DNA glycosylase activity assay based on streptavidin paramagnetic bead substrate capture.
A method to detect DNA glycosylase activity is described. The substrate used was an oligodeoxyribonucleotide with a unique hypoxanthine base, but has general application to any DNA glycosylase or endonuclease. The oligodeoxyribonucleotide was labeled at the 5' end with 32P and at the 3' end with a biotin linkage and annealed to a complementary oligodeoxyribonucleotide. The hypoxanthine base was excised in solution using the MPG protein, a human DNA glycosylase. Following cleavage of the phosphodiester linkage by NaOH, the oligodeoxyribonucleotide was attached to streptavidin-coated, paramagnetic beads. Binding of the labeled oligodeoxyribonucleotide to the beads was indicative of the kinetics of the reaction. As a control, half of the reaction products were loaded on to a denaturing polyacrylamide gel. Comparable values for steady-state kinetic constants were obtained using both methods. This nonelectrophoretic technique is a rapid assay of DNA glycosylase activity for both purified proteins and crude extracts. This method can be directly adapted for high-throughput techniques.
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Department of Biology, Beckman Research Institute, City of Hope National Medical Center, 1450 East Duarte Road, Duarte, California 91010, USA.
O'Connor
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D045647:DNA Glycosylases; D006801:Humans; D007700:Kinetics; D008280:Magnetics; D009699:N-Glycosyl Hydrolases / Q000032:analysis* / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D019809:Streptavidin / Q000737:chemistry*
D009838:Oligodeoxyribonucleotides; D019809:Streptavidin; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases