Vanyushin B F, Dobritsa A P
Biochim Biophys Acta. 1975 Sep 12;407(1):61-72. doi: 10.1016/0005-2787(75)90023-4.
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.
在[甲基-³H]甲硫氨酸存在的情况下培养短短芽孢杆菌S甲硫氨酸营养缺陷型突变体的细胞时，发现几乎所有掺入DNA中的放射性都存在于5-甲基胞嘧啶和N⁶-甲基腺嘌呤中。对从DNA中分离出的嘧啶等密度区带的分析表明，放射性仅存在于单核苷酸和二核苷酸中，并且R-m⁵C-R和R-m⁵C-T-R寡核苷酸中5-甲基胞嘧啶的含量相等。通过胰DNA酶水解从DNA中分离出二核苷酸进行分析，可以确定与5-甲基胞嘧啶相邻的嘌呤残基的性质，并表明5-甲基胞嘧啶位于G-m⁵C-A和G-m⁵C-Tr片段中。修饰胞嘧啶残基的短短芽孢杆菌S DNA甲基化酶识别GCA/TGC简并核苷酸序列，该序列是具有二重对称轴的以下互补结构的一部分：(5')...N'-G-C-T-G-C-N... (3') (3')...N-C-G-A-C-G-N'... (5')（甲基化的胞嘧啶残基用星号表示）。修饰胞嘧啶的DNA甲基化酶活性是从短短芽孢杆菌细胞中分离出来的；它能够在体外甲基化同源和异源DNA。因此细菌细胞中的DNA可能会发生甲基化不足。这种酶在相同的核苷酸序列中对天然和变性DNA中的胞嘧啶残基进行甲基化。体外和体内胞嘧啶残基甲基化的特异性不取决于底物DNA的性质。短短芽孢杆菌不同变体（R、S、P⁺、P⁻）的DNA甲基化酶在相同的核苷酸序列中甲基化胞嘧啶残基。这意味着短短芽孢杆菌不同变体细胞中DNA胞嘧啶残基甲基化的特异性是相同的。
