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-β10 loop” capping 8-oxoG in the active site of bacterial Fpg is very short in AthFpg. Deletion of a segment encompassing residues 213 to 229 in Escherichia coli Fpg (EcoFpg) and corresponding to the “αF-β10 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.

Tautomerization-dependent recognition and excision of oxidation damage in base-excision DNA repair

“…These structures identified a 13-amino acid residue loop that plays important roles in the recognition of both THF and Tg. For oxoG-recognizing Fpg proteins, the similar lesion recognition loop is much longer, although the lesion recognition loops of Bacillus stearothermophilus Fpg (BstFpg) and Lactococcus lactis Fpg were either visible or disordered in different structures (36,38,39); for Arabidopsis thaliana Fpg which does not excise oxoG, its corresponding loop was found to be in one uniform conformation in the apo and THF-bound structures (40). For the Tg-recognizing MvNei1, only one conformation was observed for the corresponding loop in the apo, THF-and Tg-bound structures of MvNei1, which mostly resembles the THF-bound NEIL1 structure (33,34).…”

Two glycosylase families diffusively scan DNA using a wedge residue to probe for and identify oxidatively damaged bases

“…Crystal structures of Fpg with either 8-oxoG (10,16) or FapyG (41) show that the lesion is stabilized in the binding pocket by the αF-β9/10 loop. However, FapyG is recognized even when this loop is deleted (42). There are currently no structures of bacterial Nei or Nth with lesions in the substrate binding pocket, but the structure of a viral ortholog of human NEIL1, MvNei, has been solved with Tg or 5-hydroxyuracil in the binding site pocket (43).…”

Removal of oxidatively generated DNA damage by overlapping repair pathways

“…The prokaryotic Fpg protein, referred also as MutM, which is known to repair 8oxoG and FapyG [70] belongs to the Fpg/Nei family of DNA glycosylases [65]. However, the eukaryotic Fpg homolog NEIL1 does not excise 8-oxoG, but recognizes and processes its oxidation products Sp and Gh [71, 72].…”