Reactive oxygen and nitrogen species (RONS) generated from endogenous and environmental sources can result in detrimental oxidation of DNA bases. The hyperoxidized products of guanine, guanidino- and spiriminodi-hydantoins (Gh and Sp), are particularly toxic and mutagenic. The endonuclease VIII-like (NEIL) family of DNA repair glycosylases have been shown to have high activity toward the removal of Gh and Sp and are capable of excising these lesions in a variety of contexts, such as duplex, single stranded, bubble, and bulge DNA. Notably, NEIL1 and NEIL3 are also capable of excising the hydantoins from G-quadruplexes (G4) under certain conditions. Due to the prevalence of G4 structures in both oncogene and DNA repair enzyme promoter regions, understanding how glycosylases process damaged bases in a G4 context will provide insight into the impact of damage and its repair on gene expression. In this study, we conducted binding and kinetic studies to determine the ability of NEIL1 and NEIL3 to target and remove Gh lesions from G4 DNA. The features of NEIL enzyme action were examined in G4 sequences from the <i>VEGF, KRAS</i>, and <i>RAD17</i> promoters with the lesions located in different positions. Under single turnover conditions, the observed production curves for NEIL-catalyzed base removal are biphasic, indicating a fraction of substrate is processed rapidly followed by a fraction that is removed relatively slowly. The extent of substrate processing at these two distinct rates differs due to the sequence and location of the Gh lesion. However, the most striking observation is the impact on G-quad structure on the overall extent of Gh removal. In some cases, such as VEGF, a significant amount of substrate remains unprocessed despite enzyme being present in excess. Binding studies suggest this may be a consequence of NEIL enzyme binding to the G4 in an unproductive manner that does not support efficient Gh excision. These results provide insight into the interaction and glycosylase activity of NEIL with G4’s and suggest a possible dynamic relationship between DNA repair and gene regulation.

Excision of oxidatively damaged bases in G-quadruplexes by the DNA glycosylases NEIL1 and NEIL3

2019 Fall National Meeting

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