04. DNA Replication — Prokaryotes

Question 1

What proteins are involved in prokaryotic DNA replication?

Answer 1

• DnaA — initiates DNA replication
  
  • Recognizes 4 DnaA boxes in OriC
  • Will start replication if DNA is negatively supercoiled
  • They form an initial complex
  • Single-stranded binding (SSBs) proteins — protect DNA from degradation
    
    • prevents internal paring
• DnaB (helicase)
  
  • DnaC (the clamp loader) escorts the helicase to DnaA
  • Moves towards the duplex part of the replication fork
  • encircles both strands of separated DNA
• DNA primase (DnaG/RNA polymerase)
  
  • Functions with helicase to form RNA primers on both strands
• DNA polymerase III
  
  • catalyzes the formation of phosphodiester bonds between nucleotides
  • core polymerase — nucleotide addition, 3’-5’ exonuclease activity
  • beta clamp — increases processivity
• RNase H
  
  • Removes RNA primer nucleotides except for the last one
• DNA polymerase I
  
  • removes the last ribonucleotide
  • fills the gap with deoxyribonucleotides
  • exonuclease activity in both direction
• DNA ligase
  
  • reassembles phosphodiester bonds between fragments of DNA on lagging strand
• Topoisomerase II
  
  • Introduces negative supercoils to undo helicase

Question 2

What is the replisome?

Answer 2

The combination of all the proteins that work at the replication fork.

Question 3

Describe how DNA replication is initiated in prokaryotes.

Answer 3

• A specific region of DNA (called oriC) is recognized by proteins (DnaA)
• Contain 4 DnaA boxes and 3 13-mers.
• If both strands are methylated (via Dam methylase), replication can occur.