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

Tipo de material: Texto

TextoSeries ; Journal of Molecular Biology, 51(2), p.393-409, 1970Trabajos contenidos:

Kelly Jr, T. J
Smith, H. O

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Resumen: 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 percent)and pGpApCpNp . (40 percent), 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 percent)and TpC (40 percent). 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.

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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 percent)and pGpApCpNp . (40 percent), 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 percent)and TpC (40 percent). 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.

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