Specific suppression of depurination at deoxyguanylate residues by silver ions: A useful reaction for the identification of deoxyadenylate residues in DNA

Abstract

The original form of the Maxam and Gilbert DNA sequencing method [l] utilized alkylation of DNA by dimethylsulphate followed by the kinetically rapid depurination of the polynucleotide chain at the positively charged 3-methyl deoxyadenylate residues as a specific reaction for the location of deoxyadenylate residues. Experience showing that this method is unreliable has led to its replacement by the less specific but more controllable acid depurination reaction [2]. This latter version of the sequencing method suffers from a minor disadvantage when compared to the primed synthesis methods [3,4] in that the deoxyadenylate residues must be identified by a comparison between 2 electrophoretic channels, of which one contains fragments cleaved only at deoxyguanylate residues and the other of which contains fragments cleaved at both deoxyguanylate and deoxyadenylate residues. Because of the high density of bands in the channel containing fragments cleaved at both types of purine nucleotide residue problems may arise in the interpretation of autoradiograms of DNA sequencing gels when long polypurine tracts are present. These problems are particularly severe when the sequence of interest does not lie close to a convenient site for end labelling. I describe here a simple and reliable modification to the conditions of the depurination reaction which confers a high degree of specificity for deoxyadenylate residues upon the reaction. This specific reaction depends upon the selective suppression of depurination at deoxyguanylate residues by high [Ag’]. Details of this method are provided in fig.1. The monovalent silver cation (Ag’) has been widely used in studies of DNA base compositional heterogeneity to facilitate the separation of DNA species on the basis of differential buoyant density shifts in Cs2S04 [5]. Although the primary sites of binding of this ligand to DNA have not been unambiguously identified, 2 discrete modes of binding have been distinguished [6,7]. Type I binding of Ag+ to DNA is relatively insensitive to pH changes over the neutral range and does not involve the displacement of protons from the bases [6,7]. The most probable sites for type I binding are the nucleophilic nitrogen atoms at which proton&ion of the nucleotides o&_trs under acidic conditions (N-7 of deoxyguanylate, N-3 of deoxycytidylate and N-l or N-7 of deoxyadenylate). Type II binding of Ag’ to DNA is sensitive to pH changes and results in the displacement of H’ from the DNA. Since the most readily displaced H’ on DNA are bound at N-3 of thymidylate and N-l of deoxyguanylate residues these nitrogen atoms are prime candidates for type II binding sites. The amino groups of adenine, guanine and cytosine are potential secondary type II binding sites. Under the conditions of high [Ag’] and low pH used here, the net decrease in the rate of depurination of pBR322 DNA (A/G = 1) due to the added Ag’ is -80% (see fig.2). Depurination must therefore be inhibited at both deoxyguanylate and deoxyadenylate residues. The hydrolysis of N-glycoside bonds in DNA proceeds via a unimolecular reaction and that the rate of hydrolysis is greatly enhanced by protonation of the base [8]. The rate of reaction for any individual nucleotide therefore depends upon the average electron density at the glycosidic nitrogen. Providing the type I site for silver binding on deoxyadenylate residues is the same as the site of protonation (N-l) the suppression of depurination at these