Rapid kinetic studies of site-specific DNA binding and uracil flipping by uracil DNA glycosylase (UDG)

The DNA repair enzyme UDG catalyzes the hydrolysis of premutagenic uracil residues from DNA by flipping the uracil base from the DNA helix. The kinetics of site-specific binding and uracil flipping steps were studied in the absence of glycosidic bond cleavage using a stable DNA substrate analog containing 2'fluoro-2'-deoxyuridine. The real-time kinetics of binding and uracil flipping were measured by following the fluorescence changes of a 2-aminopurine base located adjacent to the target uracil. Con form at i on al changes in UDG were followed by monitoring tryptophan fluorescence. These studies show that i) site-specific binding is tight and diffusion-con t r oiled (k<,n 10s s"1, K,i <50 nM), ii) a highly fluorescent, stable base-flipped intermediate is formed, and iii) binding to uracil-DNA. but not other DNA leads to a conformational change in UDG that is overall rate-limiting for all steps leading to. and including, glycosidic bond cleavage.