Purification of a site-specific endonuclease, I-Sce II, encoded by intron 4 alpha of the mitochondrial coxI gene of Saccharomyces cerevisiae
| Autor: | C M, Wernette, R, Saldahna, P S, Perlman, R A, Butow |
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| Rok vydání: | 1990 |
| Předmět: |
Endodeoxyribonucleases
Saccharomyces cerevisiae Proteins Base Sequence Genes Fungal Molecular Sequence Data Saccharomyces cerevisiae Chromatography Ion Exchange DNA Mitochondrial Chromatography Affinity Introns Mitochondria Substrate Specificity Electron Transport Complex IV Molecular Weight Kinetics Oligodeoxyribonucleotides Chromatography Gel Deoxyribonucleases Type II Site-Specific |
| Zdroj: | The Journal of biological chemistry. 265(31) |
| ISSN: | 0021-9258 |
| Popis: | We have purified to near homogeneity a site-specific, double-stranded DNA endonuclease (I-Sce II) encoded by intron 4 alpha (aI4 alpha) of the yeast mitochondrial coxI gene. Our purification starts with a high salt extract of mitochondria isolated from a yeast strain that overproduces the enzyme because of a block in splicing of aI4 alpha. The final step of purification is an affinity column consisting of covalently bound double-stranded DNA multimers of a synthetic sequence, 5'-TTGGTCATCCAGAAGTAT-3', which contains the I-Sce II cleavage/recognition site. Typical yields of enzyme are 3-5% with a specific activity of approximately 500,000 units/mg, where 1 unit of activity cleaves 50 ng of DNA substrate/h at 30 degrees C. I-Sce II has a monomer molecular mass of 31 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Active enzyme purifies as a 55-kDa species, which we presume to be a homodimer. I-Sce II monomer comigrates with an in vivo synthesized mitochondrial translation product made in the strain that overproduces the enzyme. We conclude that I-Sce II is derived by proteolytic processing of a precursor polypeptide, p62, encoded by an in-frame fusion of coxI exons 1-4 with the downstream aI4 alpha reading frame. I-Sce II is most active at pH 7.5 and at 20-30 degrees C. Endonuclease activity is sensitive to salt and is dependent upon Mg2+ or Mn2+, but is unaffected by inclusion of ATP or GTP. I-Sce II is the first intron-encoded protein to be purified and characterized from yeast mitochondria. |
| Databáze: | OpenAIRE |
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