BENDING AND CIRCULARIZATION OF SITE-SPECIFIC AND STEREOISOMERIC CARCINOGEN-DNA ADDUCTS

The potent tumorigen and mutagen (+)-7(R),8(S)-dihydroxy-9(S),10(R)-ep oxy-7,8,9,10- tetrahydrobenzo[a]pyrene ((+)-anti-BPDE) is a metabolite of benzo[a]pyrene that binds predominantly to the exocyclic amino gro up of guanine residues in DNA in vivo and in vitro. While the (-)-7S,8 R,9R, 10S enantiomer, (-)-anti-BPDE, also reacts with DNA to form simi lar covalent N-2-deoxyguanosyl adducts, this diol epoxide is nontumori genic and its mutagenic activities are different from those of (+)-ant i- BPDE. In this work, Tit ligase-induced cyclization methods have bee n employed to demonstrate that the (+)-anti-[BP]-N-2-dG lesions (G) c ause significantly greater amounts of bending and circularization of t he one-base overhang undecamer duplex 5'-d(CACAT[G]TACAC).d(TGTACATGT GG) than the stereoisomeric oligonucleotide duplex with G = (-)-anti- [RP]-N-2-dG. In the case of the (+)-anti-BPDE-modified oligonucleotide s, the ratio of circular to linear DNA multimers reaches values of 8-9 for circle contour sizes of 99-121 base pairs, while for the (-)-anti -[BP]-N-2-dG-modified DNA this ratio reaches a maximum value of only s imilar to 1 at 154-176 base pairs. Assuming a planar circle DNA model, the inferred bending angles for 90-92% of the observed circular ligat ion products range from 30 to 51 degrees per (+)-trans-anti-[BP]-N-2-d G lesion and from 20 to 40 degrees per (-)-trans-anti-[BP] -N-2-dG les ion. In the case of unmodified DNA, the probability of circular produc t formation is at least 1 order of magnitude less efficient than in th e BPDE-modified sequences and about 90% of the circular products exhib it bending angles in the range of 14 -19 degrees. In the most abundant circular products observed experimentally, the bending angles are 40 degrees and 26 +/- 2 degrees per (+)-anti-[BP]- or (-)-anti-[BP]-modif ied 11-mer; these values correspond to a net contribution of 21-26 deg rees and 5-19 degrees, respectively, to the observed overall bending p er lesion. The coexistence of circular DNA molecules of different size s and, therefore, different average bending angles per lesion, suggest that the lesions induce both torsional flexibility and flexible bends , which permit efficient cyclization, especially in the case of (+)-tr ans-[BP]-N-2-dG adducts. The NMR characteristics of (+)-trans-[BP]-N-2 -dG lesion in the 11-mer duplex 5'-d(CACAT[G]TACAC).d-(GTGTACATGTG) i ndicate that all base pairs are intact, except at the underlined base pairs. This suggests a distortion in the normal conformation of the du plex on the 5'-side of the modified guanosine residue, which may be du e to bending enhanced base pair opening and bending induced by the bul ky carcinogen residue. The implications of base sequence-dependent fle xibilities and conformational mobilities of anti[BP]-N-2-dG lesions on DNA replication and mutation are discussed.