Mutation of a Unique Aspartate Residue Abolishes the Catalytic Activity but Not Substrate Binding of the Mouse N-Methylpurine-DNA Glycosylase (MPG)

Rabindra Roy,Tapan Biswas,J.Ching Lee,Sankar Mitra

doi:10.1074/jbc.275.6.4278

Abstract

N-Methylpurine-DNA glycosylase (MPG) initiates base excision repair in DNA by removing a variety of alkylated purine adducts. Although Asp was identified as the active site residue in various DNA glycosylases based on the crystal structure, Glu-125 in human MPG (Glu-145 in mouse MPG) was recently proposed to be the catalytic residue. Mutational analysis for all Asp residues in a truncated, fully active MPG protein showed that only Asp-152 (Asp-132 in the human protein), which is located near the active site, is essential for catalytic activity. However, the substrate binding was not affected in the inactive Glu-152, Asn-152, and Ala-152 mutants. Furthermore, mutation of Asp-152 did not significantly affect the intrinsic tryptophan fluorescence of the enzyme and the far UV CD spectra, although a small change in the near UV CD spectra of the mutants suggests localized conformational change in the aromatic residues. We propose that in addition to Glu-145 in mouse MPG, which functions as the activator of a water molecule for nucleophilic attack, Asp-152 plays an essential role either by donating a proton to the substrate base and, thus, facilitating its release or by stabilizing the steric configuration of the active site pocket.

Highlights

Unrepaired, damaged bases could be mutagenic [2] and cause cytotoxicity by blocking DNA replication [3]
Some activity was observed in crude E. coli extract containing the Asn-152 mutant protein (Table II) using a less sensitive assay, we conclude that the purified enzyme lacks detectable methylpurine-DNA glycosylase (MPG) activity
Aspartic acid was shown to be the catalytic residue in the cases of most DNA glycosylases and DNA glycosylase/lyases, including E. coli alkA, the MPG ortholog, uracil-DNA glycosylase, MutY, E. coli endonuclease III, hNTH1, Fpg, human 8-oxoguanine-DNA glycosylase-1, yeast 8-oxoguanine-DNA glycosylase-1, and yeast 8-oxoguanine-DNA glycosylase-2 (16 – 23)

Summary

EXPERIMENTAL PROCEDURES

Construction of Site-specific Mutants of MPG—The mutants were generated using Stratagene’s Chameleon double-stranded site-directed mutagenesis kit. Overexpression and Purification of MPG Proteins—The wild type (N⌬100C⌬18) and mutant (Asn-152, Ala-152, and Glu-152) MPG proteins were expressed as a glutathione S-transferase fusion protein in E. coli MV1932 (alkA-tagϪ) and purified as described previously. N-Methylpurine DNA-Glycosylase Assay—The wild type (5 ng) and three mutant enzymes (1 ␮g) were individually incubated with 32Plabeled 1,N6-ethenoadenine (⑀A)-containing duplex oligonucleotide (ϳ10,000 cpm; the sequence is shown in Table IB) substrate for various times when necessary at 37 °C in an assay buffer (25 mM HEPES-KOH, pH 7.9, 0.5 mM EDTA, 0.5 mM dithiothreitol, 150 mM NaCl, and 10% glycerol) in a total volume of 20 ␮l. The binding reaction (50 ␮l) was performed with 4 fmol of 5Ј-32P-labeled oligonucleotide (ϳ15,000 cpm; the sequence is the same as of the substrate oligonucleotide), 5 ng of plasmid DNA, and varying amounts of the wild type N⌬100C⌬18 MPG or mutant proteins (Asn-152, Ala-152, and Glu152) in the assay buffer. Lane M, protein molecular mass markers; lane 1, MPG wild type (N⌬100C⌬18), 2 ␮g; lane 2, mutant MPG, Asn-152, 2 ␮g; lane 3, mutant MPG, Ala-152, 2 ␮g; lane 4, mutant MPG, Glu-152, 2 ␮g

Specific activity

RESULTS

TABLE III

DISCUSSION