Turnbull J F, Adams R L
Nucleic Acids Res. 1976 Mar;3(3):677-95. doi: 10.1093/nar/3.3.677.
DNA methylase has been purified 405-fold from Krebs II ascites cells. The purified enzyme is homogeneous on SDS-poly acrylamide gel electrophoresis (molecular weight about 80,000) and the only product of the reaction with DNA is 5-methyl cytosine. Both native and denatured DNA are methylated by the enzyme; with calf thymus DNA the double stranded form is the better substrate but the enzyme preferentially methylates single stranded E.coli DNA even in "native" preparations. Our results do not support a mechanism whereby the enzyme methylates DNA by binding irreversibly and "walking" along it. By measuring maximum levels of methylation of DNAs from different sources we have estimated the proportion of unmethylated sites present in them. Homologous ascites DNA can be methylated, but only to about 5% of the level of the best substrate, undermethylated mouse L929 cell DNA. DNA isolated from growing cells or tissues is a better substrate than DNA from normal liver or pancreas, or from stationary cells.
已从克雷布斯II腹水细胞中纯化出405倍的DNA甲基化酶。纯化后的酶在SDS - 聚丙烯酰胺凝胶电泳上呈均一状态(分子量约为80,000),与DNA反应的唯一产物是5 - 甲基胞嘧啶。天然DNA和变性DNA均可被该酶甲基化;对于小牛胸腺DNA,双链形式是更好的底物,但即使在“天然”制剂中,该酶也优先甲基化单链大肠杆菌DNA。我们的结果不支持该酶通过不可逆结合并沿DNA“移动”来甲基化DNA的机制。通过测量不同来源DNA的最大甲基化水平,我们估算了其中未甲基化位点的比例。同源腹水DNA可以被甲基化,但仅达到最佳底物(甲基化不足的小鼠L929细胞DNA)水平的约5%。从生长中的细胞或组织中分离出的DNA比从正常肝脏、胰腺或静止细胞中分离出的DNA是更好的底物。