A metal ion-dependent mechanism promoting gain of function in NEIL1 variants

Maintenance of genomic integrity following ionizing radiation (IR) and chemical exposures is primarily a function of the base excision repair (BER) pathway. This genome surveillance is initiated by DNA glycosylases, with the endonuclease VIII-like 1 (NEIL1) glycosylase, apurinic/apyrimidinic site (AP) lyase being responsible for the release of IR-induced DNA lesions. Suppressed expression of NEIL1 leads to increased IR cytotoxicity. Previous analyses of a single nucleotide polymorphic (SNP) variant of NEIL1, Thr103Ala (T103A), revealed compromised catalytic activities. This investigation was designed to determine the mechanisms underlying the deficiencies of T103A.

Mutant and variant NEIL1 enzymes were constructed and characterized for their ability to catalyze incision of DNAs containing site-specific base damages. Molecular dynamics (MD) simulations of wild-type (WT) and T103A variant NEIL1 using the NEIL1 crystal structure, addressed mechanisms conferring catalytic activity changes.

The glycosylase and AP lyase activities of NEIL1 T103A could be partially restored in the presence of Mg²⁺. The catalytic activities of T103S were also stimulated by this metal. Similar increases of the reaction rates of T103A and T103S were observed in the presence of Ca²⁺. In contrast, the activities of WT NEIL1 and T103V mutant were not stimulated by the addition of these metals. MD simulations reveal that the surface loop (residues 102-110) displays enhanced conformational fluctuations in the T103A variant compared to that region in WT NEIL1. Mg²⁺ hexahydrate coordinated to carbonyl oxygens of loop residues dampens loop dynamics.

This study is the first report of the metal-dependent stimulation of compromised activities in a DNA glycosylase variant. The MD simulations suggest that Mg²⁺ stabilizes the surface loop dynamics in the T103A variant, with the effects of the T to A substitution and Mg²⁺ presence being transmitted over a distance greater than 26 Å to the NEIL1 active site.