[New rare cutting restriction endonuclease SmII from Streptococcus milleri recognises 5'-ATTTAAAT-3']

New restriction endonuclease (restrictase) Smil of type II was detected in the bacterial strain Streptococcus milleri. Cellular lysate enzyme cut T7 and adenovirus-2 DNAs at site 5'-ATTT decreases AAAT-3' but not lambda DNA which does not contain this sequence. Intense aeration inhibited the growth of S. milleri. The content of restrictase in the cells was the greatest during the logarithmic growth phase. A total of 20,000 units of Smil were isolated from 4 g of cells by cellular extract fractionation with ammonium sulfate and subsequent chromatography on columns with Bio Gel A 0.5 m, heparin agarose, and phosphocellulose. Purified enzyme cut the synthetic oligonucleotide duplex in the center of the recognized site 5'-ATTT decreases AAAT-3'. Smil restrictase is a true isoschisomer of rare-cutting Swal enzyme. Smil belongs to a small group of enzymes which recognize octanucleotide sites and can be used for large-block fragmentation of DNA. Comparison of specificities of rare-cutting and other restrictases suggests that the enzymes recognizing octanucleotides can evolutionally originate from enzymes recognizing both hexanucleotides and tetranucleotides.