Hsieh Chih-Lin
Department of Urology, University of Southern California, 1441 Eastlake Ave,, Rm 5420, Norris Cancer Center, Los Angeles, CA 90033, USA.
BMC Biochem. 2005 Mar 30;6:6. doi: 10.1186/1471-2091-6-6.
Though Dnmt1 is considered the primary maintenance methyltransferase and Dnmt3a and Dnmt3b are considered de novo methyltransferases in mammals, these three enzymes may work together in maintaining as well as establishing DNA methylation patterns. It has been proposed that Dnmt1 may carry out de novo methylation at sites in the genome with transient single-stranded regions, such as replication origins, and then spread methylation from these nucleation sites in vivo, even though such activity has not been reported.
In this study, we show that Dnmt3a does not act on single-stranded substrates in vitro, indicating that Dnmt3a is not likely to initiate DNA methylation at such proposed nucleation sites. Dnmt3a shows similar methylation activity on unmethylated and hemimethylated duplex DNA, though with some substrate preference. Unlike Dnmt1, pre-existing cytosine methylation at CpG sites or non-CpG sites does not stimulate Dnmt3a activity in vitro and in vivo.
The fact that Dnmt3a does not act on single stranded DNA and is not stimulated by pre-existing cytosine methylation indicates that the de novo methylation activity of Dnmt3a is quite different from that of Dnmt1. These findings are consistent with a model in which Dnmt3a initiates methylation on one of the DNA strands of duplex DNA, and these hemimethylated sites then stimulate Dnmt1 activity for further methylation.
尽管在哺乳动物中，Dnmt1被认为是主要的维持甲基转移酶，而Dnmt3a和Dnmt3b被认为是从头甲基转移酶，但这三种酶可能共同作用以维持和建立DNA甲基化模式。有人提出，Dnmt1可能在基因组中具有瞬时单链区域的位点（如复制起点）进行从头甲基化，然后在体内从这些成核位点扩散甲基化，尽管尚未有此类活性的报道。
在本研究中，我们表明Dnmt3a在体外不作用于单链底物，这表明Dnmt3a不太可能在这些提议的成核位点启动DNA甲基化。Dnmt3a对未甲基化和半甲基化的双链DNA表现出相似的甲基化活性，不过有一定的底物偏好。与Dnmt1不同，CpG位点或非CpG位点预先存在的胞嘧啶甲基化在体外和体内均不刺激Dnmt3a的活性。
Dnmt3a不作用于单链DNA且不受预先存在的胞嘧啶甲基化刺激这一事实表明，Dnmt3a的从头甲基化活性与Dnmt1的截然不同。这些发现与一种模型一致，即Dnmt3a在双链DNA的一条链上启动甲基化，然后这些半甲基化位点刺激Dnmt1活性以进行进一步甲基化。
