M.BssHII, a multispecific cytosine-C5-DNA-methyltransferase with unusual target recognizing properties

A new multispecific cytosine-C5-DNA-methyltransferase (C5-MTase), M.BssHII, was identified in Bacillus stearothermophilus H3. The M.BssHII gene was cloned and sequenced. The amino acid sequence deduced shows the characteristic building plan of a C5-MTase. By sequencing bisulfite-treated DNA methylat...

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Bibliographic Details
Published in:Journal of molecular biology 1996-04, Vol.257 (5), p.949-959
Main Authors: Schumann, J, Walter, J, Willert, J, Wild, C, Koch, D, Trautner, T A
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Bacillus stearothermophilus
Base Sequence
Cloning, Molecular
Conserved Sequence
DNA (Cytosine-5-)-Methyltransferases - chemistry
DNA (Cytosine-5-)-Methyltransferases - genetics
DNA (Cytosine-5-)-Methyltransferases - metabolism
DNA - metabolism
DNA-Cytosine Methylases - metabolism
Electrophoresis, Polyacrylamide Gel
Geobacillus stearothermophilus - enzymology
Methylation
Molecular Sequence Data
Mutagenesis
Point Mutation
Recombinant Fusion Proteins - chemistry
Recombinant Fusion Proteins - metabolism
Sequence Alignment
Sequence Analysis
Sequence Deletion
Substrate Specificity
Sulfites - metabolism
Sulfites - pharmacology
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Summary:A new multispecific cytosine-C5-DNA-methyltransferase (C5-MTase), M.BssHII, was identified in Bacillus stearothermophilus H3. The M.BssHII gene was cloned and sequenced. The amino acid sequence deduced shows the characteristic building plan of a C5-MTase. By sequencing bisulfite-treated DNA methylated by M.BssHII and by restriction enzyme analysis, we defined the following methylation targets of M.BssHII: ACGCGT/CCGCGG (MluI/SacII), PuGCGCPy (HaeII), PuCCGGPy (Cfr10I) and GCGCGC (BssHII). The relative location of the specificity determinants in the C5-MTase was derived from the analysis of M.BssHII derivatives carrying deletions within the variable region "V" and chimeric C5-Mtases constructed between M.BssHII and the related monospecific enzyme M.phi3TII. Four of the M.BssHII specificities (MluI, SacII, Cfr10I and BssHII) could be associated with amino acid segments within the variable region "V". The determinant for HaeII activity had to be assigned to sequences defining the enzyme core, the first example of a C5-MTase in which a sequence-specific methylation potential is mediated by structures outside of the variable region. Another intriguing result came from the analysis of one particular chimera made between M.BssHII and M.phi3TII. This construct showed a relaxation of the methylation capacity, both with respect to the target recognized and the targeting of methylation within this sequence.
ISSN:0022-2836
DOI:10.1006/jmbi.1996.0214