MONOFUNCTIONAL ADENINE N-3 ADDUCTS OF MELPHALAN - OCCURRENCE AT A MUTATIONAL HOTSPOT SEQUENCE AND RESISTANCE TO REMOVAL BY ALKA PROTEIN

Previous work showed that a CTAAA sequence in the supF gene of the shu ttle plasmid pZ189 was a hotspot for mutagenesis by the aromatic nitro gen mustards melphalan and chlorambucil, and indirect evidence suggest ed adenine N-3 adducts as premutagenic lesions. In order to characteri ze the adducts Formed at this sequence more directly, a substrate was prepared in which the three adjacent adenines in the CTPAAA sequence w ere H-3-labeled. Following treatment of this substrate with [C-14]melp halan, thermolabile adducts were depurinated and analyzed by HPLC. Onl y a single peak bearing both H-3 and C-14 label was detected and it co eluted with the single major adduct formed by the reaction of melphala n with free adenine base. Various spectrometric analyses of this speci es were all consistent with its identification as a monofunctional ade nine N-3 adduct of melphalan. There was no evidence for any bifunction al adducts involving the labeled adenines. There was little if any rel ease of the adenine N-3 adduct of melphalan by Escherichia coli AlkA p rotein, under conditions where 3-methyladenine was quantitatively rele ased. 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. (C) 1998 Wiley-Liss , Inc.