A New Class of DNA Glycosylase/Apurinic/Apyrimidinic Lyases That Act on Specific Adenines in Single-stranded DNA (1998)

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. 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), 1The abbreviations used are: PAP, pokeweed antiviral protein; RIP, ribosome inactivating protein; AP, apurinic/apyrimidinic; ss, single-stranded; ODN, oligodeoxyribonucleotide; UDG, uracil-DNA glycosylase; bp, base pair; PAGE, polyacrylamide gel electrophoresis. 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 (1Barbieri L. Battelli M.G. Stirpe F. Biochim. Biophys. Acta. 1993; 1154: 237-282Crossref PubMed Scopus (846) Google Scholar). The molecular mechanism of inactivation, elucidated in a cell-free system by Endo and colleagues (2Endo Y. Mitsui K. Motizuki M. Tsurugi K. J. Biol. Chem. 1987; 262: 5908-5912Abstract Full Text PDF PubMed Google Scholar), is the removal of a specific adenine of the 28 S rRNA. This damage, which has been shown to occur in RIP-treated cells, has been generally accepted as responsible for cytotoxicity (1Barbieri L. Battelli M.G. Stirpe F. Biochim. Biophys. Acta. 1993; 1154: 237-282Crossref PubMed Scopus (846) Google Scholar). However, inPlasmodium falciparum-infected erythrocytes, intoxication by gelonin was reported to be associated with the elimination of the parasite 6-kb extrachromosomal (mitochondrial) DNA (3Nicolas E. Goodyer I.D. Taraschi T.F. Biochem. J. 1997; 327: 413-417Crossref PubMed Scopus (39) Google Scholar). Moreover, some reports of anti-viral or anti-tumor activities of RIPs also suggest the possibility of additional cytotoxic pathways (4Teltow G.J. Irvin J.D. Aron G.M. Antimicrob. Agents Chemother. 1983; 23: 390-396Crossref PubMed Scopus (39) Google Scholar, 5McGrath M.S. Hwang K.M. Caldwell S.E. Gaston I. Luk K.-C. Wu P. Ng V.L. Crowe S. Daniels J. Marsh J. Deinhart T. Lekas P.V. Vennari J.C. Yeung H.-W. Lifson J.D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2844-2848Crossref PubMed Scopus (320) Google Scholar, 6Tumer N.E. Hwang D.-J. Bonness M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3866-3871Crossref PubMed Scopus (130) Google Scholar). The weak activity of RIPs in cleaving and linearizing supercoiled, double-stranded DNA in vitro (7Li M.-X Yeung H.-W. Pan L.-P. Chan S.I. Nucleic Acids Res. 1991; 19: 6309-6312Crossref PubMed Scopus (106) Google Scholar, 8Go T.T.M. Yeung H.W. Fong W.P. Life Sci. 1992; 51: 1347-1353Crossref PubMed Scopus (31) Google Scholar, 9Ling J. Liu W.-Y. Wang T.P. FEBS Lett. 1994; 345: 143-146Crossref PubMed Scopus (69) Google Scholar, 10Ling J. Liu W.-Y. Wang T.P. Bioorg. Chem. 1994; 22: 395-404Crossref Scopus (9) Google Scholar) 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 (11Roncuzzi L. Gasperi-Campani A. FEBS Lett. 1996; 392: 16-20Crossref PubMed Scopus (73) Google Scholar, 12Nicolas E. Beggs J.M. Haltiwanger B.M. Taraschi T.F. FEBS Lett. 1997; 406: 162-164Crossref PubMed Scopus (40) Google Scholar). 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 (12Nicolas E. Beggs J.M. Haltiwanger B.M. Taraschi T.F. FEBS Lett. 1997; 406: 162-164Crossref PubMed Scopus (40) Google Scholar). 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 3H-labeled sodium borohydride, unlike the fragment generated by the action on rRNA (10Ling J. Liu W.-Y. Wang T.P. Bioorg. Chem. 1994; 22: 395-404Crossref Scopus (9) Google Scholar), it was suggested that RIPs do not act by a DNA glycosylase mechanism (13Ling J. Li X.-D. Wu X.-H. Liu W.-Y. Biol. Chem. Hoppe-Seyler. 1995; 376: 637-641Crossref PubMed Scopus (19) Google Scholar). 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 (9Ling J. Liu W.-Y. Wang T.P. FEBS Lett. 1994; 345: 143-146Crossref PubMed Scopus (69) Google Scholar). Stirpe and co-workers (14Barbieri L. Valbonesi P. Bonora E. Gorini P. Bolognesi A. Stirpe F. Nucleic Acids Res. 1997; 25: 518-522Crossref PubMed Scopus (270) Google Scholar), however, suggested that DNA breakage could spontaneously occur because of the weakening produced by the removal of adenines. In this investigation, our primary aim was to study the mechanism of DNA degradation of ssDNA by different RIPs, to reconcile, if possible, the recent descriptions of RIPs as polynucleotide:adenosine nucleosidases (15Barbieri L. Gorini P. Valbonesi P. Castiglioni P. Stirpe F. Nature. 1994; 372: 624Crossref PubMed Scopus (108) Google Scholar), polynucleotide:adenosine glycosidases (16Barbieri L. Valbonesi P. Gorini P. Pession A. Stirpe F. Biochem. J. 1996; 319: 507-513Crossref PubMed Scopus (70) Google Scholar, 17Olivieri F. Prasad V. Valbonesi P. Srivastava S. Ghosal-Chowdhury P. Barbieri L. Bolognesi A. Stirpe F. FEBS Lett. 1996; 396: 132-134Crossref PubMed Scopus (54) Google Scholar), and endonucleases (7Li M.-X Yeung H.-W. Pan L.-P. Chan S.I. Nucleic Acids Res. 1991; 19: 6309-6312Crossref PubMed Scopus (106) Google Scholar, 8Go T.T.M. Yeung H.W. Fong W.P. Life Sci. 1992; 51: 1347-1353Crossref PubMed Scopus (31) Google Scholar, 9Ling J. Liu W.-Y. Wang T.P. FEBS Lett. 1994; 345: 143-146Crossref PubMed Scopus (69) Google Scholar, 11Roncuzzi L. Gasperi-Campani A. FEBS Lett. 1996; 392: 16-20Crossref PubMed Scopus (73) Google Scholar, 12Nicolas E. Beggs J.M. Haltiwanger B.M. Taraschi T.F. FEBS Lett. 1997; 406: 162-164Crossref PubMed Scopus (40) Google Scholar). The results reveal a new class of DNA glycosylase/AP lyases that act on specific adenines in ssDNA. Gelonin and ricin were purchased from Sigma. PAP was purchased from Worthington Biochemical Corporation (Freehold, NJ). The proteins were dialyzed against 10 mm HEPES, pH 7.5, prior to use. Protein concentration was determined by reaction with bicinchonic acid (Pierce) using albumin as a standard. The pUC18 DNA plasmid and the 100-bp ladder were obtained from Life Technologies, Inc. The oligodeoxyribonucleotides 28GR-A25 (5′-GTTGGGTCTCGCCTGGGTTTTCCCAGTC-3′), 28P-A14 (5′-TGGCGTCTGGGGGATGTGCTGCTCGGCG-3′), 28GR-U25, and 28P-U14 (with uracil in place of adenine) were purchased from Biosource International (Camarillo, CA). [α-32P]dATP, [γ-32P]ATP, and SequenaseTM T7 polymerase were from Amersham Pharmacia Biotech; T4 polynucleotide kinase was from Promega (Madison, WI); HindIII, Asp700, and G-25 Quickspin columns were from Boehringer Mannheim; uracil-DNA glycosylase (UDG) was from New England Biolabs, Inc. (Beverly, MA); TrevigelTM500 was from Trevigen (Gaithersburg, MD); and SDS-PAGE precast gradients gels (4–20%) were from Bio-Rad. Linear pUC18 DNA was prepared by incubation with HindIII, phenol/chloroform extraction, and ethanol precipitation. The concentration was determined by absorption spectroscopy. The HindIII-Asp700 restriction fragments generated from pUC18 were labeled with [α-32P]dATP and Sequenase™ T7 polymerase. The 791-bp fragment labeled at the 3′ end of the HindIII site was isolated on a preparative, nondenaturing, 4% polyacrylamide gel. In some instances, 7 mol % of the radioactive fragment was added to unlabeled, linear pUC18 DNA. ssDNA was prepared by heat denaturation as described in Ref. 12Nicolas E. Beggs J.M. Haltiwanger B.M. Taraschi T.F. FEBS Lett. 1997; 406: 162-164Crossref PubMed Scopus (40) Google Scholar. The ODNs were 5′ end-labeled with T4 polynucleotide kinase and [γ-32P]ATP. The reaction mixture was loaded onto a G-25 Quickspin column equilibrated in 10 mm HEPES, pH 7.0, to remove the unincorporated label. In the assays with ODNs, activity was assayed by the inclusion of 1 mol %32P-ODN with the unlabeled ODN. The amounts of protein and DNA are indicated in the figure legends. Reactions were carried out in 10 mm HEPES, pH 7.5, 0.1 mm ZnSO4, and the products were resolved by gel electrophoresis in 16 mmHEPES-KOH, 16 mm sodium acetate, 0.8 mm EDTA (18Povirk L.F. Houlgrave C.W. Biochemistry. 1988; 27: 380-385Crossref Scopus (122) Google Scholar). For the inhibition study with NaBH4, a stock solution (1 m) was freshly prepared immediately prior to use. Assays were performed in the presence of 10 mm NaBH4or 10 mm NaCl. To assay for DNA glycosylase activity in the of a with mm was performed electrophoresis. the radioactive fragment was in the reaction assays were by of for onto a gel for electrophoresis in mm mm mm and To the of the DNA cleavage the products of the PubMed Scopus Google Scholar) on the radioactive fragment were as 1 of gelonin or PAP or of ricin, in 10 mm HEPES, pH 0.1 was at for 1 with the oligonucleotide in a reaction of 10 at of To assay for DNA glycosylase activity in the of a with 10 mm for at was Assays were by of for onto a gel for electrophoresis in and The assay was from Ref. S. Nucleic Acids Res. 1997; 25: PubMed Scopus Google Scholar. The were added a for at of the proteins in the reaction used but with 10 or at the were with as and to SDS-PAGE on a polyacrylamide gel of sodium pH 4% and The gel was and by and were using a and with RIPs have been to their are proteins with a molecular of about are proteins of of by a is to the class (1Barbieri L. Battelli M.G. Stirpe F. Biochim. Biophys. Acta. 1993; 1154: 237-282Crossref PubMed Scopus (846) Google Scholar). In this RIPs were Gelonin and PAP to class ricin to class have previously reported that ssDNA was the substrate for the activity of gelonin and that this activity was by (12Nicolas E. Beggs J.M. Haltiwanger B.M. Taraschi T.F. FEBS Lett. 1997; 406: 162-164Crossref PubMed Scopus (40) Google Scholar). 1 that PAP and ricin gelonin ssDNA in the presence of On a the activity was gelonin PAP on gels has been used to the activity of RIPs on supercoiled (7Li M.-X Yeung H.-W. Pan L.-P. Chan S.I. Nucleic Acids Res. 1991; 19: 6309-6312Crossref PubMed Scopus (106) Google Scholar, 8Go T.T.M. Yeung H.W. Fong W.P. Life Sci. 1992; 51: 1347-1353Crossref PubMed Scopus (31) Google Scholar, 9Ling J. Liu W.-Y. Wang T.P. FEBS Lett. 1994; 345: 143-146Crossref PubMed Scopus (69) Google Scholar, J. Li X.-D. Wu X.-H. Liu W.-Y. Biol. Chem. Hoppe-Seyler. 1995; 376: 637-641Crossref PubMed Scopus (19) Google Scholar) or linear (12Nicolas E. Beggs J.M. Haltiwanger B.M. Taraschi T.F. FEBS Lett. 1997; 406: 162-164Crossref PubMed Scopus (40) Google Scholar) ssDNA. The of nicked and linear forms or was of aim was to the removal of adenines by RIPs described by Barbieri (14Barbieri L. Valbonesi P. Bonora E. Gorini P. Bolognesi A. Stirpe F. Nucleic Acids Res. 1997; 25: 518-522Crossref PubMed Scopus (270) Google Scholar) is the in the degradation of ssDNA. The presence of abasic is by their to with Res. PubMed Scopus Google Scholar). 1 the of RIPs on ssDNA of reaction with 1 A. The of a only with was of the existence of abasic but 1 that the inclusion of 10 mm sodium the incubation of RIPs with DNA the degradation in 1 A. The presence of 10 mm not that the inhibition was not because of with RIPs and DNA. of the degradation of ssDNA not The inhibition of cleavage by 1 suggested that the DNA breakage by the RIPs in 1 was because of a reaction at the abasic by an associated activity J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). were in these as they were reported to be for the assay of enzymes as and for electrophoresis of DNA abasic because they DNA breakage (18Povirk L.F. Houlgrave C.W. Biochemistry. 1988; 27: 380-385Crossref Scopus (122) Google Scholar, T. P. 1995; PubMed Scopus Google Scholar). The results in 1 indicated a of to single-stranded the if was To the by the DNA glycosylase activity of the RIPs, the of the products be with of the products obtained by the PubMed Scopus Google Scholar). To this a 3′ end-labeled radioactive fragment of pUC18 DNA was in the reaction The reaction were that the was performed on abasic but not in not cleave these not In which a of the of the it was that the by to at protein a specific set of Gelonin and ricin cleavage adenines and PAP, in to adenines and also adenines and The of adenines of the and the of in 1 which were of a of degradation. incubation of DNA with RIPs, also the substrate 1 M.-X Yeung H.-W. Pan L.-P. Chan S.I. Nucleic Acids Res. 1991; 19: 6309-6312Crossref PubMed Scopus (106) Google Scholar, 8Go T.T.M. Yeung H.W. Fong W.P. Life Sci. 1992; 51: 1347-1353Crossref PubMed Scopus (31) Google Scholar, 9Ling J. Liu W.-Y. Wang T.P. FEBS Lett. 1994; 345: 143-146Crossref PubMed Scopus (69) Google Scholar, 11Roncuzzi L. Gasperi-Campani A. FEBS Lett. 1996; 392: 16-20Crossref PubMed Scopus (73) Google Scholar, 12Nicolas E. Beggs J.M. Haltiwanger B.M. Taraschi T.F. FEBS Lett. 1997; 406: 162-164Crossref PubMed Scopus (40) Google Scholar, J. Li X.-D. Wu X.-H. Liu W.-Y. Biol. Chem. Hoppe-Seyler. 1995; 376: 637-641Crossref PubMed Scopus (19) Google Scholar). The products generated these the as the products generated by This indicated that the cleavage at the 3′ of the abasic sites. lyases cleave the 3′ of the abasic site via a reaction J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). The results in suggested that the RIPs were DNA glycosylases that an associated weak DNA glycosylases and DNA glycosylase/AP lyases is that the an as the to the of the base J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar), the a from the a or an associated The glycosylase/AP intermediate be by of the imino intermediate with J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar) and by SDS-PAGE a 5′ end-labeled is used as substrate S. Nucleic Acids Res. 1997; 25: PubMed Scopus Google Scholar, J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, Biochemistry. 1997; PubMed Scopus Google Scholar, J. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, T.P. Biochemistry. 1996; PubMed Scopus Google Scholar). To this to the of RIPs, their activity on 28GR-A25 and 28P-A14 was ODNs were by of fragments of the DNA used in and were for the presence of adenines and that to be for gelonin or ricin and PAP, 28GR-A25 and 28P-A14 only these adenines. The activity was at a of that products were by incubation of RIPs with of gelonin with 28GR-A25 produced a to the produced by incubation of the glycosylase with of PAP with 28P-A14 produced a to the produced by incubation of with 28P-U14 not to the of electrophoresis of abasic site M.S. M.S. S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), were of glycosylase activity of the RIPs on The preference of gelonin for of of and of PAP for of of was as from the of To the of adenine the products were with the incubation of gelonin with 28GR-A25 and with totally the a labeled that with the of the incubation of with and with incubation of PAP with 28P-A14 and with produced a that with the of the incubation of with 28P-U14 The activity of gelonin on 28P-A14 was as was the activity of PAP on 28GR-A25 Ricin a glycosylase activity on of was from that RIPs remove adenine from single-stranded with to that with an was of activity on the Because it has been in the for the glycosylase/AP J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar) that the imino intermediate could be base removal that the possibility of was using a assay shown to be a substrate for RIPs in was used as the The of to the used in was in the of the used in Ref. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google to the of the assay for the glycosylases and the glycosylase/AP lyases. The was than was used to the of the intermediate Biochemistry. 1997; PubMed Scopus Google Scholar). at the assay indicated that 10 mm was a concentration of or to be used with gelonin and PAP because the presence of 10 mm the incubation totally the cleavage was obtained with 10 mm not This concentration was also used with ricin, the observation that 10 mm reduced the glycosylase 10 mm was determined to be the concentration to inhibit the not The of the proteins is shown in A. The of reduced by were with the of and The of was not given the weak glycosylase activity of the presence of not to be an for in the of gelonin of molecular were also as reaction with RIPs, of the previously by gel were by SDS-PAGE not of via a base DNA and proteins that were at pH have been previously J. Biol. PubMed Scopus Google Scholar). 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PubMed Scopus Google Scholar), the activity was ODNs were for glycosylase activity but were not To our this is the of the of activity of a native DNA glycosylase/AP on an ODN. of a at the of the 5′ of a site has been shown to inhibit the but not the glycosylase by T4 S. M. M. Y. T. T. E. Biochemistry. 1995; PubMed Scopus Google Scholar). results suggest that the of enzymes as DNA glycosylases or DNA glycosylase/AP lyases not on assays using as the The mechanism for the DNA glycosylase/AP J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar) that the of the imino intermediate is to be or the DNA a resulting in of the of the intermediate the reaction could the of of products in A. The cleavage of the 3′ of the S. M. M. Y. T. T. E. Biochemistry. 1995; PubMed Scopus Google Scholar, T. Y. M. S. E. K. 1995; Full Text PDF PubMed Scopus Google Scholar, T. E. 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Acta. 1993; 1154: 237-282Crossref PubMed Scopus (846) Google Scholar). possible of RIPs could be in adenine because enzymes that cleavage of the in or related are to The observation of their or F. Barbieri L. Gorini P. Valbonesi P. Bolognesi A. L. FEBS Lett. 1996; PubMed Scopus Google Scholar) that they could be in of has been shown to the of RIPs T. J.M. E. Nature. 1996; PubMed Scopus Google Scholar). Although DNA glycosylases be as against potentially of DNA, RIPs described as glycosylase/AP lyases could have a function by the of In to in 1995; Full Text PDF Scopus (69) Google Scholar), RIPs are for their Biol. 1995; PubMed Scopus Google Scholar) or anti-viral in Lifson J.D. A. Antimicrob. Agents Chemother. 1994; PubMed Scopus (69) Google Scholar). reports have suggested that their not be because of inhibition of protein synthesis (3Nicolas E. Goodyer I.D. Taraschi T.F. Biochem. J. 1997; 327: 413-417Crossref PubMed Scopus (39) Google Scholar, G.J. Irvin J.D. Aron G.M. Antimicrob. Agents Chemother. 1983; 23: 390-396Crossref PubMed Scopus (39) Google Scholar, 5McGrath M.S. Hwang K.M. Caldwell S.E. Gaston I. Luk K.-C. Wu P. Ng V.L. Crowe S. Daniels J. Marsh J. Deinhart T. Lekas P.V. Vennari J.C. Yeung H.-W. Lifson J.D. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2844-2848Crossref PubMed Scopus (320) Google Scholar, 6Tumer N.E. Hwang D.-J. Bonness M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3866-3871Crossref PubMed Scopus (130) Google Scholar). The of inhibition of DNA synthesis by PAP (4Teltow G.J. Irvin J.D. Aron G.M. Antimicrob. Agents Chemother. 1983; 23: 390-396Crossref PubMed Scopus (39) Google Scholar) or elimination of the parasite 6-kb extrachromosomal (mitochondrial) DNA of P. by gelonin (3Nicolas E. Goodyer I.D. Taraschi T.F. Biochem. J. 1997; 327: 413-417Crossref PubMed Scopus (39) Google Scholar) are with a DNA activity of The of the activities on DNA of proteins such as the of PAP that but not ribosomes N.E. Hwang D.-J. Bonness M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3866-3871Crossref PubMed Scopus (130) Google Scholar) a the of their anti-viral DNA glycosylases are described as enzymes of the DNA repair Biochem. J. 1997; PubMed Scopus Google Scholar). In the it has that in that the of the genome be of to Nature. 1997; PubMed Scopus Google Scholar). The of the that normal in has been shown to and cytotoxicity J. 1996; PubMed Scopus Google Scholar). The fact that of the glycosylases were produced by to the of the possible natural existence of such enzymes J. 1996; PubMed Scopus Google Scholar). in DNA glycosylase that to of proteins, to RIPs, are to remove normal bases, could be to RIPs are a of enzymes function is and activities do not to be because of the of of protein synthesis which they were On the basis of activities on in it been to as polynucleotide:adenosine nucleosidases (15Barbieri L. Gorini P. Valbonesi P. Castiglioni P. Stirpe F. Nature. 1994; 372: 624Crossref PubMed Scopus (108) Google Scholar) or polynucleotide:adenosine glycosidases (14Barbieri L. Valbonesi P. Bonora E. Gorini P. Bolognesi A. Stirpe F. Nucleic Acids Res. 1997; 25: 518-522Crossref PubMed Scopus (270) Google Scholar, L. Valbonesi P. Gorini P. Pession A. Stirpe F. Biochem. J. 1996; 319: 507-513Crossref PubMed Scopus (70) Google Scholar). to be to the of these proteins their and cytotoxic pathways are for and for in the