NH2-terminal Proline Acts as a Nucleophile in the Glycosylase/AP-Lyase Reaction Catalyzed by Escherichia coli Formamidopyrimidine-DNA Glycosylase (Fpg) Protein

Abstract

Formamidopyrimidine-DNA glycosylase (Fpg) protein plays a prominent role in the repair of oxidatively damaged DNA in Escherichia coli. The protein possesses three enzymatic activities, hydrolysis of the N-glycosidic bond (DNA glycosylase), ␤-elimination (AP lyase), and ␦-elimination; these functions act in a concerted manner to excise oxidized deoxynucleosides from duplex DNA. Schiff base formation between the enzyme and substrate has been demonstrated (Tchou, J., and Grollman, A. P. (1995) J. Biol. Chem. 270, 11671-11677); this protein-DNA complex can be trapped by reduction with sodium borohydride. By digesting the stable, covalently linked intermediate with proteases and determining the accurate mass of the products by negative electrospray ionization-mass spectrometry, we show that the N-terminal proline of Fpg protein is linked to DNA and, therefore, is identified as the nucleophile that initiates the catalytic excision of oxidized bases from DNA. This experimental approach may be applicable to the analysis of other protein-DNA complexes.Fpg 1 protein, a DNA base excision repair enzyme with Nglycosylase and AP lyase activities (1-3), efficiently removes 8-oxoguanine (8-oxoGua) and formamidopyrimidines from oxidatively damaged DNA (4 -7). We have shown previously (8) that this reaction involves an imino-enzyme-substrate (Schiff base) intermediate and that the amino group involved is located within a 72-amino acid fragment of Fpg protein containing the N terminus. The four-cysteine zinc finger motif located near the C terminus (9, 10) is utilized in binding oxidatively damaged DNA (10, 11). The 8-oxo moiety is a critical structural determinant by which Fpg protein recognizes duplex DNA substrates containing 8-oxoguanine (5).It has been proposed that Schiff bases participate in the catalytic action of AP endonucleases (12) and DNA glycosylases that possess AP lyase activity (13-16). Sodium borohydride and cyanoborohydride have been used to trap Schiff base intermediates as covalently linked DNA-protein complexes in reactions catalyzed by Fpg protein (8, 16) and by bacteriophage T4 endonuclease V, Micrococcus luteus UV endonuclease and Escherichia coli endonuclease III (15, 16). A mechanism involving nucleophilic attack on C1Ј of the modified deoxynucleoside targeted for excision (8,(15)(16)(17) has been proposed to explain the catalytic action of these enzymes. Nucleophilic attack, facilitated by protonation of the base, effects cleavage of the N-glycosidic bond. The AP lyase activity of Fpg protein is a concerted reaction involving ␤-and ␦-elimination reactions (2, 18, 19), producing a gap in one strand of the duplex demarcated by 3Ј-and 5Ј-phosphate termini (2, 18).Several nucleophiles capable of forming Schiff base intermediates (20) are located within the 72-residue N-terminal fragment of Fpg protein. Basic nitrogen functional groups are found in the N-terminal proline, the free amino group of Lys-56, and eight arginine residues. Following enzymatic digestion of the covalently linked complex, we used elect...

DNA Lesion Recognition by the Bacterial Repair Enzyme MutM

“…The glycosylase reaction proceeds via nucleophilic substitution at C1Ј by the ␣-amino group of Pro-2 (33,34). The ␣-nitrogen atom of Pro-2 lies close to C1Ј but is not well aligned for an in-line S N 2-type displacement of oxoG.…”

The crystal structure of human endonuclease VIII-like 1 (NEIL1) reveals a zincless finger motif required for glycosylase activity

“…Four selenium sites were located by using CNS (44), including that coming from the substituted N-terminal formylmethionine (fMet). Because the Fpg͞Nei family members require processing of the N-terminal fMet to use the second proline residue for enzyme catalysis (13)(14)(15)45), we expected to find only three selenium sites. A glycosylase assay revealed that SeMet-NEIL1C⌬56 exhibited a reduced activity on a 5,6-dihydrouracilcontaining substrate (data not shown), suggesting partial or incomplete processing of N-terminal fMet in the SeMet enzyme.…”

“…6 and 12). Catalysis by both E. coli Nei (EcoNei) and Fpg (EcoFpg) is by means of the N-terminal proline, which forms a Schiff base with the oxidized lesion (13)(14)(15). Both EcoNei and EcoFpg are trifunctional enzymes containing glycosylase, ␤,␦ lyase, and 5Ј phosphodiesterase activities (16)(17)(18)(19).…”

Identification and Characterization of a Novel Human DNA Glycosylase for Repair of Cytosine-derived Lesions

“…Consequently, DNA strand breaks containing 3Ј-phospho-␣,␤-unsaturated aldehyde groups are generated (7). However, E. coli possesses two other oxidized base-specific DNA glycosylases, namely MutM and Nei, which form a distinct class because they utilize N-terminal Pro as the active site to carry out ␤␦-elimination and generate 3Ј-phosphate at the DNA cleavage site (8,9). We recently identified two orthologs of MutM/Nei in the human genome database and named them Nei homologs NEH1 or NEH2 (10).…”