A sequence specific endonuclease from Micrococcus radiodurans |
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Authors: | Altaf A. Wani,Ralph E. Stephens,Steven M. D Ambrosio,Ronald W. Hart |
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Affiliation: | 1. Department of Radiology, School of Medicine, The Ohio State University, Columbus, OH 43210 U.S.A.;2. Department of Pharmacology, School of Medicine, The Ohio State University, Columbus, OH 43210 U.S.A. |
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Abstract: | A new sequence specific endonuclease, MraI has been purified from Micrococcus radiodurans. This enzyme cleaves bacteriophage λ DNA at three sites, adenovirus type 2 DNA at more than 12 sites and has a unique site on ΦX174 DNA. It has no sites on SV40, PM2 and pBR322 DNA. The three sites on phage λ DNA are different from those cleaved by SmaI, XmaI and XorII. The sites of cleavage are located at 0.424, 0.447 and 0.834 fractional lengths on the physical map of λ DNA. MraI is shown to be an isoschizomer of SacII and SstII recognizing the palindromic nucleotide sequence ′5-CCGC↓GG-3′. The enzyme shows an absolute requirement of Mg2+, but is active in the absence of added 2-mercaptoethanol. The enzyme shows activity at a broad range of temperature and pH with an optimum at 45°C and pH 7.0. MraI represents the first restriction enzyme from a bacterium whose DNA lacks modified methylated bases. |
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Keywords: | Endonuclease DNAase DNA mapping (M. radiodurans) PMSF phenylmethanesulfonyl fluoride |
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