Impact of 7,8-Dihydro-8-oxoguanine on Methylation of the CpG Site by Dnmt3a
Impact of 7,8-Dihydro-8-oxoguanine on Methylation of the CpG Site by Dnmt3a
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DOI：
10.1021/bi801947f
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发表时间：
2009-02-17
期刊：
影响因子：
2.9
通讯作者：
Gromova, Elizaveta S.
中科院分区：
文献类型：
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作者：
Maltseva, Diana V.;Baykov, Alexander A.;Gromova, Elizaveta S.
7，8-二氢-8-氧代鸟嘌呤（8-oxoG）是一种普遍存在的氧化性DNA损伤，由活性氧对DNA的损伤引起。8-oxoG损伤可能在癌发生过程中异常DNA甲基化模式的形成中起作用。在这项研究中，我们评估了8-oxoG对小鼠Dnmt 3a DNA甲基转移酶（Dnmt 3a-CD）催化结构域的9个30-mer寡脱氧核苷酸双链体的甲基化和复合物形成的影响。8-oxoG对半甲基化双链体甲基化率的影响从25倍降低到1.8倍增加不等，具体取决于病变相对于Dnmt 3a-CD识别位点（CpG）和靶胞嘧啶（C）的位置。当8-oxoG取代紧邻靶胞嘧啶下游的识别位点内的鸟嘌呤时，观察到最显著的效果。荧光素标记的双链体的荧光偏振实验显示，每个双链体结合两个分子的Dnmt 3a-CD，产生S形结合曲线。表现出最高表观结合协同性的双链体与Dnmt 3a-CD形成最不稳定的1：2复合物，并且以最低速率甲基化。动力学分析揭示了非常稳定的非生产性酶-底物复合物的形成与半甲基化的双链体，作为自杀底物的Dnmt 3a-CD。与非生产性结合相比，靶胞嘧啶下游CpG位点内8-oxoG的存在显著降低了生产性结合。我们提出，8-oxoG位于邻近的目标胞嘧啶干扰甲基化通过削弱DNA的亲和力Dnmt 3a-CD，从而有利于非生产性的结合模式。
7,8-Dihydro-8-oxoguanine (8-oxoG) is a ubiquitous oxidative DNA lesion resulting from injury to DNA via reactive oxygen species. 8-oxoG lesions may play a role in the formation of aberrant DNA methylation patterns during carcinogenesis. In this study, we assessed the effects of 8-oxoG on methylation and complex formation of nine 30-mer oligodeoxynucleotide duplexes by the catalytic domain of murine Dnmt3a DNA methyltransferase (Dnmt3a-CD). The effects of 8-oxoG on the methylation rate of hemimethylated duplexes varied from a 25-fold decrease to a 1.8-fold increase, depending on the position of the lesion relative to the Dnmt3a-CD recognition site (CpG) and target cytosine (C). The most significant effect was observed when 8-oxoG replaced guanine within the recognition site immediately downstream of the target cytosine. Fluorescence polarization experiments with fluorescein-labeled duplexes revealed that two molecules of Dnmt3a-CD bind per duplex, generating sigmoid binding curves. Duplexes exhibiting the highest apparent binding cooperativity formed the least stable 1:2 complexes with Dnmt3a-CD and were methylated at the lowest rate. Kinetic analyses disclosed the formation of very stable nonproductive enzyme-substrate complexes with hemimethylated duplexes that act as suicide substrates of Dnmt3a-CD. The presence of 8-oxoG within the CpG site downstream of the target cytosine markedly diminished productive versus nonproductive binding. We propose that 8-oxoG located adjacent to the target cytosine interferes with methylation by weakening the affinity of DNA for Dnmt3a-CD, thereby favoring a nonproductive binding mode.