Cloning and expression in Escherichia coli of a human cDNA encoding the DNA repair protein N-methylpurine-DNA glycosylase

D Chakravarti,G.C Ibeanu,K Tano,S Mitra

doi:10.1016/s0021-9258(18)98467-x

Abstract

A 871-base pair cDNA encoding the human N-methylpurine-DNA glycosylase (MPG) was cloned from a HeLa S3 cDNA expression library in a pUC vector by phenotypic screening of MPG-negative (tag- alkA-) Escherichia coli cells exposed to methylmethane sulfonate. The active MPG is expressed as a 31-kDa fusion protein. The human cDNA-encoded MPG releases 3-methyladenine, 7-methylguanine, and 3-methylguanine from DNA and thus has a substrate range similar to that of the indigenous enzyme and the E. coli AlkA protein. The cDNA hybridizes with distinct restriction fragments of mammalian DNAs but not with E. coli or yeast DNA. A search in the GenBank data bank failed to show any other cloned DNA with a similar sequence. Although the human protein has 62% sequence homology with the corresponding rat enzyme, only a few amino acid residues are conserved between the human protein and the E. coli and yeast MPGs. However, a conserved glutamine residue in all MPGs that release 3-alkyladenine and an arginine residue in eukaryotic MPGs and E. coli AlkA that act additionally on N-alkylguanines suggest that these residues are involved in recognition of adenine and guanine adducts in DNA, respectively. Although the 1.1-kilobase mRNAs of MPG from human and rodents are similar in size, liver and cultured cells of rat have much lower levels of MPG mRNA than do human and mouse cells. A hamster cell line variant isolated as being resistant to methylmethane sulfonate does not have a higher level of MPG mRNA than the parent cell line.

Highlights

From the $University of Tennessee-Oak RidgeGraduate School of Biomedical Sciences,and the 7lBiology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831
The human protein has62% sequence homology withthecorresponding rat enzyme, only a few amino acid residues are conserved between the human protein and the E. coli and yeast MPGs
N-MethylpurineDNA Glycosylase Assay-MPG activity in E. coli extracts was assayed by measuring the release of [3H]N-methylpurines from [3H]methyl-labeled DNA substrate, which was prepared by treating sonicated calf thymus DNA with [3H]dimethylsulfate, as described earlier (Washington et al, 1988).Crude extracts of plasmidcontaining E. coli wereprepared by sonicating the bacteria suspended in a buffer containing 70 mM Tris-C1 (pH 8.01, 1 mM EDTA, 1mM dithiothreitol, and 5%glycerol and followed by centrifugation (10,000 X g, 5min) to removecell debris

Summary

EXPERIMENTAL PROCEDURES

Bacterial strains and plasmids-The Tag- alM- E. coli mutant, MV 1932 (Volkert, 1986), was used for screening a cDNA expression. E. coli DH5a (Bethesda Research Laboratories) were used for construction of the cDNA library (Tano et al, 1990). Alk A lSS4 modified minimum essential medium containing 10%fetal calf serum. CHO cells weregrown in F-12 medium containing 5% fetal calf serum. Subcloning and Sequencing-The MPG cDNA isolated from the plasmid pPG23 insert was ligated to pUC18 inbothorientation. The cDNA in pPG23 was sequenced without further subcloningby the Sanger "dideoxy" chain terminationmethod for duplex DNA sequencing S. Biochemical Corp.) The terminal regions of the cDNA were serum albumin as standard (Smiteht al., 1985).The methylated bases sequenced with universal primers corresponding to vector sequences. N-MethylpurineDNA Glycosylase Assay-MPG activity in E. coli extracts was assayed by measuring the release of [3H]N-methylpurines from [3H]methyl-labeled DNA substrate, which was prepared

RESULTS

65 LeuGlu Phe Phe A s pG l nP r oA l aV a l

Pu I L A I

Findings

DISCUSSION