E. coli restriction-modification system
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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.