Sun Jingjing, Antczak Nicole M, Gahlon Hailey L, Sturla Shana J
Department of Health Sciences and Technology, ETH Zürich Zürich 8092 Switzerland
Department of Biological Engineering, Massachusetts Institute of Technology 77 Massachusetts Avenue Cambridge MA 02139 USA.
Chem Sci. 2022 Feb 16;13(15):4295-4302. doi: 10.1039/d1sc05648d. eCollection 2022 Apr 13.
DNA glycosylase enzymes recognize and remove structurally distinct modified forms of DNA bases, thereby repairing genomic DNA from chemically induced damage or erasing epigenetic marks. However, these enzymes are often promiscuous, and advanced tools are needed to evaluate and engineer their substrate specificity. Thus, in the present study, we developed a new strategy to rapidly profile the substrate specificity of 8-oxoguanine glycosylases, which cleave biologically relevant oxidized forms of guanine. We monitored the enzymatic excision of fluorophore-labeled oligonucleotides containing synthetic modifications 8-oxoG and FapyG, or G. Using this molecular beacon approach, we identified several hOGG1 mutants with higher specificity for FapyG than 8-oxoG. This approach and the newly synthesized probes will be useful for the characterization of glycosylase substrate specificity and damage excision mechanisms, as well as for evaluating engineered enzymes with altered reactivities.
DNA糖基化酶能够识别并去除结构各异的DNA碱基修饰形式，从而修复化学诱导损伤的基因组DNA或消除表观遗传标记。然而，这些酶往往具有混杂性，因此需要先进的工具来评估和设计它们的底物特异性。因此，在本研究中，我们开发了一种新策略，用于快速分析8-氧代鸟嘌呤糖基化酶的底物特异性，该酶可切割具有生物学相关性的鸟嘌呤氧化形式。我们监测了含有合成修饰8-氧代鸟嘌呤（8-oxoG）、FapyG或鸟嘌呤（G）的荧光团标记寡核苷酸的酶促切除过程。使用这种分子信标方法，我们鉴定出了几种对FapyG比对8-oxoG具有更高特异性的人8-氧代鸟嘌呤DNA糖基化酶1（hOGG1）突变体。这种方法和新合成的探针将有助于表征糖基化酶的底物特异性和损伤切除机制，以及评估具有改变反应活性的工程酶。
