Topoisomerases are enzymes that catalyze DNA unwinding and scissions to resolve topological entanglements possibly arising during DNA replication/transcription. Chemicals which disrupt or inhibit topoisomerase-mediated DNA unwinding can induce breaks that subsequently lead to programmed cell death. Herein we perform experiments guided by the following considerations. First, topoisomerase 1 initiates DNA cleavage utilizing the hydroxyl group of tyrosine 723 on its catalytic site as a nucleophile to attack the electrophilic phosphate on the DNA sugar-phosphate backbone. Secondly, the grape polyphenol resveratrol displays both topoisomerase inhibitory and Cu2+-dependent DNA-cutting activities, which contribute to its DNA replication/transcription inhibitory/anti-tumorigenic effects. Lastly, resveratrol contains a tyrosine-like phenolic ring; thus, upon binding to DNA whether resveratrol could act as a tyrosine mimetic to unwind and cut DNA via its hydroxyl groups warrants investigation. Polyphenol-DNA interactions (PDIs) were investigated using UV-visible spectral analysis; additionally, PDI mediated DNA changes were further analyzed by agarose gel electrophoresis using 3 supercoiled plasmid DNAs (pBR322, pSJ3, pHOT-1) as substrates. Resveratrol mediates time- and temperature-dependent, Cu2+-independent, non-enzymatic cleavage of supercoiled plasmid DNA into open, circular DNA products. Varying degree of unwinding of supercoiled DNA nucleolytic activity was also observed with other polyphenols including, piceatannol, quercetin, myricetin and EGCG. Interestingly, we found that piceatannol mediated Cu2+-independent DNA-cleavage activity was abolished by EDTA. The PDI-mediated nucleolytic cleavage of supercoiled DNA reported herein shows that polyphenolic phytochemicals display genome-active, nuclear effects by directly targeting the DNA topology which in turn could impact macromolecular processes associated with faithful replication and transmission of genetic information.

中文翻译：

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通过植物化学物质/ DNA相互作用控制DNA的结构和功能：白藜芦醇/吡啶甲酚可诱导不依赖Cu2 +的超螺旋质粒DNA裂解。

拓扑异构酶是催化DNA解链和剪切以解决在DNA复制/转录过程中可能出现的拓扑纠缠的酶。破坏或抑制拓扑异构酶介导的DNA解旋的化学物质可诱导断裂，继而导致程序性细胞死亡。在此，我们根据以下注意事项进行实验。首先，拓扑异构酶1利用其催化位点上的酪氨酸723的羟基作为亲核试剂来起始DNA裂解，以攻击DNA糖-磷酸主链上的亲电磷酸盐。其次，葡萄多酚白藜芦醇既表现出拓扑异构酶抑制作用，又表现出依赖Cu2 +的DNA切割活性，这有助于其DNA复制/转录抑制/抗致瘤作用。最后，白藜芦醇含有一个类似酪氨酸的酚环。因此，在与DNA结合后，白藜芦醇是否可以作为酪氨酸模拟物通过其羟基解开并切割DNA，值得研究。使用紫外可见光谱分析法研究了多酚-DNA相互作用（PDI）。此外，使用3个超螺旋质粒DNA（pBR322，pSJ3，pHOT-1）作为底物，通过琼脂糖凝胶电泳进一步分析PDI介导的DNA变化。白藜芦醇介导时间依赖性和温度依赖性，Cu2 +依赖性，超酶切的非酶切酶将超螺旋质粒DNA切割成开放的环状DNA产物。还观察到了其他多酚（包括皮卡季诺醇，槲皮素，杨梅素和EGCG）的超螺旋DNA核酸分解活性的解开程度不同。有趣的是，我们发现EDTA消除了皮卡季诺醇介导的不依赖Cu2 +的DNA切割活性。