Structural Characterization of a Viral NEIL1 Ortholog Unliganded and Bound to Abasic Site-containing DNA

Abstract

Endonuclease VIII (Nei) is a DNA glycosylase of the base excision repair pathway that recognizes and excises oxidized pyrimidines. We determined the crystal structures of a NEIL1 ortholog from the giant Mimivirus (MvNei1) unliganded and bound to DNA containing tetrahydrofuran (THF), which is the first structure of any Nei with an abasic site analog. The MvNei1 structures exhibit the same overall architecture as other enzymes of the Fpg/Nei family, which consists of two globular domains joined by a linker region. MvNei1 harbors a zincless finger, first described in human NEIL1, rather than the signature zinc finger generally found in the Fpg/Nei family. In contrast to Escherichia coli Nei, where a dramatic conformational change was observed upon binding DNA, the structure of MvNei1 bound to DNA does not reveal any substantial movement compared with the unliganded enzyme. A protein segment encompassing residues 217-245 in MvNei1 corresponds to the "missing loop" in E. coli Nei and the "␣F-␤10 loop" in E. coli Fpg, which has been reported to be involved in lesion recognition. Interestingly, the corresponding loop in MvNei1 is ordered in both the unliganded and furan-bound structures, unlike other Fpg/Nei enzymes where the loop is generally ordered in the unliganded enzyme or in complexes with a lesion, and disordered otherwise. In the MvNei1⅐tetrahydrofuran complex a tyrosine located at the tip of the putative lesion recognition loop stacks against the furan ring; the tyrosine is predicted to adopt a different conformation to accommodate a modified base.All organisms must cope with the generation of potentially lethal or mutagenic oxidative DNA base damage produced by endogenous free radicals. The enzymes that recognize and initiate the repair of these lesions are the DNA glycosylases, which are found ubiquitously in all three kingdoms of life (for reviews see Refs. 1-4). Some of these enzymes are bifunctional, i.e. they catalyze the hydrolysis of the N-glycosyl bond linking a base to a deoxyribose (glycosylase activity) and subsequently cleave the DNA 3Ј to the apurinic/apyrimidinic site (lyase activity), whereas others are monofunctional and only carry out the glycosylase reaction, generating abasic sites as products. Structural studies indicate that the DNA glycosylases that recognize oxidative DNA damages fall into two family groups: the helixhairpin-helix superfamily and the Fpg/Nei family (for reviews see Refs. 1, 5, 6). The helix-hairpin-helix superfamily includes a diverse group of enzymes with varying substrate specificities which nonetheless share a helix-hairpin-helix motif that consists of two ␣-helices connected by a hairpin loop, followed by a Gly/Pro-rich loop and a conserved catalytic aspartate residue (7). Glycosylase members of the second family, the Fpg/Nei family, share a two-domain architecture: The N-terminal domain consists of a two-layered ␤-sandwich with two ␣-helices, whereas the C-terminal domain contains four ␣-helices, of which two are involved in a conserved helix-two-turn-helix (H2TH)...

Two glycosylase families diffusively scan DNA using a wedge residue to probe for and identify oxidatively damaged bases

“…This search process must be robust and efficient so that glycosylases can meet the constant challenge presented by oxidative DNA damage. For Fpg, Nei, and Nth, base interrogation and recognition begin by insertion of the wedge residue, and thus damage recognition occurs before base extrusion into the enzyme's substrate binding pocket in keeping with our previous results (18) as well as recent crystallographic studies (20)(21)(22)51) and ensemble kinetics analysis (44). Although our modeling suggests that glycosylases only "spot check" for damage through wedge residue insertion, diffusive motion is highly redundant, meaning that the glycosylase revisits individual bases many times during a trajectory.…”

RNA editing changes the lesion specificity for the DNA repair enzyme NEIL1

“…Red open arrow indicates lesion recognition loop of Fpg. (B) Sequence alignment of Fpg/Nei family of DNA repair glycosylases indicating the position of the hNEIL1 recoding site and lesion recognition loop as identified by Imamura, Wallace, and Doublie (18,22). Fig.…”

“…From an analysis of these structures, we realized the NEIL1 recoding site is located in the previously identified lesion recognition loop of this family of DNA repair enzymes ( Fig. 2) (22). This observation suggested to us that RNA editing might regulate NEIL1 activity by modulating efficiency and/or specificity of damaged base removal.…”

“…In addition, while structural data for a complex of human NEIL1 with damaged DNA has not been reported, complexes of related repair glycosylases with DNA containing damaged bases have been structurally characterized (22,23). From an analysis of these structures, we realized the NEIL1 recoding site is located in the previously identified lesion recognition loop of this family of DNA repair enzymes ( Fig.…”

Tautomerization-dependent recognition and excision of oxidation damage in base-excision DNA repair

“…Although the crystal structure of NEIL1 protein is determined (32), a complex structure of NEIL1 bound to duplex DNA has not been reported. Recent studies have determined several complex structures of MvNei1 (mimivirus Nei1), a viral ortholog of human NEIL1 (33,34). However, MvNei1 and NEIL1 have apparent differences in their 3D structures and enzymatic properties (5).…”