E. coli restriction-modification system

Brief notes of relevance to me taken from Escherichia coli & Salmonella

• Restriction systems are only found in unicellular organisms.
  • Either the cell modifies certain sequences so they are not restricted (classic R-M systems)
  • or certain foreign modifications are restricted.
• Mcr Systems
  • McrBC - cleaves hm5C, m5C and m4C (hydroxymethylated or methylated cytosine at positions 4 or 5). The modified bases must be paired about 40-80bp apart. (genes mcrB and mcrC)
  • McrA - cleaves DNA methylated by HpaII and SssI methylases. (encoded by the prophagelike e14 element)

• Classic R-M systems

A classic R-M system includes an endonuclease that cleaves a specific DNA sequence and a DNA methyltransferase that methylates either adenosyl or cytosyl residues within the same DNA sequence.

• Type I R-M systems
  • Relatively rare.
  • One, three-subunit protein acts as both endonuclease and methylase.
  • Requires AdoMet as a cofactor.
  • Endonuclease activity also requires Mg²⁺ and ATP.
  • E. coli has a type 1 restriction system encoded by hsdR, hsdM, and hsdS.

• Type II R-M systems
  • Relatively abundant - these are the restriction enzymes found in the NEB catalog etc.
  • Separate nuclease and methylation enzymes.
  • Requires AdoMet as a cofactor.
  • Endonuclease activity also requires Mg²⁺
  • Little homology between restriction and methylation enzymes despite the shared DNA recognition sequence.
  • Most recognition sequences are close to symmetric.
  • Endonucleases normally act as homodimers, acting on sense and anti-sense strands.
  • A subgroup of Type II restriction enzymes (Type IIS) cleave a fixed distance away from the recognition sequence.

• Type III R-M systems
  • Relatively rare.
  • Hetero-oligomeric protein catalyzes both the restriction and modification reactions.
  • Requires AdoMet as a cofactor for modification.
  • Endonuclease activity also requires Mg²⁺ and ATP.
  • Encoded by mod and res genes in E. coli.
  • Only one strand is methylated unlike the other systems.
  • The restriction enzyme requires two recognition sites in inverse orientation.

• Regulation
  • There is no evidence of transcriptional regulation of Type I R-M systems in E. coli
  • However, there must be some regulation as the modification subunit is always expressed before the restriction subunit.
  • There is little clear information about the regulation of Type II R-M systems, in part because many of them come from poorly understood bacteria.
  • Type II R-M regulation is also not very well understood. However, the modification enzyme is again expressed prior to the restriction enzyme.