DNA base flipping analytical pipeline

Abstract

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.