A Low-Activity Polymorphic Variant of Human NEIL2 DNA Glycosylase.

Zarina I Kakhkharova,Dmitry O Zharkov,Inga R Grin

doi:10.3390/ijms23042212

Abstract

Human NEIL2 DNA glycosylase (hNEIL2) is a base excision repair protein that removes oxidative lesions from DNA. A distinctive feature of hNEIL2 is its preference for the lesions in bubbles and other non-canonical DNA structures. Although a number of associations of polymorphisms in the hNEIL2 gene were reported, there is little data on the functionality of the encoded protein variants, as follows: only hNEIL2 R103Q was described as unaffected, and R257L, as less proficient in supporting the repair in a reconstituted system. Here, we report the biochemical characterization of two hNEIL2 variants found as polymorphisms in the general population, R103W and P304T. Arg103 is located in a long disordered segment within the N-terminal domain of hNEIL2, while Pro304 occupies a position in the β-turn of the DNA-binding zinc finger motif. Similar to the wild-type protein, both of the variants could catalyze base excision and nick DNA by β-elimination but demonstrated a lower affinity for DNA. Steady-state kinetics indicates that the P304T variant has its catalytic efficiency (in terms of kcat/KM) reduced ~5-fold compared with the wild-type hNEIL2, whereas the R103W enzyme is much less affected. The P304T variant was also less proficient than the wild-type, or R103W hNEIL2, in the removal of damaged bases from single-stranded and bubble-containing DNA. Overall, hNEIL2 P304T could be worthy of a detailed epidemiological analysis as a possible cancer risk modifier.

Highlights

Received: 8 December 2021DNA repair enzymes play a central role in the protection of living cells from genotoxic insults
Three of them—NEIL1, NEIL2, and NEIL3—are H2TH proteins. They are homologous to E. coli endonuclease VIII (Nei) and formamidopyrimidine-DNA glycosylase (Fpg), two enzymes participating in the repair of oxidative DNA damage
We have characterized the biochemical properties of two polymorphic variants of human DNA glycosylase NEIL2, R103W, and P304T

Summary

Introduction

DNA repair enzymes play a central role in the protection of living cells from genotoxic insults. BER is initiated by excision of damaged bases by one of a group of enzymes called DNA glycosylases, which recognize the lesions and hydrolyze their N-glycosidic bond [1,4]. Most DNA glycosylases belong to the following three major structural superfamilies that are characterized by the presence of conserved structural elements: the α/β-fold, the helix–hairpin–helix motif, or the helix–. Three of them—NEIL1, NEIL2, and NEIL3—are H2TH proteins. They are homologous to E. coli endonuclease VIII (Nei) and formamidopyrimidine-DNA glycosylase (Fpg), two enzymes participating in the repair of oxidative DNA damage. The roles of Nei and Fpg are different in the following ways: while Fpg is specific for oxidized purines, mostly

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