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 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.
Ohtsubo
T
T
Department of Biochemistry, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.
Matsuda
O
O
Iba
K
K
Terashima
I
I
Sekiguchi
M
M
Nakabeppu
Y
Y
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins; D001483:Base Sequence; D004260:DNA Repair; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D043382:DNA-Formamidopyrimidine Glycosylase; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005091:Exons; D006147:Guanine / Q000031:analogs & derivatives / Q000032:analysis; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000235:genetics*; D014176:Protein Biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D016335:Zinc Fingers
D029681:Arabidopsis Proteins; D018744:DNA, Plant; D004268:DNA-Binding Proteins; D043382:DNA-Formamidopyrimidine Glycosylase; D029968:Escherichia coli Proteins; D006147:Guanine; D009699:N-Glycosyl Hydrolases; C024829:8-hydroxyguanine; C468610:DNA-formamidopyrimidine glycosylase, E coli; C468606:MMH-1 protein, Arabidopsis