrated samples → expect A‑DNA. Alternating purine‑pyrimidine repeats + high salt → Z‑DNA possible. Use assay type: Detect specific sequence → PCR or restriction digest. Quantify repeat number (forensics) → STR profiling. --- 👀 Patterns to Recognize Promoter motifs: TATA box (−30), −10 (Pribnow) and −35 elements in prokaryotes; CpG islands often upstream of housekeeping genes. GC‑rich stretches → higher melting temperature, often found in exon regions. Repeated “GGGTT” or “CAG” motifs → prone to forming Z‑DNA or hairpins. STR patterns in forensic loci: identical repeat numbers = same individual; differences = polymorphism. DNA‑binding protein footprints → protected regions in DNase‑I hypersensitivity assays. --- 🗂️ Exam Traps “DNA polymerase can synthesize DNA in the 3′→5′ direction.” – misleading; only exonuclease activity works 3′→5′ for proofreading. “All RNA contains uracil instead of thymine, but mitochondrial RNA contains thymine.” – false; mitochondrial RNA also uses uracil. “Supercoiling only occurs in prokaryotes.” – eukaryotic chromatin also experiences supercoiling, managed by topoisomerases. “Every DNA‑binding protein interacts with the major groove.” – some proteins bind the minor groove or the backbone (non‑specific interactions). “Z‑DNA is always pathological.” – Z‑DNA can be regulatory; its presence is sequence‑dependent, not inherently harmful. ---