DNA base flipping analytical pipeline
DNA base flipping analytical pipeline
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DOI：
10.1093/biomethods/bpx010
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发表时间：
2017-01
影响因子：
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通讯作者：
Peng Zhang;Florian D. Hastert;A. Ludwig;K. Breitwieser;Maria Hofstätter;M. C. Cardoso
中科院分区：
文献类型：
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作者：
Peng Zhang;Florian D. Hastert;A. Ludwig;K. Breitwieser;Maria Hofstätter;M. C. Cardoso
DNA碱基修饰和突变在整个生命王国的所有基因组中都被观察到。参与它们建立和去除的蛋白质被证明使用碱基翻转机制来接近它们的底物。为了更好地理解蛋白质如何翻转DNA碱基来修饰或去除它们，我们优化并开发了一系列方法来逐步检测从蛋白质- DNA相互作用开始的过程，碱基翻转本身以及随后的DNA碱基修饰或切除。由于甲基胞嘧啶是研究得最好的DNA修饰，在这里我们将重点放在编写、修改和读取这个DNA碱基的过程上。利用多色电泳迁移率转移测定，我们发现甲基胞嘧啶修饰剂Tet1表现出很少的DNA序列特异性，仅对甲基化CpG含有DNA有轻微的偏好。氯乙醛处理和高分辨率熔融温度分析的结合使我们能够检测由甲基胞嘧啶修饰剂Tet1和甲基胞嘧啶修饰剂m.h pai诱导的碱基翻转。最后，我们证明了高分辨率的熔融温度分析可以用于检测DNA底物上的糖基化酶、甲基转移酶和二氧合酶的活性。总之，这个DNA碱基翻转分析管道（BaFAP）提供了一个完整的工具箱，用于快速、灵敏地分析结合、翻转、修饰或切除DNA碱基的蛋白质。
Abstract 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.