Zhang Peng, Hastert Florian D, Ludwig Anne K, Breitwieser Kai, Hofstätter Maria, Cardoso M Cristina
Cell Biology and Epigenetics, Department of Biology, Technische Universität Darmstadt, Germany.
Max Delbrück Center for Molecular Medicine, Berlin, Germany.
Biol Methods Protoc. 2017 Aug 11;2(1):bpx010. doi: 10.1093/biomethods/bpx010. eCollection 2017 Jan.
DNA base modifications and mutations are observed in all genomes throughout the kingdoms of life. Proteins involved in their establishment and removal were shown to use a base flipping mechanism to access their substrates. To better understand how proteins flip DNA bases to modify or remove them, we optimized and developed a pipeline of methods to step-by-step detect the process starting with protein-DNA interaction, base flipping itself and the ensuing DNA base modification or excision. As methylcytosine is the best-studied DNA modification, here we focus on the process of writing, modifying and reading this DNA base. Using multicolor electrophoretic mobility shift assays, we show that the methylcytosine modifier Tet1 exhibits little DNA sequence specificity with only a slight preference for methylated CpG containing DNA. A combination of chloroacetaldehyde treatment and high-resolution melting temperature analysis allowed us to detect base flipping induced by the methylcytosine modifier Tet1 as well as the methylcytosine writer M.HpaII. Finally, we show that high-resolution melting temperature analysis can be used to detect the activity of glycosylases, methyltransferases and dioxigenases on DNA substrates. Taken together, this DNA base flipping analytical pipeline (BaFAP) provide a complete toolbox for the fast and sensitive analysis of proteins that bind, flip and modify or excise DNA bases.
在生命王国的所有基因组中都观察到了DNA碱基修饰和突变。参与其建立和去除的蛋白质被证明使用碱基翻转机制来接触它们的底物。为了更好地理解蛋白质如何翻转DNA碱基以对其进行修饰或去除，我们优化并开发了一套方法流程，用于逐步检测从蛋白质-DNA相互作用、碱基翻转本身以及随后的DNA碱基修饰或切除开始的过程。由于甲基胞嘧啶是研究最深入的DNA修饰，在这里我们重点关注写入、修饰和读取这种DNA碱基的过程。使用多色电泳迁移率变动分析，我们表明甲基胞嘧啶修饰酶Tet1几乎没有DNA序列特异性，仅对含有甲基化CpG的DNA略有偏好。氯乙醛处理和高分辨率熔解温度分析相结合，使我们能够检测到甲基胞嘧啶修饰酶Tet1以及甲基胞嘧啶写入酶M.HpaII诱导的碱基翻转。最后，我们表明高分辨率熔解温度分析可用于检测糖苷酶、甲基转移酶和双加氧酶对DNA底物的活性。综上所述，这个DNA碱基翻转分析流程（BaFAP）为快速、灵敏地分析结合、翻转和修饰或切除DNA碱基的蛋白质提供了一个完整的工具箱。
