N-2-AMINOFLUORENE AND N-2 ACETYLAMINOFLUORENE ADDUCTS - THE LOCAL SEQUENCE CONTEXT OF AN ADDUCT AND ITS CHEMICAL-STRUCTURE DETERMINE ITS REPLICATION PROPERTIES

The strong rat liver carcinogen, N-2-acetylaminofluorene, forms mainly two types of guanine adducts at the C-8 position, the acetylaminofluo rene adduct (dGuo-C8-AAF) and the aminofluorene adduct (dGuo-C8-AF). W e have constructed different oligonucleotides bearing a single AF lesi on at each of the guanine residues of the NarI mutagenesis hot spot (G (1)G(2)CG(3)CC) and analysed the structural distortion induced by this DNA adduct according to the sequence context. At position G(1) and G( 2), the deformation induced by the AF adduct is smaller than the defor mation induced by the corresponding acetylated form of this adduct (i. e. the AAF adduct at the G(1) and G(2)), whereas both AF and AAF adduc ts induce a similar structural change when bound to G(3). Single-stran ded oligonucleotides modified with AF adducts were used in primer exte nsion replication assays using purified DNA polymerases (PolIII holoen zyme, Klenow fragment(exo+ and exo-), Sequenase 2.0) and the data comp ared to the AAF containing substrates. Translesion synthesis (complete bypass) is found with all tested polymerases when AF adducts are boun d to G(1) or G(2) while little or no bypass is seen when the AF adduct is bound to G(3). On the other hand, irrespective of its position wit hin the NarI sequence, AAF adducts completely block DNA synthesis. The results described in this paper show that the sole knowledge of the c hemical structure of an adduct neither determines uniquely the conform ational change it induces at the DNA level nor its replication propert ies. Indeed, although AF adducts are in most cases non-distorting addu cts and as a consequence non-replication blocking lesions (as exemplif ied by adducts at G(1) or G(2)), some AF adducts (as at position G(3)) behave almost as AAF adducts in terms of the structural distortion in duced and its replication blocking property These findings stress the strong modulation by the local sequence context of the structural and biological consequences of a given adduct.