title: Phosphate ethylation interference assay.
abstract: INTRODUCTIONIn the ethylation interference assay, ethyl nitrosourea is used to ethylate phosphates within (32)P-end-labeled DNA molecules that contain a protein recognition site; the reaction is optimized to yield approximately one ethyl group per DNA molecule. The DNA is then incubated with barely saturating concentrations of a DNA-binding protein, but the ethylated phosphates interfere with proteins coming into close proximity to the minor groove or phosphate backbone of the binding site on the DNA molecule. Because ethylation makes the DNA susceptible to piperidine-induced cleavage, the protein recognition site can be identified by analyzing the cleavage products (from modified DNA molecules with bound protein versus modified DNA molecules without bound protein) on a polyacrylamide/urea gel alongside a sequencing ladder. Two points are critical for success with this assay. First, only about 10% of the molecules should be ethylated, which avoids having a significant number of molecules with multiple ethylations. This can be determined by modifying the DNA until ~10% of the starting probe is converted to a ladder of bands. It is critical that each DNA molecule be modified only once, so that it is clear that the modification being detected--the one nearest to the (32)P-labeled end--is the one responsible for interference. Second, only barely saturating amounts of protein should be bound to the DNA. Barely saturated amounts are used because although the ethylation decreases binding, it does not always abolish it. High concentrations of protein may overcome the deleterious effect of the modification.
authors.forename: Michael
authors.initials: M
authors.lastname: Carey
authors.forename: Stephen T
authors.initials: ST
authors.lastname: Smale