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

Abstract

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.