Human endonuclease VIII-like (NEIL) proteins in the giant DNA Mimivirus

Abstract

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.

The mouse ortholog of NEIL3 is a functional DNA glycosylase in vitro and in vivo

“…S1). Unlike EcoFpg, EcoNei, and NEIL1 that cleave their substrates via β,δ-elimination leaving the majority of cleavage products with a phosphate at the 3′ end (9, 11), but like MvNei2 (14), MmuNeil3 wt, and MmuNeil3Δ324 cleaved their substrates primarily via β-elimination leaving cleavage products with an α,β-unsaturated aldehyde (Fig. 2).…”

“…Neil3 also lacks the catalytic proline at its N terminus, but has instead a valine residue. Although the N-terminal valine is present in another Fpg/Nei family member from Acanthamoeba polyphaga mimivirus (MvNei2) which has robust glycosylase activity (14), there is no direct evidence so far that this residue functions in catalysis. Therefore, it was difficult to predict if Neil3 actually had glycosylase activity.…”

Structural Characterization of Viral Ortholog of Human DNA Glycosylase NEIL1 Bound to Thymine Glycol or 5-Hydroxyuracil-containing DNA

“…To minimize the error because of the uncertainty in the DNA concentration after ethanol precipitation, the labeled oligonucleotide for the glycosylase assay was diluted 10-fold with the corresponding unlabeled damaged oligonucleotide (26). For the AP lyase assay, the labeled oligodeoxynucleotide was used undiluted.…”

“…Expression and purification of MvNei1 variants were performed using a published protocol (26). For the kinetics experiments, all MvNei1 variants were stored at Ϫ20°C in a storage buffer (20 mM Hepes-KOH, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, and 50% (v/v) glycerol).…”

“…In at least one of these cases, the discrepancy may be attributed to a difference in the position of the lesion within the DNA oligonucleotide (29). Among the commercially available substrates tested, MvNei1 recognizes and removes thymine glycol (5,6-dihydro-5,6-dihydroxythymine (Tg)) and 5-hydroxyuracil (5-OHU) and displays negligible activity toward 8-oxoG (26). Tg is the most common oxidation product of thymine (30,31), and 5-OHU is generated by the oxidative deamination of cytosine (32,33) (Fig.…”