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

Abstract

Purpose: 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.

Materials and methods: 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.

Results: 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.

Conclusion: 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.