Molecular beacons with oxidized bases report on substrate specificity of DNA oxoguanine glycosylases |
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Authors: | Jingjing Sun Nicole M. Antczak Hailey L. Gahlon Shana J. Sturla |
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Affiliation: | Department of Health Sciences and Technology, ETH Zürich, Zürich 8092 Switzerland.; Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge MA 02139 USA ; Department of Chemistry, Skidmore College, 815 North Broadway, Saratoga Springs NY 12866 USA |
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Abstract: | DNA glycosylase enzymes recognize and remove structurally distinct modified forms of DNA bases, thereby repairing genomic DNA from chemically induced damage or erasing epigenetic marks. However, these enzymes are often promiscuous, and advanced tools are needed to evaluate and engineer their substrate specificity. Thus, in the present study, we developed a new strategy to rapidly profile the substrate specificity of 8-oxoguanine glycosylases, which cleave biologically relevant oxidized forms of guanine. We monitored the enzymatic excision of fluorophore-labeled oligonucleotides containing synthetic modifications 8-oxoG and FapyG, or G. Using this molecular beacon approach, we identified several hOGG1 mutants with higher specificity for FapyG than 8-oxoG. This approach and the newly synthesized probes will be useful for the characterization of glycosylase substrate specificity and damage excision mechanisms, as well as for evaluating engineered enzymes with altered reactivities.A three-color fluorescent molecular beacon assay for rapid profiling of substrate specificity of hOGG1 variants, and for engineering proteins to map genomic modifications. |
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