Structural and biochemical studies of a plant formamidopyrimidine-DNA glycosylase reveal why eukaryotic Fpg glycosylases do not excise 8-oxoguanine

Abstract

Formamidopyrimidine-DNA glycosylase (Fpg; MutM) is a DNA repair enzyme widely distributed in bacteria. Fpg recognizes and excises oxidatively modified purines, 4,6-diamino-5-formamidopyrimidine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 8-oxoguanine (8-oxoG), with similar excision kinetics. It exhibits some lesser activity toward 8-oxoadenine. Fpg enzymes are also present in some plant and fungal species. The eukaryotic Fpg homologs exhibit little or no activity on DNA containing 8-oxoG, but they recognize and process its oxidation products, guanidinohydantoin (Gh) and spiroiminohydantoin (Sp). To date, several structures of bacterial Fpg enzymes unliganded or in complex with DNA containing a damaged base have been published but there is no structure of a eukaryotic Fpg. Here we describe the first crystal structure of a plant Fpg, Arabidopsis thaliana (AthFpg), unliganded and bound to DNA containing an abasic site analog, tetrahydrofuran (THF). Although AthFpg shares a common architecture with other Fpg glycosylases, it harbors a zincless finger, previously described in a subset of Nei enzymes, such as human NEIL1 and Mimivirus Nei1. Importantly the "αF-β9/10 loop" capping 8-oxoG in the active site of bacterial Fpg is very short in AthFpg. Deletion of a segment encompassing residues 213-229 in Escherichia coli Fpg (EcoFpg) and corresponding to the "αF-β9/10 loop" does not affect the recognition and removal of oxidatively damaged DNA base lesions, with the exception of 8-oxoG. Although the exact role of the loop remains to be further explored, it is now clear that this protein segment is specific to the processing of 8-oxoG.

Figure 1

Ribbon diagram of unliganded AthFpgΔ88. Helices are shown in purple and β-strands in yellow. Spheres represent missing residues.

Figure 2

(A) Ribbon diagram of the AthFpgΔ88/THF complex. The strictly conserved Arg (Arg 267) of the zincless finger, the conserved Asn (Asn 186) of the H2TH motif, the catalytic proline (Pro 2) and glutamate (Glu 3) are highlighted in red. Spheres indicate missing residues. (B) Schematic representation of AthFpgΔ88-DNA interactions. Position 0 and (0) are assigned to THF and its opposite base, respectively, and numbering is positive towards the 5’-end. The arrows represent hydrogen bonds and point towards the acceptor. H₂O indicates water-mediated interactions.

Figure 2

(A) Ribbon diagram of the AthFpgΔ88/THF complex. The strictly conserved Arg (Arg 267) of the zincless finger, the conserved Asn (Asn 186) of the H2TH motif, the catalytic proline (Pro 2) and glutamate (Glu 3) are highlighted in red. Spheres indicate missing residues. (B) Schematic representation of AthFpgΔ88-DNA interactions. Position 0 and (0) are assigned to THF and its opposite base, respectively, and numbering is positive towards the 5’-end. The arrows represent hydrogen bonds and point towards the acceptor. H₂O indicates water-mediated interactions.

Figure 3

Close-up view of the everted THF. AthFpgΔ88 residues are shown in yellow and the DNA in gray. A red dashed line indicates the distance between the amino group of Pro 2 and C’1 of THF. A simulated annealing omit map (green mesh) is contoured at 3σ.

Figure 4

Close-up view of the “void filling” triad. The residues forming the intercalation triad (Met 78, Arg 126 and Phe 128) are shown in cyan and the DNA is shown in green.

Figure 5

Close-up view of the recognition loop. Superposition of the AthFpgΔ88/THF complex (light blue) with the αF-β10 loop of the BstFpg/8-oxoG complex (dark gray, PDB code 1R2Y, [51]). THF is shown in light blue and 8-oxoG in dark gray.

Figure 6

Glycosylase/lyase activity assays and activity profile on γ-irradiated DNA of wild-type EcoFpg and EcoFpgΔ213-229. (A) The glycosylase assay was performed by incubating 10 nM of double-stranded substrate containing 8-oxoG:C, MeFapy:C, Gh:C, and Sp1:C with 50 nM of EcoFpgWT and EcoFpgΔ213-229. (B) The lyase assay was performed with a double-stranded substrate containing an abasic site opposite C. Each glycosylase reaction was incubated for 1, 5, 10, and 15 min at 37ºC, whereas lyase reactions were incubated for 0.5, 1, 2.5, and 5 min. (C) Activity profile of wild-type EcoFpg and EcoFpgΔ213-229 on γ-irradiated DNA. GC/MS analysis was used to identify and quantify the amount of damaged DNA bases released by each enzyme. The enzyme:substrate ratio was the same for each experiment.

Figure 6

Glycosylase/lyase activity assays and activity profile on γ-irradiated DNA of wild-type EcoFpg and EcoFpgΔ213-229. (A) The glycosylase assay was performed by incubating 10 nM of double-stranded substrate containing 8-oxoG:C, MeFapy:C, Gh:C, and Sp1:C with 50 nM of EcoFpgWT and EcoFpgΔ213-229. (B) The lyase assay was performed with a double-stranded substrate containing an abasic site opposite C. Each glycosylase reaction was incubated for 1, 5, 10, and 15 min at 37ºC, whereas lyase reactions were incubated for 0.5, 1, 2.5, and 5 min. (C) Activity profile of wild-type EcoFpg and EcoFpgΔ213-229 on γ-irradiated DNA. GC/MS analysis was used to identify and quantify the amount of damaged DNA bases released by each enzyme. The enzyme:substrate ratio was the same for each experiment.

Figure 6

Glycosylase/lyase activity assays and activity profile on γ-irradiated DNA of wild-type EcoFpg and EcoFpgΔ213-229. (A) The glycosylase assay was performed by incubating 10 nM of double-stranded substrate containing 8-oxoG:C, MeFapy:C, Gh:C, and Sp1:C with 50 nM of EcoFpgWT and EcoFpgΔ213-229. (B) The lyase assay was performed with a double-stranded substrate containing an abasic site opposite C. Each glycosylase reaction was incubated for 1, 5, 10, and 15 min at 37ºC, whereas lyase reactions were incubated for 0.5, 1, 2.5, and 5 min. (C) Activity profile of wild-type EcoFpg and EcoFpgΔ213-229 on γ-irradiated DNA. GC/MS analysis was used to identify and quantify the amount of damaged DNA bases released by each enzyme. The enzyme:substrate ratio was the same for each experiment.

Figure 7

DNA glycosylase activity of AthFpgΔ88 WI187-188IY and EcoFpg IY170-171WI. Excess active enzyme (50 nM) was incubated with double-stranded substrate (10 nM) containing 8-oxoG, MeFapyG, Gh, or Sp1 opposite C. Each glycosylase reaction was incubated for 1, 5, 10, and 15 min at 37ºC. AthFpgΔ88 and wild-type EcoFpg were used in the same condition to allow the comparison.

Figure 7

DNA glycosylase activity of AthFpgΔ88 WI187-188IY and EcoFpg IY170-171WI. Excess active enzyme (50 nM) was incubated with double-stranded substrate (10 nM) containing 8-oxoG, MeFapyG, Gh, or Sp1 opposite C. Each glycosylase reaction was incubated for 1, 5, 10, and 15 min at 37ºC. AthFpgΔ88 and wild-type EcoFpg were used in the same condition to allow the comparison.