Expression and purification of the modification-dependent restriction enzyme BisI and its homologous enzymes.

The methylation-dependent restriction endonuclease (REase) BisI (G(m5)C ↓ NGC) is found in Bacillus subtilis T30. We expressed and purified the BisI endonuclease and 34 BisI homologs identified in bacterial genomes. 23 of these BisI homologs are active based on digestion of (m5)C-modified substrates. Two major specificities were found among these BisI family enzymes: Group I enzymes cut GCNGC containing two to four (m5)C in the two strands, or hemi-methylated sites containing two (m5)C in one strand; Group II enzymes only cut GCNGC sites containing three to four (m5)C, while one enzyme requires all four cytosines to be modified for cleavage. Another homolog, Esp638I cleaves GCS ↓ SGC (relaxed specificity RCN ↓ NGY, containing at least four (m5)C). Two BisI homologs show degenerate specificity cleaving unmodified DNA. Many homologs are small proteins ranging from 150 to 190 amino acid (aa) residues, but some homologs associated with mobile genetic elements are larger and contain an extra C-terminal domain. More than 156 BisI homologs are found in >60 bacterial genera, indicating that these enzymes are widespread in bacteria. They may play an important biological function in restricting pre-modified phage DNA.

The type I restriction endonuclease R.EcoR124I: over-production and biochemical properties.

In this paper we describe a two-plasmid system which allows over-production of the R.EcoR124I restriction endonuclease. The endonuclease has been purified to homogeneity in milligram amounts and has been shown to be fully active for both restriction and modification. Unexpectedly, the enzyme was found to require only ATP and Mg2+ for ATPase activity and DNA cleavage; S-adenosyl methionine (SAM), which has been described as a cofactor of type I restriction enzymes, is not required by R.EcoR124I. However, SAM was found to stimulate the rate of ATPase activity and DNA cleavage. This may occur through an increase in specific DNA binding by R.EcoR124I in the presence of SAM, as indicated by our surface plasmon resonance experiments. These functional differences from the well described R.EcoKI restriction endonuclease are reflected in a possible structural difference between the two enzymes, namely that the stoichiometry of R.EcoR124I appears to be R1M2S1 while that of R.EcoKI is R2M2S1. Supercoiled DNA with one or two SR124I recognition sites is cleaved by the same mechanism inferring co-operation between specifically bound and excess enzymes. Nicked-circle DNA is an intermediate of cleavage reaction. Cleavage of DNA was inhibited by an increased degree of negative supercoiling, which may reflect an increased difficulty for the enzyme to translocate the DNA. Hemi-methylated DNA was the preferred substrate for methylation.

BsuCI, a type-I restriction-modification system in Bacillus subtilis.

A type-I R-M system was identified in B. subtilis. The genes comprising the system have striking similarity to type-I R-M systems observed in Enterobacteriaceae.