A new interference footprinting method for analysing simultaneously protein contacts to phosphate and guanine residues on DNA

摘要

DNA binding proteins interact with may functional groups of the base and phosphate moiteties of DNA. Essential contacts to phoshate residues are usually determined by ethylation interference footprints, first introduced by Gilbert and co-workers (1). To ethylate the phosphates of DNA the chemical probe N-ethyl-N-Nitrosourea (ENU) is used which, according to Sun and Singer (2), forms stable ethyl phosphotriesters to-70% of the total ethylation products. The remaining 30% accounts for base ethylation, i. e. at the O~6 position of guanine(-12%), at the N-7 position of guanine (-11%), and at the N-3 position of adenine (-4.2%) (2). N-7 ethylguanine is a direct ethylationproduct, but it can also be obtained by transethylation from O6-ethylguanine (3). Here we show that it is possible to use ENU to selectively determine also essential protein contacts to guanine residues.