Polynucleotide : adenosine glycosidase is the sole activity of ribosome-inactivating proteins on DNA

Polynucleotide: adenosine glycosidases (PNAG) are a class of plant and bact erial enzymes commonly known as ribosome-inactivating proteins (RIP), They are presently classified as rRNA N-glycosidases in the enzyme nomenclature [EC 3.2.2.22], Several activities on nucleic acids, other than depurination , have been attributed to PNAG: in particular modifications induced in circ ular plasmids, including linearisation and topological changes, and cleavag e of guanidinic residues, Here we describe a chromatographic procedure to o btain nuclease-free PNAG; by dye-chromatography onto Procion Rad derivatize d Sepharose(R). Highly purified enzymes depurinate extensively pBR322 circu lar, supercoiled DNA at neutral pH and exhibit neither DNase nor DNA glycol yase activities, do not cause topological changes, and adenine is the only base released from DNA and rRNA, even at very high enzyme concentrations. A scanning force microscopy (SFM) study of pBR322 treated with saporin-S6 co nfirmed that (i) this PNAG binds extensively to the plasmid, (ii) the distr ibution of the bound saporin-Sg molecules along the DNA chain is markedly v ariable, (iii) plasmids already digested with saporin-S6 do not appear frag mented or topologically modified. The observations here described demonstra te that polynucleotide:adenosine glycosidase is the sole enzymatic activity of the four ribosome-inactivating proteins gelonin, momordin I, pokeweed a ntiviral protein from seeds and saporin-S6, These proteins belong to differ ent families, suggesting that the findings here described may be generalize d to all PNAG.