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

Research output: Contribution to journalArticlepeer-review

7 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1098-1112
Number of pages15
JournalJournal of Molecular Biology
Volume431
Issue number6
DOIs
Publication statusPublished - 15 Mar 2019

Keywords

  • DNA glycosylase
  • DNA repair
  • NEIL1
  • stopped-flow kinetics
  • substrate recognition
  • VIRAL ORTHOLOG
  • SEARCH
  • STRUCTURAL-CHARACTERIZATION
  • COLI ENDONUCLEASE-VIII
  • 8-OXOGUANINE
  • FAMILY
  • REPAIR
  • EXCISION
  • LESION RECOGNITION
  • FPG PROTEIN

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