Blaisdell Jeffrey O, Wallace Susan S
Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, University of Vermont, Burlington, VT, USA.
Nucleic Acids Res. 2007;35(5):1601-11. doi: 10.1093/nar/gkm021. Epub 2007 Feb 8.
Current methods to measure the fraction of active glycosylase molecules in a given enzyme preparation are slow and cumbersome. Here we report a novel assay for rapidly determining the active fraction based on molecular accessibility of a fluorescent DNA minor groove binder, 4',6-diamidino-2-phenylindole (DAPI). Several 5,6-dihydrouracil-containing (DHU) DNA substrates were designed with sequence-dependent DAPI-binding sites to which base excision repair glycosylases were covalently trapped by reduction. Trapped complexes impeded the association of DAPI in a manner dependent on the enzyme used and the location of the DAPI-binding site in relation to the lesion. Of the sequences tested, one was shown to give an accurate measure of the fraction of active molecules for each enzyme tested from both the Fpg/Nei family and HhH-GPD Nth superfamily of DNA glycosylases. The validity of the approach was demonstrated by direct comparison with current gel-based methods. Additionally, the results are supported by in silico modeling based on available crystal structures.
目前用于测量给定酶制剂中活性糖基化酶分子比例的方法既缓慢又繁琐。在此，我们报告一种基于荧光DNA小沟结合剂4',6-二脒基-2-苯基吲哚（DAPI）的分子可及性来快速测定活性比例的新方法。设计了几种含5,6-二氢尿嘧啶（DHU）的DNA底物，其具有序列依赖性DAPI结合位点，碱基切除修复糖基化酶通过还原共价捕获于该位点。捕获的复合物以依赖于所用酶以及DAPI结合位点相对于损伤的位置的方式阻碍DAPI的结合。在所测试的序列中，有一个序列被证明能准确测量来自Fpg/Nei家族和HhH-GPD Nth超家族的DNA糖基化酶中每种测试酶的活性分子比例。通过与当前基于凝胶的方法直接比较证明了该方法的有效性。此外，结果得到基于现有晶体结构的计算机模拟的支持。
