In Depth Notes on DNA and RNA Structure, Stabilization, and Topoisomerases

Current Agenda

• Topics to Cover:
  • Structure of DNA and RNA
  • Physico-chemical forces stabilizing DNA/RNA
  • Degrees of freedom in DNA/RNA structures
  • RNA intra-strand base pairing
  • Supercoiling and topoisomerases
  • DNA packaging

A Closer Look at DNA Structure

• Structure Details:
  • DNA consists of two antiparallel polynucleotide strands.
  • Strands are wound in a right-handed manner around a central axis.
  • Bases are positioned in the core, while the sugar-phosphate backbone is exterior, forming major and minor grooves.
  • Approx. 10 base pairs (bp) per turn with a helical twist of 36° per bp.
  • The helix pitch (rise per turn) is 34 Å.
• Classical Form: B-DNA
• Base Pairing: Complementary base pairing identified by Watson & Crick in 1953 leads to a double-stranded, double helix.

DNA Forms

• A-DNA:
  • Rare, typically found dehydrated.
  • Helix rotates 200° perpendicular to the axis.
  • Has a deep major groove and a flat minor groove. Left-handed helix with greater bp spacing compared to B-DNA.
• Z-DNA:
  • Forms left-handed helix, deeper major groove.
  • Transient in nature; biological function remains unclear.
  • Can result from G-C rich segments converting to Z-form by rotation.

RNA Structure and Properties

• A-RNA (RNA-II): 11 base pairs/turn, helical pitch of 30.9 Å.
• DNA-RNA Hybrid: Has A-form characteristics, 10.9 base pairs/turn, helical pitch of 31.3 Å.
• RNA Secondary Structures:
  • Typically single-stranded, can form hairpin structures.
  • Examples include yeast tRNA and 5S RNA from Haloarcula marismortui.

Stabilization Forces for DNA

• Key Stabilization Mechanisms:
  • Hydrogen bonding: Watson-Crick base pairing yields thermodynamically stable interactions.
  • Stacking interactions: van der Waals forces between bases.
  • Cationic shielding: contributes to structural stability.

Denaturation vs. Reannealing of DNA

• Denaturation Conditions:
  • High temperature, low ionic concentration, extreme pH.
• Renaturation Conditions:
  • Low temperature, appropriate ionic concentration and pH.
• G+C Content: DNA with high G+C content has a higher melting temperature (Tm).

DNA Flexibility and Structure

• Conformational Flexibility: DNA can breathe, bubble, bend, and melt due to limited rotational freedom of phospho-ribose segments.
• Base rotation between anti and syn conformations is limited due to steric hindrance.
• Flexibility is crucial for sequence-specific recognition by proteins.

DNA Packaging and Chromosome Structure

• Human Genome: Each chromosome contains approx. 1 x 10^8 base pairs; stretched out, DNA measures 2 meters.
• Packaging involves coiling to manage length while maintaining information accessibility.

Supercoiling and Topoisomerases

• Supercoiling: Results from twisting DNA; can be positive (overwound) or negative (underwound).
• Linking Number (Lk): Defined as the total number of times one strand wraps around another; changes with supercoiling.
• Topoisomerases: Enzymes that regulate DNA supercoiling:
  • Type I: Change linking number by 1, do not require ATP.
  • Type II: Change linking number by 2, require ATP.

Mechanisms of Topoisomerase Action

• Type I Topoisomerases:
  • Cleave one strand, allowing the other to pass through; no ATP needed.
• Type II Topoisomerases:
  • Cleave both strands simultaneously; facilitate passage and re-seal the break. Require ATP for function.

Topoisomerase Inhibitors

• Clinical Importance:
  • Certain inhibitors like Camptothecin target Type IB topoisomerases; interfere with DNA replication leading to cell death—useful in cancer therapy.
  • Ciprofloxacin and others target Type II; prevent DNA repair and replication.

Summary and Review Questions

• Understand the functions of supercoiling in DNA and its importance in genetic information storage.
• Ability to calculate linking numbers and differentiate between DNA forms (A, B, Z).
• Importance of topoisomerases in regulating DNA structure and implications for therapeutic interventions.