Catalytic Mechanism of Escherichia coli Endonuclease VIII: Roles of the Intercalation Loop and the Zinc Finger†

Endonuclease VIII (Nei), a bacterial enzyme with DNA N-glycosylase and abasic site lyase (AP1 lyase) activities, participates in base excision repair of oxidatively generated DNA damage (1, 2). Nei is homologous to another prokaryotic enzyme, formamidopyrimidine-DNA glycosylase (Fpg) (3); together with Fpg and several eukaryotic homologs, it forms the Fpg/Nei family of DNA repair glycosylases (4, 5). Despite the structural homology, Nei preferentially removes oxidatively damaged pyrimidines from substrate DNA (1, 2, 6), whereas Fpg excises oxidatively damaged purines (7, 8). During catalysis, the Nα of the N-terminal proline moiety of Nei attacks the C1’ atom of the damaged nucleoside, leading to loss of the base and formation of a Schiff base intermediate. The latter rearranges to eliminate the 3′-phosphate (β-elimination) and 5′-phosphate (δ-elimination) from the deoxyribose moiety. In bacteria, Nei likely serves as a back-up for endonuclease III, the major glycosylase with activity against oxidatively damaged pyrimidines. The three-dimensional structure of Nei (9) and its covalent complex with DNA (10) have been solved. Nei consists of two domains and possesses several defined structural motifs whose functional importance is evident from the structure. In particular, the enzyme everts the damaged nucleotide from DNA to gain access to C1’, a target for nucleophilic attack by the N-terminal proline moiety. This eversion, referred to as “base flipping”, is accompanied by the insertion of three amino acid residues (Gln-69, Leu-70 and Tyr-71, referred to here as the QLY loop or the intercalation loop) into the void left in the helix after base extrusion. The amino acids forming this short loop also participate in unstacking of the base opposite the lesion, introducing a sharp kink into DNA, and forming hydrogen bonds with the base opposite the lesion, thereby contributing to the observed opposite-base specificity. Another important structural motif is a single C-terminal Cys4-type zinc finger bearing an arginine residue (Arg-252), which is absolutely conserved among members of the Fpg/Nei family (11). Arg-252 is involved in a tight network of hydrogen bonds surrounding the lesion that hold DNA in a highly distorted conformation. The functions of several important residues (Pro-1, Glu-2, Glu-5, Lys-52, Asp-128, Asp-159, Glu-174, Arg-212, Arg-252) have been explored by site-directed mutagenesis (10, 12). In general, mutations of amino acids involved in hydrogen-bonding at the active site compromise DNA glycosylase activity of Nei but, surprisingly, do not affect AP lyase activity. Structural studies (G. Golan, D.O. Zharkov, A.P. Grollman and G. Shoham, paper in preparation) suggest that the active site of Nei possesses sufficient flexibility to compensate for the loss of a few prima facie critical interactions with the abasic substrate, allowing strand scission to take place. The substrate specificity of Nei, with regard to its preference for different lesions (lesion specificity) and for different bases opposite the lesion (opposite-base specificity), have not been systematically investigated. Approximately 20 different oxidatively damaged bases (both purine and pyrimidine derivatives) are reportedly excised by Nei (reviewed in (5)), but reports of the relative efficiency of their excision are inconsistent. The preference of Nei for the opposite base is primarily G ≥ A>(C or T) (2, 13-16), although C was found to be the preferred base opposite xanthine and 8-oxo-7,8-dihydroadenine (17). Recently, we developed a structural-bioinformatic approach designed to predict functionally important residues in DNA glycosylases (11, 18). This method was used to select residues contributing to substrate specificity of Fpg, and several site-directed Fpg mutants with altered lesion or opposite-base specificity were successfully constructed (19). Here, we applied structural and conservation considerations to select residues in Nei that are likely to contribute in the multistage process of damage recognition. We report a mutational analysis of the intercalation loop and the zinc finger in Nei, and the effect of mutations in these regions on the activity and substrate specificity of this enzyme.