Mechanism of hydroxylamine mutagenesis: tautomeric shifts and proton exchange between the promutagen N6-methoxyadenosine and cytidine

Abstract

Whereas the amino, but not imino, tautomer of the promutagen N6-methoxyadenosine (OMe6A) forms planar associates (base pairs) with the potentially complementary uridine [Stolarski, R., Kierdaszuk, B., Hagberg, C.-E., & Shugar, D. (1984) Biochemistry 23, 2906-2913], it has now been found, with the aid of 1H NMR spectroscopic techniques, that only the imino tautomer of OMe6A base pairs with the potentially complementary cytidine. The association constant for such heteroassociates is more than an order of magnitude higher than that for autoassociates of OMe6A. The formation of heteroassociates is accompanied by a marked shift in tautomeric equilibrium of OMe6A, with an increase in the population of the amino form from 18% to as high as 44% and a corresponding decrease in the population of the imino species. Furthermore, the presence of cytidine in a solution of OMe6A appreciably enhances the rate of tautomeric exchange between the two tautomeric forms. Formation of planar heteroassociates between cytidine and the imino form of OMe6A is also accompanied by proton exchange between the cytidine NH2 and the N6-H of the amino form of OMe6A. The rate constants for this exchange and for tautomeric exchange, determined by the saturation transfer technique, have been measured at various concentrations and temperatures. A model is advanced for proton exchange that takes into account the interdependence of tautomeric exchange and proton exchange, as well as the role of auto- and heteroassociates. The relevance of these results to the molecular basis of hydroxylamine and methoxyamine mutagenesis and to the phenomenon of proton exchange in other systems is briefly discussed.

High Throughput Mutagenesis for Identification of Residues Regulating Human Prostacyclin (hIP) Receptor Expression and Function

“…generally the parental plasmid is incubated directly with one of several chemicals and can be used directly for transformation after a simple purification step [18]–[21]. Chemical mutagenesis can be especially useful if low mutation rates are required or sub-cloning of the targeted region is not possible.…”

“…For this, we incubated the parental plasmid coding for the hIP receptor with 1 M hydroxyl amine for 32 min–128 min. Treatment of double stranded DNA with Hydroxyl amine is known to result in C->T and G->A transitions thus allowing for only a limited fraction of possible substitutions [21]. After a purification step, the DNA was transformed in E.coli .…”

Photochemical syn–anti Isomerization Reactions in N2-Hydroxyisocytosines—An Experimental Matrix Isolation and Theoretical Study¶

“…Such a high barrier for the rotation around the double C5N bond would prevent thermal equilibration monomolecularly (28). However, in protic solvents, these processes can be catalyzed by temporary proton exchange, leading to the amino forms of the compound (such as II-syn) in which the rotation around the single C-N bond is much easier (3,4,29,30).…”

Chemical cleavage reactions of DNA on solid support: application in mutation detection

“…The following results have been obtained: (i) The commercially available silica beads which was originally developed for DNA purification of small DNA fragments is now suitable for solid-phase chemical reactions. (ii) Typical reactions with KMnO 4 and hydroxylamine [12,13] have been successfully demonstrated on DNA bound silica support. (iii) DNA bound silica support remains intact during reactions and purification steps.…”