E. coli restriction-modification system

From OpenWetWare
Revision as of 13:04, 31 July 2005 by Barry Canton (talk | contribs)
Jump to: navigation, search

General Information Brief notes of relevance to me taken from Escherichia coli & Salmonella and ecocyc

  • 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.
  • E. Coli K12 (MG1655) restriction systems
    • According to information from Ecocyc and Escherichia coli & Salmonella, the following restriction enzymes are found in E. coli K12.
    • Contains the type I restriction enzyme EcoK encoded by hsdR - cleaves -AAC(N6)GTCG- if the second A is unmethylated.
    • Contains the Mcr systems described below. These restriction systems do not appear to have a recognition sequence, they just cut at the methylated residues.
    • Contains the Mrr system which cleaves m6A or m5c residues. The sequence specificity is unknown.
  • Mcr Systems (found in E. coli strains)
    • 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.