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An Excimer Clamp for Measuring Damaged‐Base Excision by the DNA Repair Enzyme NTH1
Author(s) -
Jun Yong Woong,
Wilson David L.,
Kietrys Anna M.,
Lotsof Elizabeth R.,
Conlon Savannah G.,
David Sheila S.,
Kool Eric T.
Publication year - 2020
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.202001516
Subject(s) - dna glycosylase , base excision repair , chemistry , dna repair , dna damage , dna , nucleotide excision repair , biochemistry , biophysics , biology
Direct measurement of DNA repair enzyme activities is important both for the basic study of cellular repair pathways as well as for potential new translational applications in their associated diseases. NTH1, a major glycosylase targeting oxidized pyrimidines, prevents mutations arising from this damage, and the regulation of NTH1 activity is important in resisting oxidative stress and in suppressing tumor formation. Herein, we describe a novel molecular strategy for the direct detection of damaged DNA base excision activity by a ratiometric fluorescence change. This strategy utilizes glycosylase‐induced excimer formation of pyrenes, and modified DNA probes, incorporating two pyrene deoxynucleotides and a damaged base, enable the direct, real‐time detection of NTH1 activity in vitro and in cellular lysates. The probe design was also applied in screening for potential NTH1 inhibitors, leading to the identification of a new small‐molecule inhibitor with sub‐micromolar potency.