Nicking activity on pBR322 DNA of ribosome inactivating proteins from Phytolacca dioica L. leaves |
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Authors: | Aceto Serena Di Maro Antimo Conforto Barbara Siniscalco Gesualdo G Parente Augusto Delli Bovi Pasquale Gaudio Luciano |
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Institution: | Dipartimento di Genetica, Biologia Generale e Molecolare, Università degli Studi di Napoli Federico II, Via Mezzocannone 8, I-80134 Napoli, Italy. |
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Abstract: | Ribosome-inactivating proteins isolated from Phytolacca dioica L. leaves are rRNA-N-glycosidases, as well as adenine polynucleotide glycosylases. Here we report that some of them cleave supercoiled pBR322 dsDNA, generating relaxed and linear molecules. PD-L1, the glycosylated major form isolated from the winter leaves of adult P . dioica plants, produces both free 3'-OH and 5'-P termini randomly distributed along the DNA molecule, as suggested by labelling experiments with alpha- 32P]dCTP and gamma- 32 P]dATP. Moreover, when the reaction is carried out under low-salt conditions, cleavage is observed mainly at a specific site, located downstream of the ampicillin resistance gene (close to position 3200), ending with the deletion of a fragment of approximately 70 nucleotides. This cleavage pattern is similar to that obtained under the same conditions with mung bean nuclease, a single-strand endonuclease. Furthermore, pBR322 DNA treated with PD-L1 shows reduced transforming activity with E . coli HB101 competent cells in comparison to untreated control plasmid DNA. |
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