Environmental toxins, endogenous metabolic products, and ionizing radiation can directly modify DNA or indirectly damage DNA through the generation of reactive oxygen and nitrogen species (RONS). This DNA damage arises at a rate of thousands per day compromising the integrity of the genome. The endonuclease VIII-like (NEIL) family of DNA repair glycosylases initiate repair of the damage produced by RONS. They exhibit particularly high activity toward the removal of hyperoxidized products of guanine, such as the guanidino- and spiriminodi-hydantoins (Gh and Sp), which mediate G-to-T and G-to-C transversion mutations and serve as replication blocks. The NEIL family is also capable of excising these lesions from a variety of DNA contexts, such as duplex, single-stranded, bubble, and bulge DNA; notably, NEIL1 and NEIL3 both have the unique ability to excise Gh and Sp from G-quadruplex (G4) DNA. Due to the location of these G4 DNA structures in various oncogene and repair enzyme promoter regions, understanding how NEIL processes damaged bases in a G4 context can provide insight into the unique roles of NEIL beyond genome maintenance. We have conducted a thorough kinetic and binding affinity analysis of the repair of Gh lesions from different G4 sequences by NEIL1 and NEIL3. Specifically, under single turnover conditions, the removal of Gh by NEIL was examined in G4 sequences from the <i>VEGF, KRAS, </i>and <i>RAD17</i> promoter regions. Interestingly, the observed production curves for NEIL-catalyzed base removal are biphasic, indicating that the substrate is processed at two distinct rates, and this is highly dependent on the sequence and location of Gh within the G4. Our most striking observation indicates that a significant amount of substrate remains unprocessed despite enzyme being present in excess. This suggests that the G4 structure makes an impact on the overall extent of Gh removal. Binding studies suggest that NEIL may be binding in an unproductive manner that does not support Gh excision. Our results showcase the interaction of NEIL with G4s and provide insight into a possible dynamic relationship between the NEIL family of enzymes, DNA repair, and gene regulation.

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

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