Three Site-Specific Endonucleases from Thermophilic Strain Bacillus Species LA are Isoschizomers of HhaI, AsuII, and HindIII

S. A. Shiryaev,¹ L. A. Zheleznaya,² and N. I. Matvienko^1,3

¹Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, 142292 Russia; fax: (7-095) 924-04-93; E-mail: protres@sovam.com

²Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142292 Russia; fax: (7-095) 135-62-19.

³To whom correspondence should be addressed.

Submitted November 28, 1996; revision submitted December 23, 1996.

Screening of thermophilic bacterial strains revealed a strain containing three site-specific endonucleases: BspLAI, BspLAII, and BspLAIII. These endonucleases were purified to functional purity by sequential chromatography. Recognition sites, DNA cleavage sites, and some properties of the endonucleases were determined. BspLAI recognizes the sequence 5´-GCG***»C-3´ on the DNA molecule and is an isoschizomer of endonuclease HhaI. BspLAII recognizes the sequence 5´-TT***»CGAA-3´ and is an isoschizomer of AsuII. BspLAIII recognizes site 5´-A***»AGCTT-3´ and is an isoschizomer of endonuclease HindIII. All the three enzymes exhibit maximal activity at 55°C. The optimal buffer is MRB, pH 7.4. They retain activity on storage for 3 weeks at room temperature and thus are highly stable.

KEY WORDS: site-specific endonuclease, isoschizomer, restriction, sequencing.

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