Lebenka A Iu, Rachkus Iu A
Biokhimiia. 1989 Jun;54(6):1009-14.
DNA-methylase Sau 3A has been isolated for the first time from Staphylococcus aureus 3A cells and purified by column chromatography on phosphocellulose PII, heparin-Sepharose and blue Sepharose. The purified enzyme methylates the GATC sequence with the formation of GATm5C as can be evidenced from the protection of DNA from digestion with restrictases Sau 3A and Bam HI, the lack of the C3H3-group incorporation into Sau 3A DNA-restricts and the formation of a single methylated base m5C. Sau 3A methylase modifies only a two-filament (but not one-filament) DNA. Thus, methylase Sau 3A modifies the both DNA chains in the recognition site during a single binding act. The 5-azacytidine-containing DNA inhibits by 95% the activity of methylase Sau 3A. Ado-met is the single methyl group donor for methylase Sau 3A. The presence of m6A in the recognition site does not affect the activity of methylase Sau 3A. The practical recommendations for the use of M. Sau 3A, alongside with M. Eco dam, for the study of dam methylation by additional methylation of the DNA in vitro in the presence of [methyl-3H]-S-adenosyl-methionine are given.
DNA甲基化酶Sau 3A首次从金黄色葡萄球菌3A细胞中分离出来，并通过磷酸纤维素PII、肝素-琼脂糖和蓝色琼脂糖柱层析进行纯化。纯化后的酶使GATC序列甲基化，形成GATm5C，这可以从DNA免受限制性内切酶Sau 3A和Bam HI消化的保护作用、Sau 3A DNA限制性片段中缺乏C3H3基团的掺入以及单个甲基化碱基m5C的形成得到证明。Sau 3A甲基化酶仅修饰双螺旋（而非单螺旋）DNA。因此，在单次结合过程中，Sau 3A甲基化酶会修饰识别位点中的两条DNA链。含5-氮杂胞苷的DNA可抑制Sau 3A甲基化酶活性达95%。腺苷甲硫氨酸是Sau 3A甲基化酶唯一的甲基供体。识别位点中m6A的存在不影响Sau 3A甲基化酶的活性。给出了在[甲基-3H]-S-腺苷甲硫氨酸存在下，将Sau 3A与Eco dam甲基化酶一起用于通过体外DNA额外甲基化研究dam甲基化的实际建议。
