Experimental Approaches to Study DNA Base Flipping
Saulius Klimasauskas and Zita Liutkeviciute
The most dramatic and localized enzyme‑induced conformational distortion to the helical structure of DNA is base flipping, in which a nucleobase is unpaired, removed from the stack and further rotated out 180˚ to assume a fully extrahelical position. Since its first demonstration in crystal structures of cytosine methyltransferase‑DNA complexes, numerous studies revealed that base flipping is a fundamental mechanism in DNA modification and repair, is involved in initiation of replication, transcription and recombination and lately has been shown to mediate sequence‑specific recognition by restriction endonucleases. Here we discuss the variety of experimental approaches that are used to study enzyme‑induced base flipping in different systems. X‑ray crystallography of protein‑DNA complexes is the sole method providing the ultimate proof of base flipping. NMR spectroscopy offers important inroads into dynamic aspects of base flipping, but its potential has not been fully exploited. An attractive method to detect and study base flipping in solution is fluorescent spectroscopy; it uses DNA substrates containing fluorescent base analogs, most often 2‑aminopurine. Chemical probing, which exploits enhanced chemical reactivity of flipped out bases in DNA, is a simple method that can be performed in a standard laboratory. Biochemical binding studies often show an enhanced affinity for substrates containing mismatched base pairs, which indirectly points to a disruption of the target base pair upon interaction with enzyme.