Methylation Interference Assay
This protocol was adapted from “Theory, Characterization, and Modeling of DNA Binding by Regulatory Transcription Factors,” Chapter 13, in Transcriptional Regulation in Eukaryotes: Concepts, Strategies, and Techniques, 1st edition, by Michael Carey and Stephen T. Smale. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2000.INTRODUCTION
Methylation interference (and methylation protection) is the major technique for studying the interaction of proteins with major-groove guanines, although it also detects adenines in the minor groove and cytosines in single-stranded DNA. It is one of the highest-resolution methods for measuring the bases involved in sequence-specific recognition by proteins. A 32P-end-labeled DNA containing a recognition site is methylated (on average once per DNA molecule) by dimethyl sulfate (DMS) and then protein is allowed to bind. Certain methylation modifications prevent the protein from binding because they are at positions where the protein comes into close proximity with the DNA. Most modifications, however, have no effect on binding. The bound and unbound DNA molecules are separated using the electrophoretic mobility shift assay (or filter binding, immunoprecipitation, or any other suitable technique). The unbound fraction will be enriched in DNA molecules containing modifications at positions that interfere with protein binding. In contrast, the bound fraction will contain modifications that do not interfere with binding of the protein. Methylation weakens the nucleotide and makes it susceptible to piperidine-induced depurination. This leads, in turn, to scission of the DNA backbone. The cleaved fragments are fractionated on a sequencing gel alongside chemical sequencing ladders to identify the affected positions.
