Monofunctional adenine N-3 adducts of melphalan: Occurrence at a mutational hotspot sequence and resistance to removal by AlkA protein

Abstract

Previous work showed that a CTAAA sequence in the supF gene of the shuttle plasmid pZ189 was a hotspot for mutagenesis by the aromatic nitrogen mustards melphalan and chlorambucil, and indirect evidence suggested adenine N-3 adducts as premutagenic lesions. In order to characterize the adducts formed at this sequence more directly, a substrate was prepared in which the three adjacent adenines in the CTAAA sequence were 3H-labeled. Following treatment of this substrate with [14C]melphalan, thermolabile adducts were depurinated and analyzed by HPLC. Only a single peak bearing both 3H and 14C label was detected and it coeluted with the single major adduct formed by the reaction of melphalan with free adenine base. Various spectrometric analyses of this species were all consistent with its identification as a monofunctional adenine N-3 adduct of melphalan. There was no evidence for any bifunctional adducts involving the labeled adenines. There was little if any release of the adenine N-3 adduct of melphalan by Escherichia coli AlkA protein, under conditions where 3-methyladenine was quantitatively released. The results support the proposal that monofunctional adenine N-3 adducts are intermediates in the generation of A.T-->T.A and A.T-->C.G transversions by aromatic nitrogen mustards.