Using Lambda Exonuclease Inhibition Assays to Map Carcinogen Binding Sites

Abstract

Previous restriction mapping studies (M.A. Mallamaci, D.P. Reed and S.A. Winkle, J. Biomolecular Structure and Dynamics, in press (1992)) have indicated that a small number of locations on the plasmid pBR322 may be high affinity binding sites for the carcinogen N-acetoxy-N-acetyl-2-aminofluorene (acetoxyAAF). PBR322 was reacted with acetoxyAAF to produce DNA with one, three or seven acetoxyAAF moieties per DNA molecule. Thus only the higher affinity binding sites are affected. Subsequent digestion with the restriction enzyme Hinf I produced fragments containing previously indicated locations of potential acetoxyAAF binding sites. Fragments thought not to contain binding sites were also examined as controls. The isolated fragments, singly 32P end-labeled, were digested with lambda exonuclease. The three fragments suspected of containing acetoxyAAF binding sites possess new lambda exonuclease inhibition sites when the fragments are obtained from acetoxyAAF reacted DNA. No such inhibition sites are found with the two fragments suggested previously not to contain acetoxyAAF binding sites. These carcinogen produced inhibition sites occur in sequences which are similar, suggesting that acetoxyAAF preferentially may target a small number of sequences.

Locating Binding Sites for the Carcinogen N-Acetoxy-N-Acetyl-2-Aminofluorene Using Restriction Enzyme Inhibition Assays

“…In our preceeding study, the binding of acetoxyAAF was shown to produce inhibition of the reaction of lambda exonuclease with fragments of pBR322 (7). In the regions of the lambda exonuclease inhibition sites (between bp 940-951; between 683-692; between 1092-1104) we observe inhibition of restriction enzyme activity.…”

“…Sequences similar to these were NOT found in regions where restriction enzyme activity was not inhibited. Since these inhibition studies were conducted at low levels ofbound carcinogen (inhibitions were observed at 1-8 adducts per DNA molecule), the sequences found at the inhibition sites may be associated with the presence of the high affinity carcinogen binding sites suggested in previous studies (6,7). Having similar sequences at the high affinity binding site regions on three different DNAs suggests that sequences such as these are, in general, the target sequences for attack by acetoxyAAF.…”

“…We have reported that certain regions of the plasmid pBR322 are hot spots for binding of the carcinogen N-acetoxy-N-acetyl-2-aminofluorene (acetoxyAAF) (6). In a subsequent study, pBR322 fragments subjected to reaction with acetoxyAAF were footprinted using lambda exonuclease (7). Lambda exonuclease inhibition sites were located on fragments previously shown to contain hotspots for acetoxyAAF binding.…”

Mass spectrometric investigation of the sequence selectivity for adduction of heterocyclic aromatic amines on single‐strand oligonucleotides

“…Some carcinogens present an adduction selectivity for particular sequences, such as benzo[ a ]pyrene which generates more adducts at the central G of sequences 5′ CGG 3′ , 5′ TGG 3′ , 5′ TGT 3′ and 5′ CGT 3′ 23, 24. Similarly, N ‐acetoxy‐ N ‐acetyl‐2‐aminofluorene (AAAF) is fixed to specific sites of pBR322 plasmid,25, 26 and a metabolite of benzo[ c ]phenanthrene has been described to bind predominantly to the hot spot of the carcinogen (+)‐(7 R, 8 S, 9 S, 10 R )‐isomer of benzo[ a ]pyrene 7,8‐diol 9,10‐epoxide ((+)‐BaP DE‐2) in the Chinese hamster hprt gene 27. In order to obtain more precise information on the flanking bases effect on the adduct formation, Vouros and co‐workers have developed a pseudo‐combinatory method based on a partial digestion of modified DNA followed by a characterization of modified oligomers (mainly trimers) by liquid chromatography coupled to mass spectrometry (LC/MS) 28.…”