A column method for determination of DNA cytosine-C5-methyltransferase activity
A column method for determination of DNA cytosine-C5-methyltransferase activity
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DOI：
10.1016/j.ab.2003.11.009
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发表时间：
2004-03-01
影响因子：
2.9
通讯作者：
Ahn, JS
中科院分区：
文献类型：
--
作者：
Kim, BY;Kwon, OS;Ahn, JS
DNA胞嘧啶5位甲基化与肿瘤发生有关。因此，DNA甲基化酶的抑制剂可以用作抗癌药物。然而，由于检测DNA甲基化的局限性，目前仅发现了少数抑制性化合物。本研究利用[H-3]-标记的S-腺苷甲硫氨酸（SAM）和SephadexG-25柱，改进了DNA胞嘧啶-C5-甲基转移酶测定系统。用HaeIII甲基化酶预处理λ DNA或人端粒酶（hTERT）的启动子区，大大减少了相应限制性内切酶HaeIII对DNA的消化（超过100倍），琼脂糖凝胶电泳进一步证实了这一结果。将该柱方法应用于另一种修饰/限制性系统EcoRI甲基化酶/内切酶，得到了类似的结果。我们的数据表明，新开发的柱方法可以有效地快速筛选大量的胞嘧啶甲基化酶抑制剂，也可以适用于其他DNA甲基化酶。(C)2003年爱思唯尔公司All rights reserved.
DNA methylation at the 5th position of cytosine has been found to be correlated with tumorigenesis. An inhibitor of DNA methylase could, therefore, be used as an anticancer drug. However, only a few inhibitory compounds have been discovered due to the limitations for assaying the DNA methylation. In this study, we describe a modification of DNA cytosine-C5-methyltransferase assay system utilizing [H-3]-labeled S-adenosyl-methionine (SAM) and Sephadex G-25 column. Pre-treatment of either lambda DNA or the promoter region of human telomerase (hTERT) with HaeIII methylase greatly reduced the digestion of the DNAs with the corresponding restriction enzyme HaeIII endonuclease (over 100-fold), and the result was further confirmed by agarose gel electrophoresis. Application of this column method to another modification/restriction system, EcoRI methylase/endonuclease, gave rise to the similar results. Our data suggest that the newly developed column method could be effective for rapid screening of large number of cytosine methylase inhibitors and could also be applicable to other DNA methylases. (C) 2003 Elsevier Inc. All rights reserved.