Title: Gelonin Is an Unusual DNA Glycosylase That Removes Adenine from Single-stranded DNA, Normal Base Pairs and Mismatches
Abstract: We reported that plant ribosome inactivating proteins (RIP) have a unique DNA glycosylase activity that removes adenine from single-stranded DNA (Nicolas, E., Beggs, J. M., Haltiwanger, B. M., and Taraschi, T. F. (1998) J. Biol. Chem. 273, 17216–17220). In this investigation, we further characterized the interaction of the RIP gelonin with single-stranded oligonucleotides and investigated its activity on double-stranded oligonucleotides. At physiological pH, zinc and β-mercaptoethanol stimulated the adenine DNA glycosylase activity of gelonin. Under these conditions, gelonin catalytically removed adenine from single-stranded DNA and, albeit to a lesser extent, from normal base pairs and mismatches in duplex DNA. Also unprecedented was the finding that activity on single-stranded and double-stranded oligonucleotides containing multiple adenines generated unstable products with several abasic sites, producing strand breakage and duplex melting, respectively. The results from competition experiments suggested similar interactions between gelonin's DNA-binding domain and oligonucleotides with and without adenine. A re-examination of the classification of gelonin as a DNA glycosylase/AP lyase using the borohydride trapping assay revealed that gelonin was similar to the DNA glycosylase MutY: both enzymes are monofunctional glycosylases, which are trappable to their DNA substrates. The k catfor the removal of adenine from single-stranded DNA was close to the values observed with multisubstrate DNA glycosylases, suggesting that the activity of RIPs on DNA may be physiologically relevant. We reported that plant ribosome inactivating proteins (RIP) have a unique DNA glycosylase activity that removes adenine from single-stranded DNA (Nicolas, E., Beggs, J. M., Haltiwanger, B. M., and Taraschi, T. F. (1998) J. Biol. Chem. 273, 17216–17220). In this investigation, we further characterized the interaction of the RIP gelonin with single-stranded oligonucleotides and investigated its activity on double-stranded oligonucleotides. At physiological pH, zinc and β-mercaptoethanol stimulated the adenine DNA glycosylase activity of gelonin. Under these conditions, gelonin catalytically removed adenine from single-stranded DNA and, albeit to a lesser extent, from normal base pairs and mismatches in duplex DNA. Also unprecedented was the finding that activity on single-stranded and double-stranded oligonucleotides containing multiple adenines generated unstable products with several abasic sites, producing strand breakage and duplex melting, respectively. The results from competition experiments suggested similar interactions between gelonin's DNA-binding domain and oligonucleotides with and without adenine. A re-examination of the classification of gelonin as a DNA glycosylase/AP lyase using the borohydride trapping assay revealed that gelonin was similar to the DNA glycosylase MutY: both enzymes are monofunctional glycosylases, which are trappable to their DNA substrates. The k catfor the removal of adenine from single-stranded DNA was close to the values observed with multisubstrate DNA glycosylases, suggesting that the activity of RIPs on DNA may be physiologically relevant. pokeweed antiviral protein ribosome inactivating protein recombinant gelonin single-stranded double-stranded oligonucleotide apurinic/apyrimidinic β-mercaptoethanol MAP, Mirabilis antiviral protein Several lines of evidence suggest that the anti-tumor, anti-viral, and anti-parasitic effects of the plant proteins such as gelonin or pokeweed antiviral protein (PAP),1 well known as ribosome inactivating proteins (RIPs) for their ability to remove an invariant adenine in a conserved loop in the 28 S rRNA (1Barbieri L. Battelli M.G. Stirpe F. Biochim. Biophys. Acta. 1993; 1154: 237-282Crossref PubMed Scopus (835) Google Scholar), are not solely due to ribosome inactivation (2Teltow G.J. Irvin J.D. Aron G.M. Antimicrob. Agents Chemother. 1983; 23: 390-396Crossref PubMed Scopus (39) Google Scholar, 3McGrath 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 (318) Google Scholar, 4Tumer N.E. Hwang D.-J. Bonness M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3866-3871Crossref PubMed Scopus (129) Google Scholar, 5Nicolas E. Goodyer I.D. Taraschi T.F. Biochem. J. 1997; 327: 413-417Crossref PubMed Scopus (39) Google Scholar). In vitro studies in search of alternative substrates that may be damaged by these enzymes revealed that they possess a single-stranded adenine DNA glycosylase activity (6Nicolas E. Beggs J.M. Haltiwanger B.M. Taraschi T.F. J. Biol. Chem. 1998; 273: 17216-17220Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). While there is still no direct evidence that this activity is physiologically relevant in plants or contributes to cytotoxicity, the ability of RIPs to damage DNA by removal of normal, non-mispaired bases in vitro distinguished them from the other members of the DNA glycosylase family, which protect the genome by removing potentially cytotoxic or mutagenic bases (7Krokan H.E. Standal R. Slupphaug G. Biochem. J. 1997; 325: 1-16Crossref PubMed Scopus (724) Google Scholar, 8David S.S. Williams S.D. Chem. Rev. 1998; 98: 1221-1261Crossref PubMed Scopus (458) Google Scholar). If the number of DNA lesions produced overwhelmed the DNA repair capacity of the cell or organism, the adenine glycosylase activity of the RIPs could be mutagenic or lethal. Recognition of the adenine DNA glycosylase activity of RIPs has been somewhat slow due to confusion in the literature, issues of possible contamination by nucleases, and the requirement of high protein/DNA ratio for activity (9Perentesis J.P. Miller S.P. Bodley J.W. BioFactors. 1992; 3: 173-184PubMed Google Scholar, 10Day P.J. Lord J.M. Roberts L.M. Eur. J. Biochem. 1998; 258: 540-545Crossref PubMed Scopus (53) Google Scholar). Stirpe and co-workers (11Barbieri L. Gorini P. Valbonesi P. Castiglioni P. Stirpe F. Nature. 1994; 372: 624Crossref PubMed Scopus (107) Google Scholar, 12Barbieri L. Valbonesi P. Bonora E. Gorini P. Bolognesi A. Stirpe F. Nucleic Acids Res. 1997; 25: 518-522Crossref PubMed Scopus (264) Google Scholar) reported that over 50 plant RIPs and the ricin-homologue, shiga-like-toxin found in Shigella dysenteria (13Barbieri L. Valbonesi P. Brigotti M. Montanaro L. Stirpe F. Sperti S. Mol. Microb. 1998; 29: 661-662Crossref PubMed Scopus (31) Google Scholar) removed adenine from various substrates, including DNA. In search of an enzyme classification that encompassed the removal of adenine from RNA and DNA, these investigators redefined RIPs as polynucleotide:adenosine nucleosidases (11Barbieri L. Gorini P. Valbonesi P. Castiglioni P. Stirpe F. Nature. 1994; 372: 624Crossref PubMed Scopus (107) Google Scholar) or polynucleotide:adenosine glycosidases (12Barbieri L. Valbonesi P. Bonora E. Gorini P. Bolognesi A. Stirpe F. Nucleic Acids Res. 1997; 25: 518-522Crossref PubMed Scopus (264) Google Scholar). Gelonin, PAP, and ricin were demonstrated to have an adenine DNA glycosylase activity on single-stranded DNA (6Nicolas E. Beggs J.M. Haltiwanger B.M. Taraschi T.F. J. Biol. Chem. 1998; 273: 17216-17220Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). We suggested that this new classification was more appropriate than those used in Refs. 11Barbieri L. Gorini P. Valbonesi P. Castiglioni P. Stirpe F. Nature. 1994; 372: 624Crossref PubMed Scopus (107) Google Scholar and 12Barbieri L. Valbonesi P. Bonora E. Gorini P. Bolognesi A. Stirpe F. Nucleic Acids Res. 1997; 25: 518-522Crossref PubMed Scopus (264) Google Scholar. Recently, Wanget al. (14Wang Y.-X. Neamati N. Jacob J. Palmer I. Stahl S.J. Kaufman J.D. Huang P.L. Huang P.L. Winslow H.E. Pommier Y. Wingfield P.T. Lee-Huang S. Bax A. Torchia D.A. Cell. 1999; 99: 433-442Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) suggested that the anti-HIV-1 and anti-tumor activity of the RIP MAP30 from Momordica charianta was a consequence of its adenine DNA glycosylase/AP lyase activity. This conclusion was made based on an extrapolation from the study with related proteins (6Nicolas E. Beggs J.M. Haltiwanger B.M. Taraschi T.F. J. Biol. Chem. 1998; 273: 17216-17220Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Direct evidence for the adenine DNA glycosylase or the AP lyase activity of MAP30 was not provided in Ref. 14Wang Y.-X. Neamati N. Jacob J. Palmer I. Stahl S.J. Kaufman J.D. Huang P.L. Huang P.L. Winslow H.E. Pommier Y. Wingfield P.T. Lee-Huang S. Bax A. Torchia D.A. Cell. 1999; 99: 433-442Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar. The novelty of the biochemical activity of this group of naturally occurring enzymes was recognized in a commentary by Putman and Tainer (15Putnam C.D. Tainer J.A. Nature Struct. Biol. 2000; 7: 17-18Crossref PubMed Scopus (12) Google Scholar). The classification of RIPs as AP lyases needed to be re-examined, however, since the conclusions drawn in Ref. 6Nicolas E. Beggs J.M. Haltiwanger B.M. Taraschi T.F. J. Biol. Chem. 1998; 273: 17216-17220Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar were partly based on results obtained using a borohydride trapping assay (16Sun B. Latham K.A. Dodson M.L. Llyod R.S. J. Biol. Chem. 1995; 270: 19501-19509Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), the accuracy of which was recently questioned (17Williams S.D. David S.S. Nucleic Acids Res. 1998; 26: 5123-5133Crossref PubMed Scopus (73) Google Scholar, 18Zharkov D.O. Grollman A.P. Biochemistry. 1998; 37: 12384-12394Crossref PubMed Scopus (89) Google Scholar, 19Noll D.M. Gogos A. Granek J.A. Clarke N.D. Biochemistry. 1999; 38: 6374-6379Crossref PubMed Scopus (112) Google Scholar, 20Williams S.D. David S.S. Biochemistry. 1999; 38: 15417-15424Crossref PubMed Scopus (42) Google Scholar). Issues of the possible contamination of RIPs by nucleases were addressed by zymography using naturally occurring RIPs and purified bacterial recombinant forms (21Barbieri L. Valbonesi P. Gorini P. Pession A. Stirpe F. Biochem. J. 1996; 319: 507-513Crossref PubMed Scopus (70) Google Scholar). The requirement for high protein/DNA ratios for activity on DNA may be a property of these proteins or could be due to the fact that, due to the newness of the discovery, the experimental conditions for the assay are not optimal. Barbieriet al. (12Barbieri L. Valbonesi P. Bonora E. Gorini P. Bolognesi A. Stirpe F. Nucleic Acids Res. 1997; 25: 518-522Crossref PubMed Scopus (264) Google Scholar) reported that the removal of adenine from DNA proceeded without cofactors, at low ionic strength, in the absence of Mg2+ and K+, with an optimal pH value of 4.0 (22Nicolas E. Beggs J.M. Haltiwanger B.M. Taraschi T.F. FEBS Lett. 1997; 406: 162-164Crossref PubMed Scopus (40) Google Scholar). Our laboratory found that the adenine DNA glycosylase of gelonin, PAP, and ricin was stimulated by zinc at physiological pH (6Nicolas E. Beggs J.M. Haltiwanger B.M. Taraschi T.F. J. Biol. Chem. 1998; 273: 17216-17220Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Kinetic studies undertaken to date to measure the rate of adenine removal from DNA utilized macromolecular substrates only (22Nicolas E. Beggs J.M. Haltiwanger B.M. Taraschi T.F. FEBS Lett. 1997; 406: 162-164Crossref PubMed Scopus (40) Google Scholar), rather than the single target oligonucleotides classically used for the characterization of DNA glycosylases. The simultaneous or consecutive splitting of many N-glycosidic bonds that occurred using the macromolecular substrates resulted in complicated kinetics (22Nicolas E. Beggs J.M. Haltiwanger B.M. Taraschi T.F. FEBS Lett. 1997; 406: 162-164Crossref PubMed Scopus (40) Google Scholar) and precluded any comparison with the DNA glycosylases. Many fundamental questions about the DNA glycosylase activity of the RIPs also require further investigation. These include whether the removal of adenines is the primary event that leads to DNA breakage, whether the breakage is RIP-mediated, and whether the activity is limited to the single-stranded regions of supercoiled DNA or also affects double-stranded DNA. In this investigation, we characterize the adenine DNA glycosylase activity of gelonin using assays and substrates (e.g. a single-target-containing oligonucleotide) that are routinely used to characterize classical DNA glycosylases. We now characterize the kinetics of the glycosylase activity of gelonin on these substrates under different buffer conditions and revisit our previous conclusion that gelonin has an associated AP lyase activity. In addition, we address the question of an unusual specificity for a widely available target (e.g. adenine) by studying the activity of gelonin on single-stranded and double-stranded oligonucleotides containing multiple adenines. The results clarify some of the confusing data described above and reveal more features that make the RIPs unusual glycosylases. Insight into the molecular mechanisms of adenine removal from DNA and the DNA cleavage that can accompany it is also provided. Plant gelonin was purchased from Sigma and resuspended in 10 mm HEPES, pH 7.0, and used within 3 weeks post-hydration. The wild type recombinant gelonin (rGel) and the mutants (rGel(C44A) and rGel(C50A)) were gifts from Drs. Stephen Carroll and Mark Better from the XOMA Corp. The production and characterization of these proteins has been previously described (23Better M. Bernhard S.L. Fishwild D.M. Nolan P.A. Bauer R.J. Kung A.H.C. Carroll S.F. J. Biol. Chem. 1994; 269: 9644-9650Abstract Full Text PDF PubMed Google Scholar). Protein concentrations were measured using the BCA™ protein assay reagent from Pierce (Rockford, IL) using bovine serum albumin as a standard. The oligodeoxynucleotides (ODN) were prepared by the Nucleic Acid Facility at Thomas Jefferson University and further purified by preparative gel electrophoresis before use. After gel purification, the ODN were concentrated using Centricon YM-3 filters (Millipore Corp., Bedford, MA). The concentration was measured spectrophotometrically. The ODN 5′-GTTGGGTCTCGCCTGGGTTTTCCCAGTC-3′, referred to as 28GR-A25 in Ref. 6Nicolas E. Beggs J.M. Haltiwanger B.M. Taraschi T.F. J. Biol. Chem. 1998; 273: 17216-17220Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, was renamed ssA25 in this investigation. ssU25 contained uracil in place of A at position 25 from the 5′ end. ssAP25, which contained an AP site at position 25 from the 5′ end, was created by treatment of ssU25 with the uracil DNA glycosylase (UDG; New England Biolabs, Inc., Beverly, CA). The ODN CssA25 5′-GACTGGGAAAACCCAGGCGAGACCCAAC-3′, complementary to ssA25, was used as the multiple adenine-containing substrate. 5′-32P-End labeling was performed using [γ-32P]ATP (NEN Life Sciences, Boston, MA)) and T4 polynucleotide kinase (Promega, Madison, WI) as described in Ref. 6Nicolas E. Beggs J.M. Haltiwanger B.M. Taraschi T.F. J. Biol. Chem. 1998; 273: 17216-17220Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar. Duplex substrates were produced by annealing ssA25 and CssA25 according to standard procedures and purification by electrophoresis under native conditions. The reactions were initiated by mixing the ODN substrate and gelonin in a 10-μl total volume of buffer I (10 mm HEPES, pH 7.0, 100 μm ZnCl2), buffer II (10 mm HEPES, pH 7.0, 2 mmZnCl2, 2% β-mercaptoethanol), or buffer III (10 mm MES, pH 5.0, 1.0 mm EDTA). Unless indicated, the concentration of gelonin was 10−7m. Substrate concentrations varied from 5 × 10−8m to 10−5m as indicated in the figure legends. 5′-32P-End-labeled substrate was added as a tracer (60 fmol, 1.25 × 105 cpm/reaction). In the competition experiments, the unlabeled substrate was replaced by an equal amount of ssU25. At the end of the desired incubation times indicated in the figure legends, the samples were post-treated either with alkali (0.2 n NaOH, 40 mm EDTA, 70 °C) when the substrate contained a single adenine, or with a reducing agent (100 mm NaBH4, 15 min on ice) when the substrate contained multiple adenines. Assays were terminated by the addition of formamide loading dye and neutralization with HCl. Substrate and product(s) were resolved by gel electrophoresis in a 15% polyacrylamide-urea gel. The distribution of the radiolabeled species in the gel was determined using a PhosphorImager 445 SI (Molecular Dynamics, Sunnyvale, CA) and ImageQuant software. Buffer II was used to determine the enzyme kinetics. Reactions, done in triplicate, contained the enzyme at 0.1 μm and the substrate ssA25 at concentrations varying from 0.5 to 10.0 μm and were stopped after 2 min. A double-reciprocal plot of the initial rateversus ODN concentration allowed the determination ofK m and V max. The catalytic constant (k cat) was calculated as the ratio ofV max to the enzyme concentration used (10−7m). The glycosylase reaction buffer used above was supplemented with the desired concentration of NaCl, NaCNBH3, or NaBH4. After various times as indicated in the figure caption, the assays were terminated by addition of SDS-polyacrylamide gel electrophoresis loading buffer. The samples were boiled for 5 min and analyzed by SDS-polyacrylamide gel electrophoresis. After elimination of the lower part of the gel that contained the free oligonucleotide, the distribution of the isotope on the gel was determined by PhosphorImager analysis. The autoradiograms were processed with Adobe Photoshop 5.0. We previously described the design of an oligonucleotide substrate and a method to study qualitatively the adenine DNA glycosylase activity of gelonin (6Nicolas E. Beggs J.M. Haltiwanger B.M. Taraschi T.F. J. Biol. Chem. 1998; 273: 17216-17220Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The same 28-mer ODN containing a single adenine at position 25 was used to determine the enzyme kinetics of gelonin. The effect of different buffering conditions on gelonin's DNA glycosylase activity was investigated. In addition to the buffer conditions we previously used (10 mm HEPES, pH 7.0, 100 μmZnCl2 (buffer I)) in Ref. 6Nicolas E. Beggs J.M. Haltiwanger B.M. Taraschi T.F. J. Biol. Chem. 1998; 273: 17216-17220Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, we found that 10 mm HEPES, pH 7.0, 2 mm ZnCl2, 2% β-mercaptoethanol (βME) (buffer II) or 10 mm MES, pH 5.0, 1 mm EDTA (buffer III) also supported the glycosylase activity. The DNA glycosylase activity obtained with these different conditions as a function of substrate concentration is shown in the autoradiogram of the denaturing polyacrylamide gel (Fig.1 A). The slower electrophoretic mobility of the product that was observed in buffer II was due to inhibition of δ-elimination by βME (24Bailly V. Verly W.G. Nucleic Acids Res. 1988; 16: 9489-9496Crossref PubMed Scopus (27) Google Scholar). The difference in the processing of the substrate in the three conditions was so remarkable that it could be ascertained without processing of the autoradiogram. The most striking difference was observed in conditions of multiple turnover ([protein] < [DNA]): at pH 7.0, in the presence of zinc, the addition of βME allowed more facile turnover so that, at a 1:50 protein/DNA ratio (mol:mol), the degradation of substrate increased from <5 to ∼60%. To avoid experimental artifacts, which can be associated with measurement of activity after a set time, the time course of the reaction was studied. Two enzyme/ODN ratios (1 or 0.02) were used (Fig. 1 B). At equimolar ratio, both buffers II and III allowed a rapid, total conversion of substrate to product. In buffer I and III at a 1:50 ratio, the low but steady progression of a degradation product suggested that the poor processing of the substrate by gelonin was not due to a single turnover mechanism as observed for the DNA glycosylase TDG (25Waters T.R. Swann P.F. J. Biol. Chem. 1998; 273: 20007-20014Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Due to the extremely inefficient turnover, buffer I and III could not be used for the determination of standard Michaelis-Menten kinetics parameters. Fig. 1 C examined the effect of varying the gelonin concentration (10−9 to 10−7m), at an equimolar gelonin/DNA ratio, on the glycosylase activity during a 2-min incubation in buffer II. The results showed that the relative rate of reaction was concentration dependent, suggesting that in the lower concentration range substrate binding was a limiting factor. As a result, a concentration of gelonin of 10−7mwas used throughout the rest of the investigation. Having defined experimental conditions that allowed efficient processing of the DNA substrate, we measured the kinetic parameters k cat andK m of the reaction. Fig.2 shows the concentration of abasic DNA produced in 2 min by 10−7m gelonin plotted against the concentration of the substrate ssA25. Adenine excision by gelonin is shown to follow Michaelis-Menten kinetics. From the double-reciprocal plots of initial velocity versus substrate concentration, k cat and K mwere estimated at 8.3 min−1 and 1.7 μm, respectively. Gelonin possesses two cysteines in positions 44 and 50 linked by a disulfide bridge (23Better M. Bernhard S.L. Fishwild D.M. Nolan P.A. Bauer R.J. Kung A.H.C. Carroll S.F. J. Biol. Chem. 1994; 269: 9644-9650Abstract Full Text PDF PubMed Google Scholar). The stimulatory effect of βME described above suggested that the low turnover efficiency observed in buffer I could be due to the disulfide bridge and that it might be possible to increase the glycosylase activity by eliminating the bridge by genetic mutation. Such an approach has been used with the RIP MAP from Mirabilis: the inhibitory activity on protein synthesis of the mutant C36S/C220S, in which the disulfide bridge was eliminated was approximately 22 times higher than that of native MAP (26Habuka N. Miyano M. Kataoka J. Tsuge H. Ago H. Noma M. J. Biol. Chem. 1991; 266: 23558-23560Abstract Full Text PDF PubMed Google Scholar). To investigate the role of the disulfide bridge between cysteines 44 and 50 in the low efficiency turnover of gelonin in buffer I, we compared the activity of the gelonin mutants C44A and C50A to that of the wild type recombinant enzyme (Fig.3). The results with the recombinant wild type protein indicated that the low turnover in buffer I and the stimulation of the activity by βME were intrinsic properties of gelonin, since they were observed with both the native (Fig. 1) and recombinant (Fig. 3) proteins. The elimination of the disulfide bridge had no effect on the gelonin turnover in buffer I, suggesting that the mechanism of stimulation by βME was not through modification of the protein. The elimination of these cysteines also had no effect on gelonin's ability to inhibit protein synthesis (23Better M. Bernhard S.L. Fishwild D.M. Nolan P.A. Bauer R.J. Kung A.H.C. Carroll S.F. J. Biol. Chem. 1994; 269: 9644-9650Abstract Full Text PDF PubMed Google Scholar). DNA glycosylase/AP lyases are glycosylases with an associated β-elimination activity that results in DNA strand breakage. A unifying hypothesis that rationalizes the apparent distinction between the two classes of glycosylases (glycosylase or glycosylase/AP lyase) has been proposed (16Sun B. Latham K.A. Dodson M.L. Llyod R.S. J. Biol. Chem. 1995; 270: 19501-19509Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The bifurcation is in the catalytic mechanism, i.e. the type of nucleophile that attacks C-1′ of the damaged base nucleoside. Bifunctional glycosylases utilize an amine nucleophile from the enzyme, while monofunctional glycosylases use a nucleophile derived from the medium. The hypothesis of a unified catalytic mechanism seemed to be supported by the borohydride-trapping assay, the principle of which is that only bifunctional glycosylases form a trappable complex with their substrate (16Sun B. Latham K.A. Dodson M.L. Llyod R.S. J. Biol. Chem. 1995; 270: 19501-19509Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). In our previous paper (6Nicolas E. Beggs J.M. Haltiwanger B.M. Taraschi T.F. J. Biol. Chem. 1998; 273: 17216-17220Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), the trapping assay with an oligonucleotide substrate was used to investigate the possibility that gelonin had an associated AP lyase activity, which was suggested by the cleavage of a single-stranded DNA fragment (∼800 bases) by gelonin, PAP, or ricin. We suggested that RIPs appeared unusual in that, while they formed a borohydride-trappable complex classifying them as DNA glycosylase/AP lyases (24Bailly V. Verly W.G. Nucleic Acids Res. 1988; 16: 9489-9496Crossref PubMed Scopus (27) Google Scholar), they could not be distinguished from monofunctional glycosylases in a strand cleavage assay using a short (28-mer) ODN substrate, since post-treatment was necessary to break the DNA at the resulting abasic site. Similar behavior was subsequently reported for the adenine DNA glycosylase, MutY, whose classification has long been a matter of controversy (17Williams S.D. David S.S. Nucleic Acids Res. 1998; 26: 5123-5133Crossref PubMed Scopus (73) Google Scholar, 18Zharkov D.O. Grollman A.P. Biochemistry. 1998; 37: 12384-12394Crossref PubMed Scopus (89) Google Scholar, 20Williams S.D. David S.S. Biochemistry. 1999; 38: 15417-15424Crossref PubMed Scopus (42) Google Scholar). It was concluded from a kinetic analysis that the slow dissociation rate of MutY from its product was suggestive of specific contacts with DNA that persisted after base removal. These contacts may result in borohydride-trappable complex formation independent of a lyase activity. These results and the observations reported in Fig. 1 prompted us to reinvestigate the origin of the gelonin-ODN complex observed in the presence of NaBH4. In particular, we investigated the formation of a trappable complex in buffer II, which supported a more efficient enzyme turnover. We (6Nicolas E. Beggs J.M. Haltiwanger B.M. Taraschi T.F. J. Biol. Chem. 1998; 273: 17216-17220Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) and others (17Williams S.D. David S.S. Nucleic Acids Res. 1998; 26: 5123-5133Crossref PubMed Scopus (73) Google Scholar) have noted that the experimental conditions for the trapping assay may not be as straightforward as proposed in the initial paper (16Sun B. Latham K.A. Dodson M.L. Llyod R.S. J. Biol. Chem. 1995; 270: 19501-19509Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), mainly because of the hypersensitivity of some glycosylases to salt. In Fig.4, we compared the effect of NaCl on the glycosylase activity of gelonin in buffers I and II. Fig. 4 Arevealed that the glycosylase activity of gelonin was less sensitive to increasing salt concentration in buffer II than in buffer I, suggesting that it was stabilized by stronger ionic interactions and/or additional hydrophobic interactions. From these results, 25 mm was selected as the concentration of reducing agent to be used in the trapping assay. A kinetic analysis of the glycosylase activity (Fig.4 B, upper panel) and of the formation of a gelonin-ODN complex (Fig. 4 B, lower panel) in the presence of 25 mm NaCNBH3 was performed.While the total removal of adenine was accomplished in 5 min, the intensity of the signal corresponding to the ODN-gelonin complex increased over the 30-min time course of the assay. No complex formation was observed when 25 mm NaCNBH3 was replaced by 25 mm NaCl (data not shown). NaBH4, a stronger reducing agent than NaCNBH3, which usually produces more effective glycosylase-DNA cross-linking (20Williams S.D. David S.S. Biochemistry. 1999; 38: 15417-15424Crossref PubMed Scopus (42) Google Scholar), was also inefficient in cross-linking gelonin to its ODN substrate when added at 25 mm during the glycosylase reaction (data not shown). Our interpretation of the absence of parallelism between the two curves was that the gelonin-ODN complex formed slowly after base removal as a result of a fortuitous encounter between a lysine and the abasic site. Using NaCNBH3 instead of NaBH4 increased the efficiency of cross-linking, because the former did not simultaneously reduce the preformed aldehydes and therefore did not inactivate the substrate. The interaction of gelonin with the abasic site was further probed by determining the conditions for the formation of a complex with an ODN with a preformed abasic site. Fig. 4C (lanes 1–4) shows that, provided that the concentration of reducing agent was reduced to 10 mm, which is consistent with the results shown in Fig.4 A, complex formation was detectable, although highly reduced, in the absence of zinc and βME. Complex formation was progressively inhibited by the addition of increasing concentrations of EDTA (lanes 5 and 6). Fig. 5 shows the glycosylase activity, as a function of time, of gelonin on the ODN CssA25 that contained multiple adenines. Despite the fact that the samples were analyzed without alkali post-treatment (lanes 2–6), smears indicative of extensive DNA degradation were observed even at the shortest time of incubation (5 min). Electrophoresis in Tris buffer can cause breakage of the backbone of DNA containing abasic sites (27Povirk L.F. Houlgrave C.W. Biochemistry. 1988; 27: 380-385Crossref Scopus (121) Google Scholar, 28Ray T. Mills R.T. Dyson P. Electrophoresis. 1995; 16: 888-894Crossref PubMed Scopus (52) Google Scholar); this can be prevented by stabilization of the abasic sites with NaBH4. To verify that the smears were due to creation of abasic sites by gelonin and determine whether the degradation was produced by gelonin during the incu