Rapid determination of the active fraction of DNA repair glycosylases: a novel fluorescence assay for trapped intermediates.

ABSTRACT: 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

SUBMITTER: Blaisdell JO 

PROVIDER: S-EPMC1865064 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature