A restriction enzyme from Hemophilus influenzae. II. Base sequence of the recognition site

Hemophilus influenzae strain Rd contains an enzyme, endonuolease R, which specifically degrades foreign DNA. With phage T7 DNA as substrate the endonuclease introduces a limited number (about 40) double-strand breaks (5′-phosphoryl, 3′-hydroxyl). The limit product has an average length of about 1000 nucleotide pairs and contains no single-strand breaks. We have explored the nucleotide sequences at the 5′-ends of the limit product by labeling the 5′- phosphoryl groups (using polynucleotide kinase) and characterizing the labeled fragments released by various nucleases. Two classes of 5′-terminal sequences were obtained: pApApCpNp ... (60%) and pGpApCpNp ... (40%), where N indicates that the base in the 4th position is not unique. The dinucleoside monophosphates at the 3′-ends were isolated after micrococcal nuclease digestion of the limit product and identified as TpT(60%) and TpC (40%). We conclude that endonuclease R of H. influenzae recognizes the following specific nucleotide sequence: 5′ ... pGpTpPy |pPupApCp ... 3′ 3′ ... pCpApPup |PypTpGp ... 5′ The implications of the twofold rotational symmetry of this sequence are discussed.