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-APopp) or adjacent to the site (AB-APadj) 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-APoppis significantly less stacked than 2-AP in the corresponding normal duplex. In general, AB-APadjon the AB strand is stacked, but AB-APadjon 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-APopp, but has little or no effect on the stacking interactions of AB-APadj. Consistent with these results, the fluorescence of AB-APoppis 18-30-fold more sensitive to an externally added quenching agent than the parent normal duplex. When uracil DNA glycosylase binds to AB-APoppin 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-APoppbecomes 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.
Center for Advanced Research in Biotechnology, University of Maryland, Biotechnology Institute and theNational Institute for Standards and Technology, 9600 Gudelsky Drive, Rockville, MD 20850, USA. stivers@carb.nist.gov
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D015075:2-Aminopurine; D020106:Acrylamide; D000178:Acrylamides; D001483:Base Sequence; D001665:Binding Sites; D002413:Cations, Divalent; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D045647:DNA Glycosylases; D005456:Fluorescent Dyes; D007700:Kinetics; D015636:Magnesium Chloride; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D009690:Nucleic Acid Conformation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D051981:Uracil-DNA Glycosidase
D000178:Acrylamides; D002413:Cations, Divalent; D005456:Fluorescent Dyes; D015636:Magnesium Chloride; D020106:Acrylamide; D015075:2-Aminopurine; D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase