On the nature of the cytosine-methylated sequence in DNA of Bacillus brevis var. G.-B.

On growing the cells of Bacillus brevis S methionine-auxotroph mutant in the presence of [Me-3H]methionine, practically all the radioactivity incorporated into DNA is found to exist in 5-methylcytosine and N6-methyladenine. The analysis of pyrimidine isopliths isolated from DNA shows that radioactivity only exists in mono- and dinucleotides and the content of 5-methylcytosine in R-m5 C-R and R-m5 C-T-R oligonucleotides is equal. The analysis of dinucleotides isolated from DNA by means of pancreatic DNAase hydrolysis allows the nature of purine residues neighbouring 5-methylcytosine to be identified and shows that 5-methylcytosine localizes in G-m5 C-A and G-m5 C-Tr fragments. B. brevis S DNA methylase modifying cytosine residues recognizes the GCA/TGC degenerate nucleotide sequence which is a part of the following complementary structure with a two-fold rotational axis of symmetry: (5')...N'-G-C-T-G-C-N... (3') (3')...N-C-G-A-C-G-N'... (5') (Methylated cytosine residues are askerisked). Cytosine-modifying DNA methylase activity is isolated from B. brevis cells; it is capable of methylating in vitro homologous and heterologous DNA. Hence DNA in bacterial cells can be undermethylated. This enzyme methylates cytosine residues in native and denatured DNA in the same nucleotide sequences. Specificity of methylation of cytosine residues in vitro and in vivo does not depend on the nature of substrate DNA. DNA methylases of different variants of B. brevis (R, S, P+, P-)) methylate cytosine residues in the same nucleotide sequences. It means that specificity or methylation of DNA cytosine residues in the cells of different variants of B. brevis is the same.