Two new site-specific endonucleases from Staphylococcus species strain D5

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) downward arrow-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 T-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 degrees 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).