Castaing B, Fourrey J L, Hervouet N, Thomas M, Boiteux S, Zelwer C
Centre de Biophysique Moléculaire, UPR 4301 affiliated to the University of Orleans, CNRS, rue Charles Sadron, 45071 Orleans Cedex 2, France.
Nucleic Acids Res. 1999 Jan 15;27(2):608-15. doi: 10.1093/nar/27.2.608.
The binding of Escherichia coli and Lactococcus lactis Fapy-DNA glyosylase (Fpg) proteins to DNA containing either cyclic or non-cyclic abasic (AP) site analogs was investigated by electrophoretic mobility shift assay (EMSA) and by footprinting experiments. We showed that the reduced AP site is the best substrate analog for the E.coli and L.lactis enzymes ( K Dapp = 0.26 and 0.5 nM, respectively) as compared with the other analogs tested in this study ( K Dapp >2.8 nM). The 1,3-propanediol (Pr) residue-containing DNA seems to be the minimal AP site structure allowing a Fpg specific DNA binding, since the ethyleneglycol residue is not specifically bound by these enzymes. The newly described cyclopentanol residue is better recognized than tetrahydrofuran (for the E.coli Fpg, K Dapp = 2.9 and 25 nM, respectively). These results suggest that the hemiacetal form of the AP site is negatively discriminated by the Fpg protein suggesting a hydrogen bond between the C4'-hydroxyl group of the sugar and a Fpg residue. High-resolution hydroxyl radical footprinting using a duplex containing Pr shows that Fpg binds to six nucleotides on the strand containing the AP site and only the base opposite the lesion on the undamaged complementary strand. This comparative study provides new information about the molecular mechanism involved in the Fpg AP lyase activity.
通过电泳迁移率变动分析（EMSA）和足迹实验，研究了大肠杆菌和乳酸乳球菌Fapy-DNA糖基化酶（Fpg）蛋白与含有环状或非环状无碱基（AP）位点类似物的DNA的结合情况。我们发现，与本研究中测试的其他类似物（KDapp>2.8 nM）相比，还原型AP位点是大肠杆菌和乳酸乳球菌酶的最佳底物类似物（KDapp分别为0.26和0.5 nM）。含1,3 - 丙二醇（Pr）残基的DNA似乎是允许Fpg特异性结合DNA的最小AP位点结构，因为乙二醇残基不会被这些酶特异性结合。新描述的环戊醇残基比四氢呋喃更易被识别（对于大肠杆菌Fpg，KDapp分别为2.9和25 nM）。这些结果表明，AP位点的半缩醛形式受到Fpg蛋白的负向识别，这表明糖的C4'-羟基与Fpg残基之间存在氢键。使用含Pr的双链体进行的高分辨率羟基自由基足迹实验表明，Fpg与含有AP位点的链上的六个核苷酸以及未受损互补链上与损伤相对的碱基结合。这项比较研究为Fpg AP裂解酶活性所涉及的分子机制提供了新信息。
