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

J. T. Stivers, K. V.V. Pankiewiçz, K. A. Watanabe

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Pages (from-to)A1352
JournalFASEB Journal
Volume12
Issue number8
StatePublished - Dec 1 1998
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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