Biochemistry Notes: DNA Secondary and Tertiary Structure

Introduction to Biochemistry and the Genotype-Phenotype Relationship

• Foundational Quote: Rosalind Franklin stated, "Science and everyday life cannot and should not be separated."
• Genetics to Phenomics Hierarchy: The relationship between genotype and phenotype is mediated through a series of biological layers and corresponding "omics" fields:
  • Genotype: DNA (Genomics) and Epigenetic modifications (Epigenomics).
  • RNA: Genetic expression (Transcriptomics).
  • Protein: Protein-protein interactions (Proteomics).
  • Metabolite: Metabolic pathways (Metabolomics).
  • Flux: Flux distributions (Fluxomics).
  • Phenotype: The visible or measurable result, which can be perturbed by diseases leading to variations in any of the above levels (e.g., disease-associated genetic variants, disease-perturbed gene expression, etc.).

Nucleic Acid Structural Hierarchy

• Primary Structure: The sequence of nucleotides in a single strand (e.g., $5'-\text{GCGCUGUGUCGA}-3'$).
• Secondary Structure: Localized folding and interactions within or between strands.
  • RNA Examples: Helix, Stem loop, Pseudoknot.
  • DNA Examples: A-form, B-form, Z-form helices.
• Tertiary Structure: Higher-order folding and three-dimensional arrangement.
  • Examples include supercoiling, nucleosomes, and the wrapping of DNA around histone proteins.
• Quaternary Structure: Interaction of nucleic acids with other macromolecules, such as the formation of the full chromatin structure or complexes like the ribosome (DNA-histone protein complexes).

Historical Context and Chargaff’s Rules

• Chargaff’s Contributions (1940s): Erwin Chargaff (Aug 1905 – Jun 2002) established rules based on base composition across species prior to the WCF (Watson-Crick-Franklin) model.
  • Rule 1: The base composition of DNA varies between different species.
  • Rule 2: The base composition of DNA from different tissues within the same species does not vary.
  • Rule 3: Within a species, base composition remains constant regardless of age, nutritional state, or environmental changes.
  • Rule 4: In all cellular DNA, the number of adenine residues equals the number of thymine residues ($\#A = \#T$) and the number of cytosine residues equals the number of guanine residues ($\#C = \#G$). Consequently, the sum of purines equals the sum of pyrimidines ($\#(\text{A+G}) = \#(\text{C+T})$).
• Outcome of Chargaff's Rules: The parity observed ($A=T$, $C=G$) implies that the two antiparallel strands of DNA are complementary.

The Elucidation of DNA Structure

• Rosalind Elsie Franklin (1920-1958):
  • Earned a P.H.D. in Chemistry in 1945 from Cambridge (born in London, England).
  • Conducted X-ray diffraction work that resulted in Photo 51 (1952).
  • Investigative process: It took approximately $100\,\text{hours}$ to produce the image and $365\,\text{days}$ to analyze it.
  • Her work supported the discovery of the B-form DNA helix.
• Watson and Crick Publication (1953): Published in Nature (No. 4316, April 25, 1953).
  • They proposed a right-handed double helix where phosphate-sugar chains are on the outside and bases are paired on the inside.
  • Critique of Pauling and Corey’s Model: Pauling and Corey had proposed a three-chain structure with phosphates near the axis. Watson and Crick argued this was unsatisfactory because:
    1. The negatively charged phosphates would repel each other.
    2. Van der Waals distances were too small.
• Additional Figures: Aaron Klug (1982 Nobel Winner) worked with Franklin's data later; Raymond Gosling was Franklin's student and co-author.

Biophysical Parameters of DNA Secondary Structure

• Stability Interactions:
  • Hydrogen Bonding: The ideal distance for H-bond interaction is approximately $3\,\text{Å}$. This distance is maintainable in both a "ladder" configuration and a "double helix."
  • Van der Waals (VDW) / Aromatic Stacking: The ideal distance for aromatic stacking is $3.4\,\text{Å}$.
  • Geometric Constraint: VDW interactions are proportional to $1/r^6$. The $3.4\,\text{Å}$ stacking distance can only be maintained in the double helix conformation, not the ladder.
• Main Features of B-form DNA:
  • Helicity: Right-handed.
  • Rise: $3.4\,\text{Å}$ per base pair.
  • Pitch: One full turn is approximately $36\,\text{Å}$ (or $3.6\,\text{nm}$).
  • Density: Approximately $10.5\,\text{bases/turn}$.
  • Conformation: Sugar is in the $C2'\text{-endo}$ pucker; glycosidic bond is in the anti conformation.
  • Solubility: Hydrophilic phosphate backbones are solvent-exposed; hydrophobic bases are packed in the interior.
• Grooves: Base pairing is offset from the helical axis, creating a Major Groove and a Minor Groove. These serve as binding sites for endogenous ligands and drugs.

Comparison of DNA Conformations (A, B, and Z)

• A-DNA:
  • Conditions: Favored under dehydrating conditions ($0-75\,\%$ humidity) or in stretches of 4 purines in a row.
  • Structure: More rigid and less stable than B-DNA; bases stack off-center.
  • Conformation: Sugar is in the $3'\text{-endo}$, anti conformation.
  • Turn: $28\,\text{Å}$ per turn.
• B-DNA:
  • Conditions: Standard physiological conditions ($75-100\,\%$ humidity).
  • Conformation: Sugar is in the $C2'\text{-endo}$, anti conformation.
  • Turn: $34-36\,\text{Å}$ per turn.
• Z-DNA:
  • Structure: Left-handed helix with a jagged/zigzag backbone.
  • Sequence: Occurs in long stretches of alternating purine-pyrimidine sequences.
  • Base Flipping: Rotation of the sugar-phosphate backbone by $180^\circ$.
  • Conformation: Alternation of syn (for purines) and anti (for pyrimidines); purine sugar is in the $C3'\text{-endo}$ pucker.

Advanced Secondary Structures and Sequences

• Palindromes and Repeats:
  • Inverted Repeat: Sequence that is self-complementary on the same strand, promoting intrastrand pairing.
  • Mirror Sequence: A sequence that reads the same forward and backward on the same strand but does not promote hairpins.
  • Hairpins and Cruciforms: Formed from inverted repeats. These leave bases unpaired in loops and are less stable than double-stranded DNA (dsDNA). They are often found near promoter regions and act as targets for regulatory proteins.
• H-DNA (Triple Helix):
  • Found in promoter regions.
  • Requirements: Palindromic polypurines or polypyrimidines and Hoogsteen base pairing.
  • Stability is lower than double-exed DNA due to the loss of some standard base pairing.
• G-Quadruplexes (Ganosine Tetraplexes):
  • Planar structures formed via Hoogsteen G-G bonding.
  • Contain a central cavity large enough for a monovalent metal cation (e.g., $K^+$), which provides stability via coordinate bonding with carbonyl oxygens.
  • Found in telomeres and promoter regions.

Telomeres and Longevity

• Telomeres: Protective "caps" at the end of DNA strands that regulate molecular longevity.
• Telomerase: An enzyme containing an RNA template with reverse transcriptase activity used to elongate telomeres.
• Outcomes of Telomere Length:
  • Long Telomeres: Maintenance of genomic stability.
  • Short Telomeres: Loss of protective mechanisms, cellular senescence, and DNA damage response.
• Disease Risks Associated with Short Telomeres:
  • Cancers: Lung adenocarcinoma, melanoma, endometrial cancer, ovarian cancer, glioma, neuroblastoma.
  • Degenerative Diseases: Alzheimer's, Coronary heart disease, Type 1 Diabetes, Liver cirrhosis, Idiopathic pulmonary fibrosis.
• Telomeric Stress Factors:
  • Oxidative Stress: Reactive oxygen species ($ROS$) cause strand breaks.
  • Chronic Inflammation: Activates shortening pathways.
• Support/Stability Factors:
  • Antioxidants: Vitamin C, Vitamin E, Polyphenols.
  • Anti-Inflammatory Agents: Omega Fatty Acids, Spermidine.
  • Statins: Possibly reduce inflammatory cytokines.

DNA Packaging and Tertiary Structure

• The Packaging Problem: Linear DNA in a typical eukaryotic cell is longer than the cell diameter ($1-5\,\mu\text{m}$). Packaging is non-random.
• Hierarchy of Packaging:
  • DNA double helix
  • Nucleosomes ($2^\circ$ structure + Histone wrapping)
  • $30\,\text{nm}$ Fibre
  • Loop
  • Rosette
  • Coil
  • Chromatids
  • Chromosome
• Circular DNA (cDNA):
  • Found in prokaryotes (e.g., E. coli) and eukaryotic mitochondria (mtDNA).
  • Distinguishable by the absence of free $5'$ or $3'$ ends.

DNA Topology and Supercoiling

• Linking Number ($L$): A topological property defined as the number of times one strand crosses the other. It remains unchanged by deformation or stretching but changes if the DNA is torn.
• Twist ($T$): A geometric property representing the number of helical turns.
• Writhe ($W$): A geometric property representing the number of turns the duplex axis makes around the superhelical axis (the helix crossing itself).
• Fundamental Equation:     $L = T + W$
• Relaxed DNA: When cDNA properties match linear B-DNA ($1\,\text{turn} \approx 10.5\,\text{bp}$), the DNA is relaxed and free from strain ($W=0$).
• Underwinding: Promotes strand separation to relieve strain, essential for cellular events requiring single-stranded DNA.
• Parameters and Calculations:
  • $L_{relaxed} = L_0 = \frac{\text{Number of base pairs}}{10.5}$
  • Superhelix Density ($\sigma$): $\sigma = \frac{\Delta L}{L_0} = \frac{L - L_0}{L_0}$
  • Practice Problem: For a closed-circular DNA of $4200\,\text{bp}$ and $L = 412$:
    • $L_0 = \frac{4200}{10.5} = 400$
    • $\Delta L = 412 - 400 = 12$
    • $\sigma = \frac{12}{400} = 0.03$
    • Since $\sigma > 0$, this DNA is overwound.
• Topoisomerases: Enzymes that change the linking number.
  • Type I: $\Delta L = 1$
  • Type II: $\Delta L = 2$

Thermodynamics of DNA Denaturation (Melting)

• Process: DNA Double Helix $\rightarrow$ Denatured Random Coil.
• Thermodynamic Parameters ($\Delta G = \Delta H - T\Delta S$):
  • Enthalpy (\Delta H): Positive ($> 0$). Dominated by hydrogen bonding ($A-T = 2$ bonds, $G-C = 3$ bonds) and $\pi$-stacking. Increases with the loss of base pairing.
  • Entropy (\Delta S): Positive ($> 0$). Denaturation leads to a gain in entropy as the highly organized double helix becomes a random coil.
• Temperature Effects:
  • Physiological Temperature: $\Delta G > 0$. Enthalpy dominates ($\Delta H > T\Delta S$). The helix is stable.
  • Elevated Temperature: $\Delta G < 0$. Entropy dominates as the $T\Delta S$ term increases in magnitude. The helix denatures.