An Autoinhibitory Role for the GRF Zinc Finger Domain of DNA Glycosylase NEIL3

Abstract

DNA damage within a cell can cause damaging effects on genome stability if not properly corrected. Exogenous factors such as UV-light and environmental toxins, and endogenous factors including reactive metabolites and oxidative stress cause DNA damage in the form of modified DNA nucleobases. Base excision repair (BER) is the primary pathway to fix these lesions and begins with recognition and excision of the damaged nucleobase by DNA glycosylase enzymes. The DNA glycosylase, endonuclease VIII-like 3 (NEIL3), removes oxidized bases from single-stranded (ss) DNA, and unhooks interstrand cross-links (ICLs), which covalently tether opposing DNA strands and block transcription and replication. NEIL3 contains two C-terminal GRF-type zinc finger (Zf-GRFs) motifs absent in other NEIL paralogs and the role of the Zf-GRFs are unknown. Here we show the first crystal structure of the C-terminus of NEIL3 which includes a flexible head-to-tail configuration, predicted to bind ssDNA in many conformations. We show via electrophoretic mobility shift assays (EMSA) that the Zf-GRF domain binds with higher affinity than individual Zf-GRF motifs. Binding assays performed by fluorescence anisotropy showed DNA length dependence and DNA binding stoichiometry of the Zf-GRF domain. Functionally we show that the Zf-GRFs inhibit base excision activity of the NEIL3 glycosylase domain (NEIL3-GD) in trans and in cis. This autoinhibitory activity contrasts GRF-Zf domains of other DNA processing enzymes, which typically bind ssDNA to enhance catalytic activity, and suggests that the C-terminal of NEIL3 is involved in DNA damage recruitment and enzymatic regulation.