Pogolotti A L, Ono A, Subramaniam R, Santi D V
Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
J Biol Chem. 1988 Jun 5;263(16):7461-4.
Experiments were performed to determine whether EcoRI methylase catalyzes the transfer of the methyl group of S-adenosylmethionine (a) directly to the N6 of adenine in DNA or (b) initially to N1 to give N1-methyladenine followed by isomerization of the N1-methylamino and 6-NH2 to give N6-methyladenine (Dimroth rearrangement). A facile synthesis of highly enriched [6-15N]deoxyadenosine and a dodecamer substrate of EcoRI methylase with [6-15N]adenine in the methylation site are reported. In the product of EcoRI enzymatic methylation, all of the isotope remains at the N6 position of the N6-methyladenine product. It is concluded that, contrary to existing chemical precedent, the methylation occurs by direct transfer from S-adenosylmethionine to the N6 of adenine in DNA.
开展了实验以确定EcoRI甲基化酶是否催化以下过程：(a) 将S-腺苷甲硫氨酸的甲基直接转移至DNA中腺嘌呤的N6位；或(b) 首先转移至N1位生成N1-甲基腺嘌呤，随后N1-甲基氨基和6-NH2发生异构化生成N6-甲基腺嘌呤（迪姆罗特重排）。本文报道了一种简便的方法，可合成高度富集的[6-15N]脱氧腺苷以及在甲基化位点含有[6-15N]腺嘌呤的EcoRI甲基化酶的十二聚体底物。在EcoRI酶促甲基化产物中，所有同位素均保留在N6-甲基腺嘌呤产物的N6位。得出的结论是，与现有的化学先例相反，甲基化是通过从S-腺苷甲硫氨酸直接转移至DNA中腺嘌呤的N6位而发生的。
