Newcastle School of Biomedical, Nutritional and Sport Sciences

DNA Structure, Function, and Replication

Prof Heath Murray
heath.murray@newcastle.ac.uk

Overview of Session

  1. Franklin & Wilkins 1952
  2. Watson & Crick 1953
  3. Meselson & Stahl 1958
  4. Kornberg 1958
  5. Initiation
  6. Unwinding
  7. Summary

This session will cover:

  • X-ray fibre diffraction (Franklin & Wilkins, 1952)
  • Double-Helix Model for DNA Structure (Watson & Crick, 1953)
  • Semi-Conservative Replication (Watson & Crick, 1953; Meselson & Stahl, 1958)
  • DNA Replication (Kornberg, 1958)
  • Initiation of DNA Replication
  • Unwinding of DNA
  • Summary of Key Concepts

Franklin & Wilkins 1952

X-ray Fibre Diffraction

  • Pioneering studies by Rosalind Franklin and Maurice Wilkins in 1952.
  • Used X-ray diffraction to determine the helical structure of DNA.
  • Key observations included helical radius of 10 Å (angstroms).

Watson & Crick 1953

Double-Helix Model for DNA Structure

  • Developed a double-helix model of DNA structure based on previous work by Franklin & Wilkins.
  • Stated that DNA is a right-handed double helix made of two anti-parallel strands:
    • One strand runs in the 5’-3’ direction, the other in the 3’-5’ direction.
    • The backbone is on the outside, composed of alternating sugar and phosphate groups.

Base Pairing

  • Defined specific base pairing:
    • Adenine (A) pairs with Thymine (T) via two hydrogen bonds.
    • Guanine (G) pairs with Cytosine (C) via three hydrogen bonds.
    • Compares base pairing to ‘steps’ on a double-sided spiral staircase.

Dimensional Properties

  • Double-helix dimensions:
    • One turn of helix = 3.4 nm
    • 10 base pairs per turn
    • Rise per base = 0.34 nm
    • Helix diameter = 2 nm
  • Total spacing defined as:
    • 34 Å for helical repeat.
    • 3.4 Å spacing per base.

Structural Stability

  • Contributes to structural integrity through:
    • Hydrophobic effect: Hydrophobic bases sheltered inside and charged phosphate backbone on outside.
    • Hydrogen bonds between complementary bases: A-T = 10 kJ/mol, G-C = 15 kJ/mol.
    • Van der Waals forces between stacked bases contributing 4 kJ/mol.

Meselson & Stahl 1958

Semi-Conservative Replication

  • Investigated how DNA replicates, confirming the semi-conservative model:
    • Each new strand is formed with one old and one new strand, determined by the sequence on the original strand.

Experimental Method

  • Utilized heavy nitrogen isotope 15N and light nitrogen isotope 14N to label DNA.
  • Separated DNA using density-gradient equilibrium sedimentation in CsCl to observe intermediate strands.
  • Results showed:
    • Heavy (15N)
    • Light (14N)
    • Intermediate strands as 50:50 15N:14N after replication.

Kornberg 1958

DNA Replication

  • Defined the synthesis process of DNA using DNA polymerases:
    • Reaction formula: (DNA)n + dNTP ightarrow (DNA){n + 1} + PPi.
  • Requires deoxyribonucleotide triphosphate (dNTP) precursors and Mg^{2+} cofactor.

Mechanism of Action

  • Assembles new DNA strands using a pre-existing template.
  • Activates chain formation of phosphodiester linkages only when complementary bases are present on the template.
  • Functionality involves:
    • 3’-OH group on the growing chain to initiate elongation which proceeds in the 5’-3’ direction.

Mistakes and Corrections

  • DNA polymerase has intrinsic 3’-5’ exonuclease activity to remove mismatched nucleotides ensuring fidelity during replication.

Initiation of Replication

  • Identifies the origin of replication (oriC) in circular genomes, like E. coli:
    • Total size: 4.6 x 10^6 bp with unique starting point.
    • Involves specific sequences for DnaA binding and assembly.

Mechanisms of Initiation

  • Assembles via:** AT-rich** regions which promote unwinding.
  • Intermediate sections assist in the melting of DNA strands leading to the replication fork formation via DnaB helicase recruitment.

Unwinding Mechanism

  • DnaB helicase is responsible for unwinding DNA:
    • ATPase-dependent translocation unzips the double-helix.
    • Single-stranded binding (SSB) proteins prevent re-annealing and maintain the stability of the single-stranded configuration of DNA during the unwinding process.

Summary

  • Franklin & Wilkins 1952: Established helical dimensions and structural insights through X-ray fibre diffraction.
  • Watson & Crick 1953: Developed the double-helix model, defining base pairs and structural DNA properties.
  • Meselson & Stahl 1958: Proved the semi-conservative nature of DNA replication through isotope labeling techniques.
  • Kornberg 1958: Outlined the mechanisms and parameters of DNA synthesis and error correction through polymerases.
  • Key Processes: DNA replication involves initiation at the oriC, subsequent unwinding by helicase, and stabilization by SSB proteins.