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.
Stivers
J T
JT
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
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