DNA

DNA Overview

  • DNA (Deoxyribonucleic Acid): Composed of four nucleotides.
  • Proteins: Formed from 20 amino acids; they are the carriers of genetic information.
  • Early Research Hypothesis: Genes made of protein.

Griffith's Experiment (1928)

  • Strains of Strepococcus pneumoniae:
    • Rough Strain: Mouse survives.
    • Smooth Strain: Mouse dies.
    • Heat-Killed Smooth Strain: Mouse survives after denaturing outer layer.
    • Rough Strain + Heat-Killed Smooth Strain: Mouse dies.
  • Phenomenon Identified: Transformation; the DNA was changed.

Avery, MacLeod, and McCarty Experiment

  1. Lysed S cells
  2. Separated Contents into:
    • Lipids
    • Proteins
    • Polysaccharides
    • Nucleic Acids
  3. Testing for Transforming Ability: Only DNA could transform.
  4. Skepticism: Many scientists still believed DNA was not the genetic material.

Hershey-Chase Experiment

  • Bacteriophages (viruses infecting bacteria): consist of DNA (or RNA) and protein.
  • Procedure: Injected chloristent dye into the protein and DNA.
  • Conclusion: Injected DNA of phage is the genetic information; DNA carries genetic material.

Structure of DNA

Nucleotide Structure (Monomer)

  • Components of Nucleotides:
    1. A phosphate group.
    2. A sugar (deoxyribose).
    3. A nitrogenous base.
  • Nitrogenous Bases:
    1. Pyrimidines: One ring.
    • Cytosine (C)
    • Thymine (T)
    • Uracil (U)
    1. Purines: Two rings.
    • Adenine (A)
    • Guanine (G)

Determining DNA Structure

  1. Rosalind Franklin (1951-1953): 3D structure of DNA.
    • DNA is helical.
    • Composed of two strands (double helix).
    • Consistent width like a circular staircase.
    • Consistent distance between turns.
    • Nucleotide bases are stacked like rungs of a ladder.
  2. Erwin Chargaff (1949):
    • Chargaff’s Rule:
      • Total purine (A + G) = total pyrimidine (C + T).
      • Amount of A = Amount of T; A/T ~ 1.
      • Amount of C = Amount of G; C/G ~ 1.
      • Note: A + T ≠ G + C.
  3. Watson and Crick Model of DNA (1953):
    • Structure explains:
      • How DNA can carry genetic information.
      • How information can be replicated.
      • Base Pairing: Purine + Purine = too wide; Pyrimidine + Pyrimidine = too narrow; Purine + Pyrimidine = width consistent with X-ray data.

Watson-Crick DNA Model

  • Double Helix Structure:
    1. Strands Composition:
    • Sugar-phosphate backbone (rails of the ladder), joined by phosphodiester linkages between 3’ and 5’.
    1. Nitrogenous Bases (Rungs):
    • Attached to backbone by covalent bonds (high variability).
  • Strand Connectivity:
    1. Held together by hydrogen bonds:
    • 2 hydrogen bonds between A and T.
    • 3 hydrogen bonds between C and G.
    1. Strands Directionality:
    • Run antiparallel (3’ → 5’ & 5’ → 3’).
    • 3’ end → free 3’ hydroxyl (OH); 5’ end → free 5’ phosphate.
    1. Base Pairing:
    • Purine always pairs with pyrimidine:
      • A & T (2 H-bonds).
      • C & G (3 H-bonds).
    • Strands are complementary (e.g., if one strand is 5’ ATGTTCA 3’, the other is 3’ TACAGTT 5’).

DNA Replication

Semi-Conservative Replication

  • Mechanism: Each new DNA molecule consists of:
    • 1 parental (old) strand.
    • 1 newly synthesized strand.
  • Steps in Replication:
    1. Separation of parental strands into templates.
    2. Formation of new strands complementary to template strands.

Initiation

  • Part 1: Origin of Replication:
    • DNA strands are separated, forming a replication bubble.
    • Replication proceeds out along replication forks.
    • DNA in Prokaryotes: Singular structure, copies itself and separates.
    • DNA in Eukaryotes: Complex design requiring multiple origins of replication.
  • Replication Fork Formation:
    1. DNA unwound by proteins.
    2. Single-strand binding proteins (SSBP) keep strands apart.
    3. DNA helicase unwinds helix by breaking hydrogen bonds.
    4. Topoisomerase: Relaxes the helix to prevent strain.
    5. RNA Primer: Short RNA sequence required to initiate replication.
    • Synthesized by RNA primase.
    • Later removed and replaced with DNA.
    1. Ends of two strands differ in directionality.

Elongation

  • Process: DNA polymerase adds nucleoside triphosphates (dNTPs).
    • Phosphate of the incoming dNTP attaches to the previous 3’ hydroxyl.
    • Directionality:
      • New strands are built in a 5’ to 3’ direction;
      • DNA template is read in a 3’ to 5’ direction.
    • Formation of New Phosphodiester Bonds occurs at the replication fork.
  • Leading and Lagging Strand Synthesis:
    1. Leading Strand:
    • Synthesized continuously in the direction of the replication fork.
    1. Lagging Strand:
    • Synthesized in short, discontinuous segments called Okazaki fragments, which are later joined by ligation activity.