DNA Replication

• 𝙀𝙑𝙄𝘿𝙀𝙉𝘾𝙀 𝙊𝙁 𝙎𝙀𝙈𝙄-𝘾𝙊𝙉𝙎𝙀𝙍𝙑𝘼𝙏𝙄𝙑𝙀 𝙍𝙀𝙋𝙇𝙄𝘾𝘼𝙏𝙄𝙊𝙉:

  Watson and Crick (1953) discovered the helical structure of DNA, aided by Rosalind Franklin's research into x-ray diffraction.

  They hypothesised that DNA must replicate conservatively (both parent strands are kept) or semi-conservatively (one parent strand is kept).
• Semi-Conservative Replication: each replicated DNA molecule contains one of the original DNA strands and the newly synthesised strand.
• 𝙈𝙀𝙎𝙀𝙇𝙎𝙊𝙉 & 𝙎𝙏𝘼𝙃𝙇:

  • proved Watson and Crick's hypothesis that DNA replicates semi-conservatively
  • used two isotopes of nitrogen (¹⁵N and ¹⁴N)
  • bacteria use the nitrogen isotopes as a growth medium to make new DNA nucleotides
  • bacteria grown in a medium containing ¹⁴N will have lighter DNA
  • can be proved by centrifugation (denser ¹⁵N settles lower)
• 𝙈𝙀𝙎𝙀𝙇𝙎𝙊𝙉 & 𝙎𝙏𝘼𝙃𝙇'𝙨 𝙀𝙓𝙋𝙀𝙍𝙄𝙈𝙀𝙉𝙏:

  1. bacteria were grown in a medium containing ¹⁵N: in centrifugation 100% of DNA contained ¹⁵N
  2. bacteria were grown in a medium containing ¹⁴N: in centrifugation 100% of DNA was ¹⁴N ¹⁵N
  3. bacteria were grown in a medium containing ¹⁴N: in centrifugation 50% of DNA was ¹⁴N ¹⁵N and 50% was ¹⁴N ¹⁴N
  4. bacteria were grown in a medium containing ¹⁴N: in centrifugation 25% of DNA was ¹⁴N ¹⁵N and 75% was ¹⁴N ¹⁴N

  
• 𝙋𝙍𝙊𝘾𝙀𝙎𝙎 𝙊𝙁 𝙎𝙀𝙈𝙄-𝘾𝙊𝙉𝙎𝙀𝙍𝙑𝘼𝙏𝙄𝙑𝙀 𝙍𝙀𝙋𝙇𝙄𝘾𝘼𝙏𝙄𝙊𝙉:

  1. DNA helicase unwinds the double helix.
  2. Both strands act as templates.
  3. Free DNA nucleotides line up in complementary pairs.
  4. DNA polymerase joins the nucleotides by phosphodiester bonds.
  5. Each new DNA molecule consists of one original/template strand and one new strand.
• DNA polymerase uses condensation reactions to join adjacent nucleotides, synthesising a new DNA strand.
• What did the Meselson-Stahl experiment show?

  It confirmed that DNA replicates semi-conservatively and ruled out the other proposed mechanisms of DNA replication.
• Why were 14N and 15N used in Meselson and Stahl’s experiment?

  Nitrogen is one of the abundant elements in DNA structure. Meselson and Stahl used the isotopes N14 and N15 in their experiment to incorporate them into the DNA of replicating bacteria and separate DNA of different densities to observe the mode of DNA replication.
• How did Meselson and Stahl turn E.coli DNA into heavy DNA?

  Meselson and Stahl cultured E.coli in nutrient broth containing the dense nitrogen isotope (15N) over many generations. As a result, 15N was incorporated into the bacterial DNA, converting it into heavy DNA.
• What is the semi-conservative model of DNA replication?

  Both strands of DNA segregate in the replication process, and each strand acts as a template to synthesise a new daughter strand. Resultingly, daughter DNA contains one parent strand and one new strand.
• How did Meselson and Stahl provide evidence for semi-conservative replication?

  • One sample of bacteria was grown in a nutrient broth containing heavy nitrogen (N15)
  • The bacteria took up the nitrogen to make new nucleotides - it was incorporated into their DNA
  • A sample of each was centrifuged and the DNA grown in the N15 settled at the bottom of the tube
  • The bacteria grown in the N15 broth were allowed to replicate in light nitrogen (N14)
  • When centrifuged, it settled in the middle of the tube.
  • Shows that a strand of the old DNA was kept and a new strand was created with the light nitrogen