Multispecific DNA methyltransferases from Bacillus subtilis phages

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Abstract
Temperate Bacillus subtilis phages SPR, φ3T, ϱ11 and SPβ code for DNA methyltransferases, each having multiple sequence specificities. The SPR wild‐type and various mutant methyltransferases were overproduced 1000‐fold in Escherichia coli and were purified by three consecutive chromatographic steps. The stable form of these multispecific enzymes in solution are monomers with a relative molecular mass (Mr) of about 50000. The methyl‐transfer kinetics of the SPR wild‐type and mutant enzymes were determined with DNA substrates carrying either none or one of the three recognition sequences (GGCC, CCGG, CCATGG). Evaluation of the catalytic properties for DNA and S‐adenosylmethionine binding suggested that the NH2‐terminal part of the protein is important for both non‐sequence‐specific DNA binding and S‐adenosylmethionine binding as well as transfer of methyl groups. On the other hand, mutations in the COOH‐terminal part lead to weaker site‐specific interactions of the enzyme. Antibodies raised against the purified SPR enzyme specifically immunoprecipitated the φ3T, ϱ11 and SPβ methyltransferases, but failed to precipitate the chromosomally coded enzymes from B. subtilis (BsuRI) and B. sphaericus (BspRI). Immunoaffinity chromatography is an efficient purification step for the related phage methyltransferases.