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Multispecific DNA methyltransferases from Bacillus subtilis phages. Properties of wild-type and various mutant
Abstract:
Temperate Bacillus subtilis phages SPR, phi 3T, rho 11 and SP beta 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 50,000. 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 phi 3T, rho 11 and SP beta methyltransferases, bu 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.
Insights
Temperate phages like Bacillus subtilis SPR encode multispecific DNA methyltransferases. The NH2-terminal region is crucial for DNA and S-adenosylmethionine binding and methyl transfer, while the COOH-terminal region affects site-specific DNA interactions.
Area of Science:
- Molecular Biology
- Enzymology
- Bacteriophage Genetics
Background:
- Temperate Bacillus subtilis phages encode DNA methyltransferases with multiple sequence specificities.
- These enzymes play a role in phage-host interactions and genome regulation.
Purpose of the Study:
- To characterize the SPR phage DNA methyltransferase and its mutants.
- To investigate the structure-function relationship of these multispecific enzymes.
- To explore the potential of immunoaffinity chromatography for purifying related phage methyltransferases.
Main Methods:
- Overproduction and purification of SPR methyltransferase in Escherichia coli using three chromatographic steps.
- Kinetic analysis of methyl-transfer reactions with various DNA substrates.
- Antibody generation and immunoprecipitation assays.
- Immunoaffinity chromatography.
Main Results:
- SPR methyltransferase exists as a monomer (Mr ~50,000) in solution.
- The NH2-terminal protein region is essential for DNA and S-adenosylmethionine binding and methyl group transfer.
- Mutations in the COOH-terminal region impair site-specific DNA binding.
- Antibodies against SPR specifically precipitated related phage methyltransferases but not chromosomal enzymes.
Conclusions:
- The NH2-terminal domain of SPR methyltransferase is critical for catalytic activity and substrate binding.
- The COOH-terminal domain mediates sequence-specific DNA recognition.
- Immunoaffinity chromatography is a viable method for purifying related phage-encoded DNA methyltransferases.