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 co llection from soil. These endonucleases were purified to functional homogen eity by sequential chromatography. Site-specific endonuclease SspD5 I recog nizes sequence 5'-GGTGA(8N/8N)down arrow-3' on DNA. Unlike Hph I, it cleave s DNA at a distance of 8 nucleotides from the recognized sequence on both c hains producing blunt-end DNA fragments, while endonuclease Hph I cleaves D NA forming mononucleotide 3'-OH protruding ends. Thus, endonuclease SspD5 I is a new type II site-specific endonuclease and a neoschizomer of endonucl ease Hph I. The advantage of this new endonuclease is that the blunt-end DN A products of this enzyme can be inserted without additional treatment into vector DNAs cleaved with endonucleases yielding DNA blunt-ends. Endonuclea se SspD5 II recognizes site 5'-ATGCA down arrow T-3' and thus is an isoschi zomer of endonuclease Nsi I. The molecular mass of SspD5 I is about 35 kD a nd that of SspD5 II is 40 kD. The enzymes exhibit maximal activity at 37 de grees 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).