MOLECULAR-CLONING OF ATMMH, AN ARABIDOPSIS-THALIANA ORTHOLOG OF THE ESCHERICHIA-COLI MUTM GENE, AND ANALYSIS OF FUNCTIONAL DOMAINS OF ITS PRODUCT

We isolated and characterized cDNAs and a genomic clone encoding an Ar abidopsis thaliana MutM homolog (AtMMH). AtMMH is a single-copy gene s panning about 3 kb in the nuclear genome, and comprises ten exons. The AtMMH gene encodes two types of mRNA (AtMMH-1 and AtMMH-2) formed by alternative splicing of exon 8. Western analysis of a crude extract fr om leaves of A. thaliana, using polyclonal antibodies against the reco mbinant proteins, demonstrated the presence in vivo of a single 44-kDa polypeptide that comigrates with the product of in vitro translation of the AtMMH-1 mRNA. AtMMH-1 protein prepared in vitro is able to nick double- stranded oligonucleotides containing 8-oxo-7,8-dihydroguanine (8-oxoG) and to bind such oligonucleotides, as does the Escherichia c oli MutM protein, which possesses 8-oxoG DNA glycosylase and apurinic/ apyrimidinic (AP) lyase activities. Deletion of six amino acids (PELPE V), which are conserved among all known MutM homologs, from the N-term inal end of the AtMMH-1 protein abolishes its nicking but not its DNA- binding activity, indicating that these residues are essential for cat alytic activity. Although the AtMMH-1 protein has a unique structure a t its C-terminal end, which consists of alternating repeats of basic a nd acidic amino acids, this structure is dispensable for activity. How ever, the adjacent amino acid sequence (residues 268 to 281) is essent ial for repair activity.