M.BSSHII, A MULTISPECIFIC CYTOSINE-C5-DNA-METHYLTRANSFERASE WITH UNUSUAL TARGET RECOGNIZING PROPERTIES

A new multispecific cytosine-C5-DNA-methyltransferase (C5-MTase), M.Bs sHII, was identified in Bacillus stearothermophilus H3. The M.BssHII g ene was cloned and sequenced. The amino acid sequence deduced shows th e 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/CCGC GG (MluI/SacII), PuGCGCPy (HaeII), PuCCGGPy (Cfr10I) and GCGCGC (BssHI I). The relative location of the specificity determinants in the C5-MT ase was derived from the analysis of M.BssHII derivatives carrying del etions within the variable region ''V'' and chimeric C5-Mtases constru cted between M.BssHII and the related monospecific enzyme M.phi 3TII. Four of the M.BssHII specificities (MluI, SacII, Cfr10I and BssHII) co uld be associated with amino acid segments within the variable region ''V''. The determinant for HaeII activity had to be assigned to sequen ces defining the enzyme core, the first example of a C5-MTase in which a sequence-specific methylation potential is mediated by structures o utside of the variable region. Another intriguing result came from the analysis of one particular chimera made between M.BssHII and M.phi 3T II. This construct showed a relaxation of the methylation capacity, bo th with respect to the target recognized and the targeting of methylat ion within this sequence. (C) 1996 Academic Press Limited