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

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 (MmuNeil3Delta324) 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.

Fig. 1.

Alignment of the structural features of Mus musculus Neil3 with representative members of the Fpg/Nei family. Domains and motifs in the scheme are described in the box below. Sequences of the conserved N terminus are shown in the bracket. MmuNeil3 wt: wild-type Mus musculus Neil3; MmuNeil3Δ324: glycosylase domain of Mus musculus Neil3; NEIL1: human Neil1; NEIL2: human Neil2; MvNei2: Mimivirus Nei2; EcoNei: Escherichia coli Nei; EcoFpg: Escherichia coli Fpg.

Fig. 2.

DNA glycosylase/lyase activity of MmuNeil3. Double-stranded substrates containing Sp1, Sp2, Gh, or an AP site (25 nM) were incubated with 25 nM active MmuNeil3 wt (lanes 6, 13, 20, and 27), MmuNeil3Δ324 (lanes 7, 14, 21, and 28), EcoFpg (lanes 2, 9, 16, and 23), EcoNei (lanes 3, 10, 17, and 24), NEIL1 (lanes 4, 11, 18, and 25) and MvNei2 (lanes 5, 12, 19, and 26) at 37 °C for 30 min. Reactions were stopped by formamide stop buffer to measure glycosylase plus lyase activities. Lanes 1, 8, 15, and 22, No enzyme control.

Fig. 3.

Quantification of base lesions recognized by MmuNeil3 in (A) double-stranded substrates and (B) single-stranded substrates. 25 nM active MmuNeil3 wt (black) or MmuNeil3Δ324 (gray) was incubated with 25 nM substrate at 37 °C for 30 min. Reactions were stopped by formamide stop buffer to measure glycosylase plus lyase activities. Sp stands for the mixture of Sp1 and Sp2. Data are expressed as means of three independent measurements. Uncertainties are standard deviations.

Fig. 4.

Substrate specificity of MmuNeil3 on γ-irradiated DNA. The amounts of damaged bases released by each enzyme as measured by GC/MS (Table S2) were normalized to show the preference of each enzyme as described in SI Materials and Methods.

Fig. 5.

Schiff base assay of purified MmuNeil3 proteins. Lanes 6 and 13, MmuNeil3 wt; lanes 7 and 14, MmuNeil3Δ324. Controls: Lanes 1 and 8, no enzyme control; lanes 2 and 9, trapping with purified EcoFpg; lanes 3 and 10, EcoNei; lanes 4 and 11, NEIL1; lanes 5 and 12, MvNei2.

Fig. 6.

Substrate specificity of MmuNeil3 in vivo. (A) Spontaneous forward mutation frequencies to rifampin resistance in E. coli. Mutants per 10⁸ cells are shown. Data are expressed as means of five independent measurements. Uncertainties are standard deviations. Statistical significance was determined by student’s t test and the p values were calculated relative to values in fpg nei mutY (DE3) cells; *, p < 0.01. (B) GC/MS measurement of the amount of FapyG in E. coli genomic DNA. Data are expressed as means of three independent measurements. Uncertainties are standard deviations. The p values were calculated relative to values of fpg nei mutY triple mutant strain; *, p < 0.01.