A simplified method forin vivofootprinting using DMS

Important in understanding the regulation of gene transcription is the elucidation of specific protein-DNA interactions that occur in vivo. One strategy for such in vivo footprinting involves the treatment of whole cells with dimethyl sulphate (DMS), which leads to methylation of guanine residues in DNA at the N7 position (1, 2). This N7 atom lies in the major groove and its susceptibility to methylation in chromatin is affected by its proximity to bound non-histone proteins (3). Subsequent treatment with piperidine breaks the DNA backbone at methylated sites that can be mapped by various methods, including a sequence specific, radioactive primer extension assay, using a DNA polymerase (1, 4, 5). We and others have used Taq DNA polymerase, since it is very thermostable, allowing for both greater specificity of priming and multiple extension cycles to increase signal. Here we demonstrate that DMS treatment is alone sufficient to terminate Taq polymerisation in our assay, thus obviating the need for the piperidine treatment step (compare lanes 1 and 2 in Fig. 1). This is probably because the incubation at 95 °C for 5 minutes that precedes the Taq polymerase reaction is sufficient to break the unstable glycosidic bonds of methylated purines (6), and leads to termination by Taq polymerase one nucleotide before a methylated purine base (Fig. 1).