Prokaryotic Replication

• E Coli grows very quickly with very few requirements
• E coli has circular chromosomes
• Steps of DNA replication:

  1. Separate DNA strands
  2. Binding of primers to complementary sequence
  3. Complementary base H-bonds
  4. Nucleotide added to the strand
  5. Proofread
  6. Repeat until the end
• DnaA

  • Recognises the origin of replication (oriC site)
  • Binds and seperates DNA at the AT-rich region
• Helicase (DnaB)

  • Loaded onto each DNA strand
  • Moves 5' to 3' to unwind the DNA
• Single-stranded DNA binding protein (SSB)

  • Binds to each DNA strand
  • Keeps the strands separated
• All DNA polymerases need a primer to add nucleotides onto
• Primers provide the 3' OH that a nucleotide is added to
• Primase (DnaG) is an RNA polymerase
• RNA polymerase = polymerase that makes RNA
• RNA polymerase do not need primers to start synthesis
• Nucleoside: base and sugar
• Nucleotide: nucleoside and phosphate
• Nucleotides are the substrate for nucleic acid synthesis
• Nucleotides generalise into NTPs or dNTPs
• Active site of DNA polymerase

  1. Correct base pairing with template
  2. Formation of a phosphodiester bond
• DNA polymerase catalyses the formation of a phosphodiester bond
• Pyrophosphate is released from the formation of a phosphodiester bond, which breaks down into two phosphates
• The free energy released from the breakdown of pyrophosphate is used to provide energy for DNA synthesis
• The breakdown of pyrophosphate removes a product of phosphodiester bond formation, therefore driving the reaction forward
• Mg2+ is a co-factor of DNA polymerase
• Proofreading

  Check 1: Do the nucleotides fit properly in the active site of the enzyme?

  Check 2: Is the nucleotide that was just added base pairing correctly?
• If the added nucleotide is not base pairing correctly, then it is removed by a 3' to 5' exonuclease
• The correct nucleotide is being added if it sits well in the active site of the enzyme
• A nucleotide that does not fit well in the enzyme can still be added, but it will be less favourable
• Nucleases are enzymes that cut phosphodiester bonds
• 3' to 5' exonucleases cut phosphodiester bonds from the 3' end
• 5' to 3' exonucleases cut phosphodiester bonds from the 5' end
• Endonucleases cut phosphodiester bonds in the middle of the strand
• The replication bubble grows as helicase moves along each strand
• DNA is synthesised on both strands at the replication fork as it opens
• Since the lagging strand runs 3' to 5', it cannot be synthesised in one go
• The lagging strand is synthesised as discontinuous Okazaki fragments
• DNA Polymerase III has 9 subunits
• Subunits of DNA polymerase III:

  • Pol III core adds the nucleotides and proofreads
  • Beta sliding clamp holds onto the DNA
  • Clamp loader grabs the lagging strand and moves it to the polymerase
• DNA synthesis stops when the beta sliding clamp falls off
• DNA polymerase III has high processivity - due to the beta sliding clamp, it can synthesise for a long time before falling off
• As the DNA strand unwinds, it undergoes positive supercoiling
• Positive supercoiling: Same direction as the double helix; tight and hard to separate
• Negative supercoiling: Opposite direction to the double helix; makes DNA easier to separate