A continuous hyperchromicity assay to characterize the kinetics and thermodynamics of DNA lesion recognition and base excision.

ABSTRACT: We report a continuous hyperchromicity assay (CHA) for monitoring and characterizing enzyme activities associated with DNA processing. We use this assay to determine kinetic and thermodynamic parameters for a repair enzyme that targets nucleic acid substrates containing a specific base lesion. This optically based kinetics assay exploits the free-energy differences between a lesion-containing DNA duplex substrate and the enzyme-catalyzed, lesion-excised product, which contains at least one hydrolyzed phosphodiester bond. We apply the assay to the bifunctional formamidopyrimidine glycosylase (Fpg) repair enzyme (E) that recognizes an 8-oxodG lesion within a 13-mer duplex substrate (S). Base excision/elimination yields a gapped duplex product (P) that dissociates to produce the diagnostic hy

SUBMITTER: Minetti CA 

PROVIDER: S-EPMC2224234 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature