Molecular cloning of AtMMH, an Arabidopsis thaliana ortholog of the Escherichia coli mutM gene, and analysis of functional domains of its product

T. Ohtsubo, O. Matsuda, K. Iba, I. Terashima, M. Sekiguchi, Y. Nakabeppu

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Abstract

We isolated and characterized cDNAs and a genomic clone encoding an Arabidopsis thaliana MutM homolog (AtMMH). AtMMH is a single-copy gene spanning 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 from leaves of A. thaliana, using polyclonal antibodies against the recombinant 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-coli MutM protein, which possesses 8-oxoG DNA glycosylase and apurinic/apyrimidinic (AP) lyase activities. Deletion of six amino acids (PELPEV), which are conserved among all known MutM homologs, from the N-terminal end of the AtMMH-1 protein abolishes its nicking but not its DNA-binding activity, indicating that these residues are essential for catalytic activity. Although the AtMMH-1 protein has a unique structure at its C-terminal end, which consists of alternating repeats of basic and acidic amino acids, this structure is dispensable for activity. However, the adjacent amino acid sequence (residues 268 to 281) is essential for repair activity.

Original languageEnglish
Pages (from-to)577-590
Number of pages14
JournalMolecular and General Genetics
Volume259
Issue number6
DOIs
Publication statusPublished - Nov 14 1998

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Molecular Cloning
Arabidopsis
Escherichia coli
Genes
Oligonucleotides
Exons
DNA Glycosylases
Acidic Amino Acids
Basic Amino Acids
Messenger RNA
Lyases
Escherichia coli Proteins
Alternative Splicing
Complex Mixtures
Recombinant Proteins
Amino Acid Sequence
Complementary DNA
Clone Cells
Genome
Amino Acids

All Science Journal Classification (ASJC) codes

  • Genetics

Cite this

Molecular cloning of AtMMH, an Arabidopsis thaliana ortholog of the Escherichia coli mutM gene, and analysis of functional domains of its product. / Ohtsubo, T.; Matsuda, O.; Iba, K.; Terashima, I.; Sekiguchi, M.; Nakabeppu, Y.

In: Molecular and General Genetics, Vol. 259, No. 6, 14.11.1998, p. 577-590.

Research output: Contribution to journalArticle

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