No abstract

To protect cells from oxidative DNA damage and mutagenesis, organisms possess multiple glycosylases to recognize the damaged bases and to initiate the Base Excision Repair pathway. Three DNA glycosylases have been identified in mammals that are homologous to the Escherichia coli Fpg and Nei proteins, Neil1, Neil2, and Neil3. Neil1 and Neil2 in human and mouse have been well characterized while the properties of the Neil3 protein remain to be elucidated. In this study, we report the characterization of Mus musculus (house mouse) Neil3 (MmuNeil3) as an active DNA glycosylase both in vitro and in vivo. In duplex DNA, MmuNeil3 recognizes the oxidized purines, spiroiminodihydantoin (Sp), guanidinohydantoin (Gh), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) and 4,6-diamino-5-formamidopyrimidine (FapyA), but not 8-oxo-7,8-dihydroguanine (8-oxoG). Interestingly, MmuNeil3 prefers lesions in single-stranded DNA and in bubble structures. In contrast to other members of the family that use the N-terminal proline as the nucleophile, MmuNeil3 forms a Schiff base intermediate via its N-terminal valine. We expressed the glycosylase domain of MmuNeil3 (MmuNeil3Δ324) in an Escherichia coli triple mutant lacking Fpg, Nei, and MutY glycosylase activities and showed that MmuNeil3 greatly reduced both the spontaneous mutation frequency and the level of FapyG in the DNA, suggesting that Neil3 plays a role in repairing FapyG in vivo.base excision repair | endonuclease VIII Like 3 | 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) | Spiroiminodihydantoin D NA glycosylases play an important role in maintaining genomic integrity by recognizing various nonhelix distorting base lesions created by ionizing radiation, alkylating, or oxidizing agents, which are often cytotoxic or mutagenic (1). DNA glycosylases cleave the N-glycosylic bond and release the base lesion from the sugar backbone, resulting in an apurinic or apyrimidinic (AP) site (2). Besides glycosylase activity, some of these enzymes also have a lyase activity to process the AP site. In this way, DNA glycosylases initiate the Base Excision Repair (BER) † pathway and create the substrates for further processing by a series of BER enzymes including phosphodiesterases, AP endonucleases, DNA polymerases, and DNA ligases to complete lesion repair (3).DNA glycosylases that recognize oxidized bases can be divided into two families based on structure and sequence homology (4, 5), the Nth family, whose members are widely distributed in bacteria, archaea, and eukaryotes (5), and the Fpg/Nei family, which is more sparsely distributed across phyla. Fpg/Nei family members are characterized by a signature helix-two turns-helix (H2TH) motif and a zinc finger (or "zincless finger") motif for DNA binding; they also have a conserved N terminus harboring a proline residue (P2) important for catalysis (6). In Escherichia coli, formamidopyrimidine DNA glycosylase (EcoFpg) mainly recognizes oxidized purines (7), and endonuclease VIII (EcoNei) mainly recognizes oxidized pyrimidines and adenin...

In prokaryotes, two DNA glycosylases recognize and excise oxidized pyrimidines: endonuclease III (Nth) and endonuclease VIII (Nei). The oxidized purine 8-oxoguanine, on the other hand, is recognized by Fpg (also known as MutM), a glycosylase that belongs to the same family as Nei. The recent availability of the human genome sequence allowed the identification of three human homologs of Escherichia coli Nei. We report here the crystal structure of a human Nei-like (NEIL) enzyme, NEIL1. The structure of NEIL1 exhibits the same overall fold as E. coli Nei, albeit with an unexpected twist. Sequence alignments had predicted that NEIL1 would lack a zinc finger, and it was therefore expected to use a different DNA-binding motif instead. Our structure revealed that, to the contrary, NEIL1 contains a structural motif composed of two antiparallel ␤-strands that mimics the antiparallel ␤-hairpin zinc finger found in other Fpg͞Nei family members but lacks the loops that harbor the zinc-binding residues and, therefore, does not coordinate zinc. This ''zincless finger'' appears to be required for NEIL1 activity, because mutating a very highly conserved arginine within this motif greatly reduces the glycosylase activity of the enzyme.

Endonuclease VIII (Nei), which recognizes and repairs oxidized pyrimidines in the Base Excision Repair (BER) pathway, is sparsely distributed among both the prokaryotes and eukaryotes. Recently, we and others identified three homologs of E. coli endonuclease VIII-like (NEIL) proteins in humans. Here, we report identification of human NEIL homologs in Mimivirus, a giant DNA virus that infects Acanthamoeba. Characterization of the two mimiviral homologs, MvNei1 and MvNei2, showed that they share not only sequence homology but also substrate specificity to the human NEIL proteins, that is, they recognize oxidized pyrimidines in duplex DNA and in bubble substrates and as well show 5′2-deoxyribose-5-phosphate lyase (dRP lyase) activity. However, unlike MvNei1 and the human NEIL proteins, MvNei2 preferentially cleaves oxidized pyrimidines in single stranded DNA forming products with a different end chemistry. Interestingly, opposite base specificity of MvNei1 resembles human NEIL proteins for pyrimidine base damages whereas it resembles E. coli formamidopyrimidine DNA glycosylase (Fpg) for guanidinohydantoin (Gh), an oxidation product of 8-oxoguanine. Finally, a conserved arginine residue in the "zincless finger" motif, previously identified in human NEIL1, is required for the DNA glycosylase activity of MvNeil. Thus, Mimivirus represents the first example of a virus to carry oxidative DNA glycosylases with substrate specificities that resemble human NEIL proteins. Based on the sequence homology to the human NEIL homologs and novel bacterial NEIL homologs identified here, we predict that Mimivirus may have acquired the DNA glycosylases through the host-mediated lateral transfer from either a bacterium or from vertebrates.

Formamidopyrimidine DNA glycosylase (Fpg) and endonuclease VIII (Nei) share an overall common three-dimensional structure and primary amino acid sequence in conserved structural motifs but have different substrate specificities, with bacterial Fpg proteins recognizing formamidopyrimidines, 8-oxoguanine (8-oxoG) and its oxidation products guanidinohydantoin (Gh), and spiroiminodihydantoin (Sp) and bacterial Nei proteins recognizing primarily damaged pyrimidines. In addition to bacteria, Fpg has also been found in plants, while Nei is sparsely distributed among the prokaryotes and eukaryotes. Phylogenetic analysis of Fpg and Nei DNA glycosylases demonstrated, with 95% bootstrap support, a clade containing exclusively sequences from plants and fungi. Members of this clade exhibit sequence features closer to bacterial Fpg proteins than to any protein designated as Nei based on biochemical studies. The Candida albicans (Cal) Fpg DNA glycosylase and a previously studied Arabidopsis thaliana (Ath) Fpg DNA glycosylase were expressed, purified and characterized. In oligodeoxynucleotides, the preferred glycosylase substrates for both enzymes were Gh and Sp, the oxidation products of 8-oxoG, with the best substrate being a site of base loss. GC/MS analysis of bases released from γ-irradiated DNA show FapyAde and FapyGua to be excellent substrates as well. Studies carried out with oligodeoxynucleotide substrates demonstrate that both enzymes discriminated against A opposite the base lesion, characteristic of Fpg glycosylases. Single turnover kinetics with oligodeoxynucleotides showed that the plant and fungal glycosylases were most active on Gh and Sp, less active on oxidized pyrimidines and exhibited very little or no activity on 8-oxoG. Surprisingly, the activity of AthFpg1 on an AP site opposite a G was extremely robust with a k obs of over 2500 min −1 .