Structure of Formamidopyrimidine-DNA Glycosylase Covalently Complexed to DNA

Abstract

Oxidative DNA damage is generated by a variety of environmental and endogenous agents, including ionizing radiation, certain chemicals, and products of aerobic metabolism (1). 8-oxoG 1 is one of the most abundant forms of oxidative DNA damage (2). Due to its ability to form a Hoogstein-type base pair with adenine (3), 8-oxoG is miscoding (4) and mutagenic, resulting in G3 T transversions in bacterial and eukaryotic cells (5, 6). The potential harmful effects of this lesion are avoided by base excision repair. In Escherichia coli, formamidopyrimidine-DNA glycosylase (Fpg, EC 3.2.2.23) removes 8-oxoG, Me-FaPy, and several structurally related lesions from damaged DNA (7,8). Fpg is a component of the "GO system" that includes MutY, a mismatch adenine-DNA glycosylase, and MutT, an 8-oxodGTPase (9, 10); E. coli strains deficient in any of these genes are strong mutators (11).Fpg shares significant sequence homology with endonuclease VIII (Nei) of E. coli (12). Both proteins belong to a family unrelated by sequence or tertiary structure to a larger family of DNA glycosylases, for which the prototype is endonuclease III (Nth) (13,14). The substrate specificity of Fpg differs significantly from Nei (7, 8, 15) but closely resembles that of the eukaryotic 8-oxoguanine-DNA glycosylase, Ogg1, a member of the Nth family (14,16,17). Fpg also possesses AP lyase activity, nicking the phosphodiester backbone of DNA at the site of the lesion. Base excision by Fpg is followed immediately by two ␤-elimination steps, resulting in a single nucleotide gap flanked by phosphate termini (7). A Schiff base intermediate, involving Pro-1 of the enzyme and C1Ј of the damaged nucleotide, forms early in the reaction sequence and can be reductively trapped by treatment with NaBH 4 forming a stable covalent complex (18,19). The mechanism of cleavage is similar to that of Nei (15,20), but not to that of Ogg1 where only one ␤-elimination occurs, and the efficiency of the elimination step is very low compared with base excision (16,17).Comparing the structures of Fpg, Nei, and Ogg1 provides a unique opportunity to analyze features of damage recognition and catalysis common to DNA glycosylases/AP lyases. The presence of DNA enhances the analytic power of the model by revealing the precise nature of enzyme-DNA interactions. The structure of the human Ogg1 catalytic domain complexed to DNA has been solved (21, 22), as has the structure of E. coli Nei covalently cross-linked to DNA by NaBH 4 (23). The structure of Fpg from Thermus thermophilus HB8 (Tth-Fpg) has recently been solved in the absence of DNA (24). Although mechanisms for lesion recognition and catalysis by Fpg have been suggested on the basis of this structure and on earlier biochemical studies of E. coli Fpg (8,18,24,25), many questions remain unanswered regarding the mode of Fpg-DNA interactions and the catalytic reaction mechanism of this important DNA repair protein.To investigate the mechanisms of Fpg-DNA interactions, we have utilized NaBH 4 reduction of the Schiff base intermediate t...

DNA Lesion Recognition by the Bacterial Repair Enzyme MutM

“…3b). Confidence in this model is strengthened by the fact that the active site structure, including the position and conformation of Glu-3, is nearly identical in several independently determined x-ray co-crystal structures of MutM (11)(12)(13) and EndoVIII (31) representing different stages of the repair reaction. The only significant point of divergence among all the DNA-bound structures, apart from the differences in covalent structure of the bound lesion, is the degree of disorder in the ␤F-␣10 loop.…”

“…2). The oxo-dG moiety is extruded from the DNA duplex and inserted into the extrahelical MutM active site, as anticipated from the oxoG-less structures (11)(12)(13) and in common with human Ogg1 (hOgg1) and other structurally characterized DNA glycosylases that act on single-base lesions. Apart from this point of similarity, the oxoG recognition mode of MutM is quite distinct from that of hOgg1.…”

“…A segment of the protein near the active site is disordered in the DNA-bound MutM structures lacking an oxoG nucleobase, yet is ordered in the absence of DNA, thus leading to the tantalizing suggestion that induced fit might contribute to lesion base recognition. Understanding the structural determinants of lesion recognition by MutM has been hampered by the fact that none of the available high resolution structures contain a damaged base (11)(12)(13).…”

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

“…However, some members of these families have common npg substrates. OGG1, MutY, Nth and their homologs have HhH structure [42][43][44]; while MutM (Fpg) and Nei have H2TH motif [44][45][46][47].…”

The crystal structure of human endonuclease VIII-like 1 (NEIL1) reveals a zincless finger motif required for glycosylase activity

“…Fpg and Nei also share common structural motifs, including helix-two-turns-helix (H2TH) and antiparallel ␤-hairpin zinc finger motifs (6,12). A comparison of EcoNei covalently complexed to DNA (20) with the Fpg structures (21)(22)(23)(24) revealed that their overall folds are very similar; however, their substrate preferences are markedly different: EcoFpg prefers 8-oxoguanine and oxidized purines (25,26), whereas EcoNei recognizes oxidized pyrimidines (19,(27)(28)(29).…”