Prigodich R V, Martin C T
Chemistry Department, Trinity College, Hartford, Connecticut 06106.
Biochemistry. 1990 Sep 4;29(35):8017-9. doi: 10.1021/bi00487a003.
This study demonstrates that the reaction of Fe(II)-EDTA and hydrogen peroxide with the single-stranded nucleic acids d(pT)70 and a 29-base sequence containing a mixture of bases results in substantial damage which is not directly detected by gel electrophoresis. Cleavage of the DNA sugar backbone is enhanced significantly after the samples are incubated at 90 degrees C in the presence of piperidine. The latter reaction is used in traditional Maxam-Gilbert DNA sequencing to detect base damage, and the current results are consistent with reaction of the hydroxyl radical with the bases in single-stranded DNA (although reaction with sugar may also produce adducts that are uncleaved but labile to cleavage by piperidine). We propose that hydroxyl radicals may react preferentially with the nucleic acid bases in ssDNA and that reaction of the sugars in dsDNA is dominant because the bases are sequestered within the double helix. These results have implications both for the study of single-stranded DNA binding protein binding sites and for the interpretation of experiments using the hydroxyl radical to probe DNA structure or to footprint double-stranded DNA binding protein binding sites.
本研究表明，Fe(II)-EDTA与过氧化氢和单链核酸d(pT)70以及含有多种碱基的29个碱基序列发生反应，会导致严重损伤，而凝胶电泳无法直接检测到这种损伤。在哌啶存在下，样品于90℃孵育后，DNA糖骨架的切割显著增强。后一种反应在传统的Maxam-Gilbert DNA测序中用于检测碱基损伤，目前的结果与羟基自由基与单链DNA中的碱基反应一致（尽管与糖的反应也可能产生未切割但对哌啶切割不稳定的加合物）。我们提出，羟基自由基可能优先与单链DNA中的核酸碱基反应，而双链DNA中糖的反应占主导，因为碱基被隔离在双螺旋结构中。这些结果对于单链DNA结合蛋白结合位点的研究以及使用羟基自由基探测DNA结构或对双链DNA结合蛋白结合位点进行足迹分析的实验解释都具有重要意义。
