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...

contributed equally to this work Endonuclease VIII (Nei) of Escherichia coli is a DNA repair enzyme that excises oxidized pyrimidines from DNA. Nei shares with formamidopyrimidine-DNA glycosylase (Fpg) sequence homology and a similar mechanism of action: the latter involves removal of the damaged base followed by two sequential b-elimination steps. However, Nei differs signi®cantly from Fpg in substrate speci®city. We determined the structure of Nei covalently crosslinked to a 13mer oligodeoxynucleotide duplex at 1.25 A Ê resolution. The crosslink is derived from a Schiff base intermediate that precedes b-elimination and is stabilized by reduction with NaBH 4 . Nei consists of two domains connected by a hinge region, creating a DNA binding cleft between domains. DNA in the complex is sharply kinked, the deoxyribitol moiety is bound covalently to Pro1 and everted from the duplex into the active site. Amino acids involved in substrate binding and catalysis are identi®ed. Molecular modeling and analysis of amino acid conservation suggest a site for recognition of the damaged base. Based on structural features of the complex and site-directed mutagenesis studies, we propose a catalytic mechanism for Nei.

The crystal structure of protein L9 from the Bacillus stearothermophilus ribosome has been determined at 2.8 A resolution using X‐ray diffraction methods. This primary RNA‐binding protein has a highly elongated and unusual structure consisting of two separated domains joined by a long exposed alpha‐helix. Conserved, positively charged and aromatic amino acids on the surfaces of both domains probably represent the sites of specific interactions with 23S rRNA. Comparisons with other prokaryotic L9 sequences show that while the length of the connecting alpha‐helix is invariant, the sequence within the exposed central region is not conserved. This suggests that the alpha‐helix has an architectural role and serves to fix the relative separation and orientation of the N‐ and C‐terminal domains within the ribosome. The N‐terminal domain has structural homology to the smaller ribosomal proteins L7/L12 and L30, and the eukaryotic RNA recognition motif (RRM).

Escherichia coli endonuclease VIII (Nei) excises oxidized pyrimidines from DNA. It shares significant sequence homology and similar mechanism with Fpg, a bacterial 8-oxoguanine glycosylase. The structure of a covalent Nei–DNA complex has been recently determined, revealing critical amino acid residues which are important for DNA binding and catalysis. Several Fpg structures have also been reported; however, analysis of structural dynamics of Fpg/Nei family proteins has been hindered by the lack of structures of uncomplexed and DNA-bound enzymes from the same source. We report a 2.8 Å resolution structure of free wild-type Nei and two structures of its inactive mutants, Nei-E2A (2.3 Å) and Nei-R252A (2.05 Å). All three structures are virtually identical, demonstrating that the mutations did not affect the overall conformation of the protein in its free state. The structures show a significant conformational change compared with the Nei structure in its complex with DNA, reflecting a ∼50° rotation of the two main domains of the enzyme. Such interdomain flexibility has not been reported previously for any DNA glycosylase and may present the first evidence for a global DNA-induced conformational change in this class of enzymes. Several local but functionally relevant structural changes are also evident in other parts of the enzyme.