Denaturation and renaturation studies of benzo[a]pyrene metabolite modified DNAs

Evidence from absorbance, fluorescence, and circular dichroism (CD) measurements strongly suggests that adduct conformations at the binding sites are grossly different before and after thermal denaturation of (+)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]py ren e [(+)-anti-BPDE] modified DNAs. This conclusion is reached through the following observations: (1) upon melting and cooling, the (+)-anti-BPDE-modified DNA exhibits pronounced hypochromic effects with concomitant spectral red shifts for the pyrenyl absorbance; (2) the pyrenyl CD spectrum reverses sign upon thermal denaturation-renaturation; (3) the fluorescence emission spectra resulting from excitations at 353 nm (10 nm to the red of hydrolyzed and unbound anti-BPDE) exhibit enhanced intensities and spectral red shifts for the thermally denatured and cooled adducts; and (4) in contrast to the absence of a shoulder prior to melting, the postmelt adducts exhibit a prominent 355-nm maximum (evidence of stacking interactions) in the excitation spectrum when 384-387-nm emission is monitored. Studies with synthetic polynucleotides further reveal that (+)-anti-BPDE-modified poly(dG).poly(dC) exhibits the greatest nonreversible renaturation at the binding sites, possibly as a consequence of pyrenyl self-stacking. This, coupled with the previous findings that this polymer suffers the most extensive (+)-anti-BPDE modification, appears to suggest that (dG)n . (dC)n regions may be responsible for such observed effects in native DNA.