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A Site-Specific Endonuclease from the Thermophilic Strain Bacillus Species F4

T. Yu. Zharmukhamedova1, N. G. Privezencova1, O. V. Shishova1, S. A. Shiryaev2, L. A. Zheleznaya3, and N. I. Matvienko2*

1School of Biology, Lomonosov Moscow State University Branch, Pushchino, Moscow Region, 142292 Russia

2Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, 142292 Russia; fax: (095) 924-0493; E-mail: nikmatv@vega.protres.ru

3Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142292 Russia; fax: (095) 135-6219

* To whom correspondence should be addressed.

Received June 29, 1998; Revision received January 10, 1999
A strain producing the site-specific endonuclease BspF4I was found during screening of thermophilic bacteria isolated from soil. The restriction endonuclease, free from contaminant nonspecific nucleases, was purified using three steps of column chromatography--on hydroxyapatite, blue agarose, and DEAE-Trisacryl. The enzyme is stable on storage and exhibits maximal activity at 48-56°C in the presence of albumin in buffer containing 10 mM Tris-HCl (pH 7.5) and 10 mM MgCl2. BspF4I recognizes the sequence 5´-GGNCC-3´ on DNA and is an isomer and not an isoschizomer of the endonuclease Sau96I. Unlike the prototype, BspF4I does not cleave the site in a defined way. A strand with purine in the center of the sequence is cleaved after the first G, as in the case of the prototype, while the strand with pyrimidine is cleaved either before or after the first G.
KEY WORDS: site-specific endonuclease, isoschizomer, thermophilic bacteria