Biochemical studies of replication-dependent DNA repair by endonuclease VIII-like III

Abstract

DNA damage within a cell can cause genome instability if not properly corrected. 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. NEIL3 unhooks interstrand cross-links (ICLs), which covalently tether opposing DNA strands and block transcription and replication and have been known to be repaired by the Fanconi Anemia pathway. NEIL3 contains two C-terminal GRF-type zinc finger (GRF-ZF) motifs absent in other NEIL paralogs and the role of the GRF-ZF were previously unknown. Here we show the substrate specificity of the NEIL3 glycosylase domain (NEIL3-GD) toward model replication forks with ICL or oxidized lesions on the leading template strand. 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 multiple conformations. We use multiple biochemical methods to show that the GRF-ZF domain binds with higher affinity than individual GRF-ZF motifs, and to calculate the DNA length dependence and DNA binding stoichiometry of the GRF-ZF domain. Functionally we show an autoinibitory role of the GRF-ZF domain toward base excision and unhooking activity of the NEIL3-GD. This autoinhibitory activity contrasts GRF-ZF domains of other DNA processing enzymes, which typically bind ssDNA to enhance catalytic activity, and suggests that the NEIL3 C-terminal is involved in DNA damage recruitment and enzymatic regulation. In contrast to the DNA-binding GRF-ZF, the internal NEIL3 Npl4-like zinc finger (NZF) binds ubiquitinated replicative CMG (Cdc45-MCM2-7-GINS) helicase and is currently being investigated for ubiquitin stoichiometry and linkage specificity. Preliminary ubiquitin binding assays show a preference toward monoubiquitin over diubiquitin species, similar to other NZF containing proteins. Thus, the work presented in this dissertation helps to reveal the role of the three domains within NEIL3 that aid in recruitment to replication-dependent damage and regulate ICL unhooking and base excision activity.