Two New Site-Specific Endonucleases from Staphylococcus Species Strain D5

S. A. Shiryaev¹, E. N. Zheleznyakova¹, N. N. Matvienko³, L. A. Zheleznaya², and N. I. Matvienko^1*

¹Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, 142292 Russia; fax: (095) 924-0493; E-mail: nikmatv@vega.protres.ru

²Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region, 142292 Russia; fax: (095) 135-6219

³Branch of the Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino, Moscow Region, 142292 Russia

^* To whom correspondence should be addressed.

Received May 27, 1999

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Staphylococcus species strain D5 containing two site-specific endonucleases, SspD5 I and SspD5 II, was found during screening of a bacterial strain collection from soil. These endonucleases were purified to functional homogeneity by sequential chromatography. Site-specific endonuclease SspD5 I recognizes sequence 5´-GGTGA(8N/8N)^v-3´ on DNA. Unlike Hph I, it cleaves DNA at a distance of 8 nucleotides from the recognized sequence on both chains producing blunt-end DNA fragments, while endonuclease Hph I cleaves DNA forming mononucleotide 3´-OH protruding ends. Thus, endonuclease SspD5 I is a new type II site-specific endonuclease and a neoschizomer of endonuclease Hph I. The advantage of this new endonuclease is that the blunt-end DNA products of this enzyme can be inserted without additional treatment into vector DNAs cleaved with endonucleases yielding DNA blunt-ends. Endonuclease SspD5 II recognizes site 5´-ATGCA^vT-3´ and thus is an isoschizomer of endonuclease Nsi I. The molecular mass of SspD5 I is about 35 kD and that of SspD5 II is 40 kD. The enzymes exhibit maximal activity at 37°C. The optimal buffer for the reaction is HRB (10 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 100 mM NaCl, and 1 mM dithiothreitol).

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

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