人们对这一代人的了解很多,移除,5-甲基胞嘧啶(5mC)在真核DNA中的作用,关于N6-甲基腺嘌呤的证据越来越多,但是对真核生物DNA中的N4-甲基胞嘧啶(4mC)知之甚少。第一个后生动物DNA甲基转移酶产生4mC(N4CMT)的基因最近被其他人报道和表征,在微小的淡水无脊椎动物中被称为bdelloid轮虫。Bdelloid轮虫是古老的,显然是无性动物,缺乏规范的5mCDNA甲基转移酶。这里,我们表征了来自Bdelloid轮虫Adinetavaga的N4CMT蛋白催化域的动力学特性和结构特征。我们发现N4CMT在首选位点产生高水平的甲基化,(a/c)CG(t/c/a),和低水平的甲基化在不受欢迎的位点,以ACGG为例。像哺乳动物从头5mCDNA甲基转移酶3A/3B(DNMT3A/3B),N4CMT在两条DNA链上甲基化CpG二核苷酸,产生半甲基化的中间体和最终完全甲基化的CpG位点,特别是在有利的对称站点的背景下。此外,像DNMT3A/3B,N4CMT甲基化非CpG位点,主要是CpA/TpG,虽然在一个较低的速度。N4CMT和DNMT3A/3B甚至更喜欢相似的CpG侧翼序列。在结构上,N4CMT的催化结构域非常类似于crescentuscenterus细胞周期调节的DNA甲基转移酶。CpG的对称甲基化,与细胞周期调节的DNA甲基转移酶相似,共同表明,N4CMT也可能在DNA复制后进行DNA合成依赖性甲基化。
Much is known about the generation, removal, and roles of 5-methylcytosine (5mC) in eukaryote DNA, and there is a growing body of evidence regarding N6-methyladenine, but very little is known about N4-methylcytosine (4mC) in the DNA of eukaryotes. The gene for the first metazoan DNA methyltransferase generating 4mC (N4CMT) was reported and characterized recently by others, in tiny freshwater invertebrates called bdelloid rotifers. Bdelloid rotifers are ancient, apparently asexual animals, and lack canonical 5mC DNA methyltransferases. Here, we characterize the kinetic properties and structural features of the catalytic domain of the N4CMT protein from the bdelloid rotifer Adineta vaga. We find that N4CMT generates high-level methylation at preferred sites, (a/c)CG(t/c/a), and low-level methylation at disfavored sites, exemplified by ACGG. Like the mammalian de novo 5mC DNA methyltransferase 3A/3B (DNMT3A/3B), N4CMT methylates CpG dinucleotides on both DNA strands, generating hemimethylated intermediates and eventually fully methylated CpG sites, particularly in the context of favored symmetric sites. In addition, like DNMT3A/3B, N4CMT methylates non-CpG sites, mainly CpA/TpG, though at a lower rate. Both N4CMT and DNMT3A/3B even prefer similar CpG-flanking sequences. Structurally, the catalytic domain of N4CMT closely resembles the Caulobacter crescentus cell cycle-regulated DNA methyltransferase. The symmetric methylation of CpG, and similarity to a cell cycle-regulated DNA methyltransferase, together suggest that N4CMT might also carry out DNA synthesis-dependent methylation following DNA replication.