Conformational Dynamics of Damage Processing by Human DNA Glycosylase NEIL1

Olga A. Kladova, Inga R. Grin, Olga S. Fedorova, Nikita A. Kuznetsov, Dmitry O. Zharkov

Результат исследования: Научные публикации в периодических изданияхстатьярецензирование

7 Цитирования (Scopus)

Аннотация

Endonuclease VIII-like protein 1 (NEIL1) is a DNA repair enzyme found in higher eukaryotes, including humans. It belongs to the helix–two turn–helix (H2TH) structural superfamily together with Escherichia coli formamidopyrimidine–DNA glycosylase (Fpg) and endonuclease VIII (Nei), and removes a variety of oxidized purine and pyrimidine bases from DNA. Structural, modeling and kinetic studies have established that the bacterial H2TH superfamily enzymes proceed through several conformational intermediates while recognizing and removing their cognate lesions. Here we apply stopped-flow kinetics with detection of intrinsic Trp fluorescence and Förster resonance energy transfer fluorescence to follow the conformational dynamics of human NEIL1 and DNA when the enzyme interacts with undamaged DNA, or DNA containing cleavable or non-cleavable abasic sites, or dihydrouracil lesions. NEIL1 processed a natural abasic site and a damaged base in DNA equally well but showed an additional fluorescently discernible step when DHU was present, likely reflecting additional rearrangements during base eversion into the enzyme's active site. With undamaged DNA and DNA containing a non-cleavable abasic site analog, (3-hydroxytetrahydrofuran-2-yl)methyl phosphate, NEIL1 was diverted to a non-productive DNA conformation early in the reaction. Our results support the view of NEIL1 as an enzyme that actively destabilizes damaged DNA and uses multiple checkpoints along the reaction coordinate to drive substrate lesions into the active site while rejecting normal bases and non-substrate lesions.

Язык оригиналаанглийский
Страницы (с-по)1098-1112
Число страниц15
ЖурналJournal of Molecular Biology
Том431
Номер выпуска6
DOI
СостояниеОпубликовано - 15 мар 2019

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