Base sequence effects in bending induced by bulky carcinogen-DNA adducts: Experimental and computational analysis

The covalent binding of bulky mutagenic or carcinogenic compounds to DNA ca n lead to bending, which could significantly alter the interactions of DNA with critical replication and transcription proteins. The impact of adducts derived from the highly reactive bay region enantiomeric (+)- and (-)-anti -7,8-diol-9,10-epoxide derivatives of benzo[a]pyrene (BPDE) are of interest because the (+)-7R,8S,9S,10R-anti-BPDE enantiomer is highly tumorigenic in rodents, while the (-)-7S,8R,9R,10S-anti-BPDE enantiomer is not. Both (+)- and (-)-anti-BPDE bind covalently with DNA predominantly by trans addition at the exocyclic amino group of guanine to yield 10S (+)- and 10R (-)-tran s-anti-[BP]-N-2-dG adducts. We have synthesized a number of different oligo nucleotides with single (+)- and (-)-trans-anti-[BP]N-2-dG adducts (G*) in the base sequence context XG*Y. where X and Y are different DNA bases. The G* residues were positioned at or close to the center of 11 base pair (simi lar to1 helical turn) or 16 base pair (similar to1.5 turns) duplexes. All b ases, except for X and Y and their partners, were identical. These sequence s were self-ligated with T4 ligase to form multimers that yield a ladder of bands upon electrophoresis in native polyacrylamide gels. The extent of be nding in each oligonucleotide was assessed by monitoring the decrease in ge l mobilities of these linear, self-ligated oligomers, relative to unmodifie d oligonucleotides of the same base sequence. The extent of global bending was then estimated using a sequence-specific three-dimensional model from w hich the values of the base-pair step parameter roll adjacent to the lesion site could be extracted. We find that (+)-trans-anti- [BP] -N-2-dG adducts . are considerably more bent than the (-) isomers regardless of sequence an d that A-T base pairs flanking the [BP]-N2-dG lesion site allow for local f lexibility consistent with adduct conformational heterogeneity. Interesting ly, the fit of computed versus observed gel mobilities using classical rept ation treatments requires enhancement of unmodified DNA flexibility in gels , compared to aqueous salt solution. The differences in bending between the two stereoisomeric adduct duplexes and the observed base sequence context effects may play a significant role in the differential processing of these lesions by cellular replication, transcription, and repair enzymes.