Transient Kinetic Methods for Mechanistic Characterization of DNA Binding and Nucleotide Flipping

Enzymes that modify nucleobases in double-stranded genomic DNA, either as part of a DNA repair pathway or as an epigenetic modifying pathway, adopt a multistep pathway to locate target sites and reconfigure the DNA to gain access. Work on several different enzymes has shown that in almost all cases base flipping, also known as nucleotide flipping, is a key feature of specific site recognition. In this chapter, we discuss some of the strategies that can be used to perform a kinetic characterization for DNA binding and nucleotide flipping. The resulting kinetic and thermodynamic framework provides a platform for understanding substrate specificity, mechanisms of inhibition, and the roles of important amino acids. We use a human DNA repair glycosylase called alkyladenine DNA glycosylase as a case study, because this is one of the best-characterized nucleotide-flipping enzymes. However, the approaches that are described can be readily adapted to study other enzymes, and future studies are needed to understand the mechanism of substrate recognition in each individual case. As more enzymes are characterized, we can hope to uncover which features of DNA searching and nucleotide flipping are fundamental features shared by many different families of DNA modifying enzymes, and which features are specific to a particular enzyme. Such an understanding provides reasonable models for less characterized enzymes that are important for epigenetic DNA modification and DNA repair pathways.