Although the biological function of DNA glycosylases is to protect the genome by removal of potentially cytotoxic or mutagenic bases, this investigation describes the existence of natural DNA glycosylases with activity on undamaged, nonmispaired bases. Gelonin, pokeweed antiviral protein, and ricin, previously described as ribosome-inactivating proteins, are shown to damage single-stranded DNA by removal of a protein-specific set of adenines and cleavage at the resulting abasic sites. Using an oligonucleotide as the substrate reveals that the reaction proceeds via the enzyme-DNA imino intermediate characteristic of DNA glycosylase/AP lyases. The adenine glycosylase activity on single-stranded DNA reported here challenges the concept that a normal base has to be in a mismatch to be specifically removed. By contrast to other glycosylases, these enzymes are expected to damage DNA rather than participate in repair processes. The significance of this DNase activity to the biological function of these plant proteins and to their toxicity to animal cells remains to be determined.Ricin and other related plant proteins such as abrin, gelonin, pokeweed antiviral protein (PAP), 1 and trichosanthin have been classified as ribosome-inactivating proteins (RIPs) in reference to the fact that they inhibit protein synthesis by inactivation of the ribosomes (1). The molecular mechanism of inactivation, elucidated in a cell-free system by Endo and colleagues (2), is the removal of a specific adenine of the 28 S rRNA. This damage, which has been shown to occur in RIPtreated cells, has been generally accepted as responsible for cytotoxicity (1). However, in Plasmodium falciparum-infected erythrocytes, intoxication by gelonin was reported to be associated with the elimination of the parasite 6-kb extrachromosomal (mitochondrial) DNA (3). Moreover, some reports of anti-viral or anti-tumor activities of RIPs also suggest the possibility of additional cytotoxic pathways (4 -6).The weak activity of RIPs in cleaving and linearizing supercoiled, double-stranded DNA in vitro (7-10) has not been given serious consideration because of concerns about contaminating nucleases in the protein preparations. More recent reports have described a preference for single-stranded (ss) DNA (11,12). The polypeptide responsible for the (zinc-activated) degradation of linear ssDNA by preparations of gelonin, from both native and recombinant bacterial sources, has been identified as gelonin by zymography (12). Conflicting conclusions have been reached on the possible mechanism of DNA degradation. On the basis of the observation that the nicked and linear forms generated by the action of RIPs on supercoiled DNA were not labeled by 3 H-labeled sodium borohydride, unlike the fragment generated by the action on rRNA (10), it was suggested that RIPs do not act by a DNA glycosylase mechanism (13). Because boiling ricin-A totally destroyed the activity on 28 S rRNA but only reduced the ability to cleave DNA, the activities were described to be independent (9). Stirp...