Gene structure and biochemical characterization of mitochondrial single-stranded DNA binding protein from Schizosaccharomyces pombe.
We studied the genomic structure and biochemical properties of Schizosaccharomyces pombe mitochondrial single-stranded DNA binding protein (mtSSB). We first determined the full-length cDNA sequence of mtSSB and clarified the exon/intron structure of the mtSSB gene (rim1), including the transcription initiation and polyadenylation sites. The cDNA of rim1 gene encoded 150 amino acids and the sequence showed homology to eukaryotic mtSSB and Escherichia coli SSB. We overexpressed mtSSB as a His-tag fusion protein in E. coli and obtained an anti-mtSSB antibody. Gel filtration analysis of S. pombe cell extracts clarified that mtSSB has a tetrameric structure. We also immunochemically detected mtSSB in S. pombe cell extract and showed that 15,000 molecules of mtSSB tetramer are present in a single S. pombe cell. Mature mtSSB lacking the presequence was overexpressed in E. coli in tetrameric soluble form. The recombinant mtSSB bound a single-stranded oligonucleotide and phiX174 virion DNA with almost identical binding activity as E. coli SSB.
Nishio
Megumi
M
Department of Biochemistry, Faculty of Science, Okayama University of Science, 1-1 Ridaicho, Okayama 700-0005, Japan.
Seki
Yuichi
Y
Ikeda
Shogo
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D040461:Gene Components; D024101:Mitochondrial Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D011485:Protein Binding; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D024101:Mitochondrial Proteins; C518884:Rim1 protein, S pombe; D029702:Schizosaccharomyces pombe Proteins