Fluorescence characteristics of site-specific and stereochemically distinct benzo[a]pyrene diol epoxide-DNA adducts as probes of adduct conformation.
Spectroscopic fluorescence quenching techniques are described for distinguishing the conformational characteristics of adducts derived from the binding of the benzo[a]pyrene metabolite anti-BPDE (the diol epoxide r7,t8-dihydroxy-t9,10epoxy-7,8,9,10-tetrahydrobenz[a]pyrene) to the exocyclic amino groups of guanine ([BP]-N(2)-dG) and adenine ([BP]-N(6)-dA) in double stranded oligonucleotides. These methods are calibrated by comparing the fluorescence quenching and UV absorbance characteristics of different, stereoisomeric anti-[BP]-N(2)-dG adducts of known adduct conformations, previously established by high-resolution NMR techniques. It is shown that intercalative adduct conformations can be distinguished from solvent-exposed adduct conformations, e.g., adducts in which the pyrenyl residues are positioned in the minor groove. These low resolution fluorescence methods are at least 4 orders of magnitude more sensitive than the high-resolution NMR techniques; the fluorescence methods are useful for distinguishing adduct conformations when either small amounts of material are available or the NMR signals are of such poor quality that high-resolution structures cannot be determined. This methodology is illustrated using a variety of anti-BPDE-modified oligonucleotides of varying adduct conformations. It is shown that the 10S (+)-trans-anti-[BP]-N(6)-dA adduct in an oligonucleotide duplex containing an N-ras protooncogene sequence, believed to be conformationally heterogeneous and disordered, is significantly more exposed to the solvent environment than the stereoisomeric, intercalated 10R adduct [Zegar et al. (1996) Biochemistry 35, 6212]. These differences suggest an explanation for the greater efficiencies of excision of the 10S adduct (relative to the 10R adduct) by human nucleotide excision repair enzymes [Buterin et al. (2000) Cancer Res. 60, 1849].
Huang
Weidong
W
Chemistry Department, New York University, New York, New York 10003-5180, USA.
Amin
Shantu
S
Geacintov
Nicholas E
NE
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000032:analysis / Q000737:chemistry*; D018736:DNA Adducts / Q000032:analysis / Q000737:chemistry*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods; D013237:Stereoisomerism
D018736:DNA Adducts; C043931:benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-DNA; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide