Rapid determination of the active fraction of DNA repair glycosylases: a novel fluorescence assay for trapped intermediates.
Current methods to measure the fraction of active glycosylase molecules in a given enzyme preparation are slow and cumbersome. Here we report a novel assay for rapidly determining the active fraction based on molecular accessibility of a fluorescent DNA minor groove binder, 4',6-diamidino-2-phenylindole (DAPI). Several 5,6-dihydrouracil-containing (DHU) DNA substrates were designed with sequence-dependent DAPI-binding sites to which base excision repair glycosylases were covalently trapped by reduction. Trapped complexes impeded the association of DAPI in a manner dependent on the enzyme used and the location of the DAPI-binding site in relation to the lesion. Of the sequences tested, one was shown to give an accurate measure of the fraction of active molecules for each enzyme tested from both the Fpg/Nei family and HhH-GPD Nth superfamily of DNA glycosylases. The validity of the approach was demonstrated by direct comparison with current gel-based methods. Additionally, the results are supported by in silico modeling based on available crystal structures.
Blaisdell
Jeffrey O
JO
Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, University of Vermont, Burlington, VT, USA.
Wallace
Susan S
SS
D004247:DNA / Q000378:metabolism; D045647:DNA Glycosylases / Q000737:chemistry* / Q000378:metabolism; D043382:DNA-Formamidopyrimidine Glycosylase / Q000737:chemistry; D043224:Deoxyribonuclease (Pyrimidine Dimer) / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D007211:Indoles / Q000737:chemistry*; D008958:Models, Molecular; D019163:Reducing Agents; D013050:Spectrometry, Fluorescence / Q000379:methods*
D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D007211:Indoles; D019163:Reducing Agents; C007293:DAPI; D004247:DNA; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; D045647:DNA Glycosylases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli