2-Aminopurine fluorescence studies of base stacking interactions at abasic sites in DNA: Metal-ion and base sequence effects

Metal-ion and sequence dependent changes in the stacking interactions of bases surrounding abasic (AB) sites in 10 different DNA duplexes were examined by incorporating the fluorescent nucleotide probe 2-aminopurine (2-AP), opposite to the site (AB-AP(opp)) or adjacent to the site (AB-AP(adj)) on either strand. A detailed study of the fluorescence emission and excitation spectra of these AB duplexes and their corresponding parent duplexes indicates that AB-AP(opp) is significantly less stacked than 2-AP in the corresponding normal duplex. In general, AB-AP(adj) on the AB strand is stacked, but AB-AP(adj) on the opposite strand shows destabilized stacking interactions. The results also indicate that divalent cation binding to the AB duplexes contributes to destabilizaton of the base stacking interactions of AB-AP(opp), but has little or no effect on the stacking interactions of AB-AP(adj). Consistent with these results, the fluorescence of AB-AP(opp) is 18-30-fold more sensitive to an externally added quenching agent than the parent normal duplex. When uracil DNA glycosylase binds to AB-AP(opp) in the presence of 2.5 mM MgCl₂, a 3-fold decrease in fluorescence is observed (K(d) = 400 ± 90 nM) indicating that the unstacked 2-AP(opp) becomes more stacked upon binding. On the basis of these fluorescence studies a model for the local base stacking interactions at these AB sites is proposed.