From the Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Philadelphia, Pennsylvania 19107
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ABSTRACT |
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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.
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INTRODUCTION |
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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 RIP-treated 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 3H-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). Stirpe and co-workers (14), 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 (15), polynucleotide:adenosine glycosidases (16, 17), and endonucleases (7-9, 11, 12). The results reveal a new class of DNA glycosylase/AP lyases that act on specific adenines in ssDNA.
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EXPERIMENTAL PROCEDURES |
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Proteins-- 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.
DNA and Oligonucleotides--
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). [-32P]dATP,
[
-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); TrevigelTM
500 was from Trevigen (Gaithersburg, MD); and SDS-PAGE precast gradients gels (4-20%) were from Bio-Rad.
Preparation of Substrates and Standards--
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
[-32P]dATP and SequenaseTM 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. 12. The ODNs were 5' end-labeled with T4
polynucleotide kinase and [
-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.
RIP Activity on pUC18 DNA-- 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 mM HEPES-KOH, 16 mM sodium acetate, 0.8 mM EDTA (18). 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 NaBH4 or 10 mM NaCl. To assay for DNA glycosylase activity in the absence of detectable cleavage, a post-treatment with alkali (0.2 M NaOH, 50 mM EDTA, 30 min, 4 °C) was performed before electrophoresis. When the radioactive fragment was included in the reaction mixture, assays were terminated by addition of formamide loading dye for direct loading onto a denaturing, 6% polyacrylamide-urea gel for electrophoresis in TBE buffer (89 mM Tris-base, 89 mM boric acid, 2 mM EDTA) and autoradiography. To determine the location of the DNA cleavage sites, the products of the Maxam-Gilbert sequencing reactions (19) on the radioactive fragment were run as markers.
RIP Activity on ODNs-- 1 µg of gelonin or PAP or 2 µg of ricin, in 10 mM HEPES, pH 7, 0.1 mM ZnSO4, was incubated at 37 °C for 1 h with the appropriate oligonucleotide in a reaction volume of 10 µl at protein/DNA molar ratios of 2:1. To assay for DNA glycosylase activity in the absence of spontaneous cleavage, a post-treatment with 10 mM spermidine for 30 min at 37 °C was performed. Assays were terminated by addition of formamide loading dye for direct loading onto a 15% polyacrylamide-urea gel for electrophoresis in TBE buffer and autoradiography.
The trapping assay was adapted from Ref. 20. The oligonucleotides were added after a preincubation for 5 min at 37 °C of the proteins in the reaction buffer used above but supplemented with 10 mM NaCl or NaBH4. After 5 min at 37 °C, the samples were post-treated with spermidine as above and subjected to SDS-PAGE on a polyacrylamide 4-20% gradient gel after addition of loading buffer (0.1 M sodium phosphate, pH 6.0, 4% SDS, 10% glycerol, 2%Illustrations-- Photographs and autoradiograms were scanned using a Hewlett Packard Scanjet 4C and processed with Adobe Photoshop 3.0.
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RESULTS |
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Degradation of Single-stranded DNA by RIPs-- RIPs have been divided into two classes according to their structure. Class I-RIPs are single-chain, basic proteins with a molecular mass of about 30,000 daltons. Class II-RIPs are neutral proteins composed of two dissimilar chains, of approximately 30,000 Da each, connected by a disulfide bridge. Their active chain is homologous to the class I-RIPs (1). In this study, three RIPs were used. Gelonin and PAP belong to class I, whereas ricin belongs to class II. We have previously reported that ssDNA was the preferred substrate for the nuclease activity of gelonin and that this activity was modulated by zinc (12). Fig. 1A shows that PAP (lane 3) and ricin (lane 4), like gelonin (lane 2), degraded ssDNA in the presence of zinc. On a molar basis, the activity was gelonin > PAP > ricin.
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Formation of Abasic Sites Is the First Step in the Degradation of
ssDNA by RIPs--
Electrophoresis on agarose gels under nondenaturing
conditions has been widely used to demonstrate the activity of various RIPs on supercoiled (7-9, 13) or linear (12) ssDNA. The appearance of
nicked and linear forms or smears was indicative of backbone cleavage.
Our first aim was to determine whether the removal of adenines by RIPs
described by Barbieri et al. (14) is the first event in the
degradation of ssDNA. The presence of abasic sites is commonly
demonstrated by their conversion to nicks upon treatment with alkali
(21). Fig. 1B shows the effect of RIPs on ssDNA under
conditions of limited reaction when compared with Fig. 1A. The appearance of a smear, only after post-treatment with alkali, was
indicative of the existence of intermediates containing abasic sites
but no nicks. Fig. 1C showed that the inclusion of 10 mM sodium borohydride (NaBH4) during the
incubation of RIPs with DNA prevented the degradation observed in Fig.
1A. The presence of 10 mM NaCl had no detectable
effect (data not shown), indicating that the inhibition was not because
of interference with ionic interactions between RIPs and DNA. Higher
concentrations of NaCl (>50 mM) inhibited the degradation
of ssDNA (data not shown). The inhibition of cleavage by
NaBH4 (Fig. 1C) suggested that the DNA breakage
caused by the RIPs in Fig. 1A was because of a
-elimination reaction at the abasic sites, possibly by an associated
AP lyase activity (22). Primary amine-containing buffers were avoided in these experiments as they were reported to be unsuitable for the
assay of enzymes utilizing aldehydes as substrates and for electrophoresis of DNA containing abasic sites, because they can cause
DNA breakage (18, 23). The results in Fig. 1B indicated a
significantly greater quantity of alkali-labile sites to authentic single-stranded breaks, suggesting the AP lyase activity, if present, was weak.
Excision of a Protein-specific Set of Adenines and Strand Scissions
at the Resulting Abasic Sites--
To identify the bases removed by
the DNA glycosylase activity of the RIPs, the electrophoretic
mobilities of the alkali-catalyzed, -elimination products can be
compared with those of the products obtained by the Maxam-Gilbert
sequencing procedure (19). To apply this strategy, a 3' 32P
end-labeled radioactive fragment of pUC18 DNA was included in the
reaction mixture. The reaction conditions were chosen so that the
alkali post-treatment was performed on intermediates containing abasic
sites but no nicks (data not shown). Electrophoresis in Tris/borate/EDTA did not cleave these intermediates (data not shown).
In Fig. 2A, which shows a
portion (C30-T105) of the autoradiogram of the sequencing gel, it was
seen that the bands revealed by alkali treatment corresponded to breaks
at sugars without adenine. Each protein had a specific set of targets.
Gelonin and ricin had similar cleavage patterns, removing adenines 34, 38, 49, 64, and 73. PAP, in addition to adenines 64 and 73, also
excised adenines 55, 67, and 82. The excision of 5 adenines of the 17 available between C30 and T105 may explain the appearance of smears in
Fig. 1A, which were suggestive of a sequence-unspecific mode
of degradation. An extended incubation of DNA with RIPs, without
chemical post-treatment, also fragmented the substrate (Fig.
1A; Refs. 7-9, 11-13). The products generated under these
conditions had the same electrophoretic mobilities as the products
generated by alkali-catalyzed
-elimination (Fig. 2B).
This indicated that the cleavage occurred at the 3' side of the abasic
sites. Glycosylase/AP lyases cleave the phosphodiester bond 3' of the
abasic site via a
-elimination reaction (22). The results in Figs. 1
and 2 suggested that the RIPs were DNA glycosylases that had an
associated weak AP lyase activity.
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Activity on ODNs and Evidence for an Imino Intermediate-- A major distinction between simple DNA glycosylases and DNA glycosylase/AP lyases is that the latter group uses an amino group as the nucleophile to attack the sugar of the damaged base nucleotide (22, 24), whereas the former use a nucleophile from the medium, most likely a hydroxide ion or an associated water molecule. The glycosylase/AP lyase-DNA covalent intermediate can be trapped by reduction of the imino intermediate with NaBH4 (22) and visualized by autoradiography after SDS-PAGE when a 5' 32P end-labeled ODN is used as substrate (20, 22, 25-28).
To apply this strategy to the characterization of RIPs, their activity on 28GR-A25 and 28P-A14 was first investigated (Fig. 3). These ODNs were designed by modification of fragments of the DNA used in Fig. 2. 28GR and 28P were chosen for the presence of adenines 38 and 82 that appear to be selective targets for gelonin or ricin and PAP, respectively (Fig. 2). 28GR-A25 and 28P-A14 contain only these adenines. The activity was measured at a RIP/ODN molar ratio of 2:1. Fig. 3A shows that no scission products were detected by direct analysis after incubation of RIPs with 28GR-A25. Incubation of gelonin with 28GR-A25 (lane 4) produced a smear similar to the one produced by incubation of the simple glycosylase UDG with 28GR-U25 (lane 2). Incubation of PAP with 28P-A14 produced a smear similar to the one produced by incubation of UDG with 28P-U14 (data not shown). These smears, ascribed to the instability of electrophoresis of abasic site containing oligonucleotides (29), were suggestive of adenine-DNA glycosylase activity of the RIPs on ODNs. The preference of gelonin for A25 of 28GR over A14 of 28P and of PAP for A14 of 28P over A25 of 28GR was as predicted from the analysis of Fig. 2. To confirm the hypothesis of adenine removal, the products were analyzed after post-treatment with the
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DISCUSSION |
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A New Class of DNA Glycosylase/AP Lyases-- On the basis of the results of our investigation, the RIPs meet the established criteria (22, 24) to be classified as DNA glycosylase/AP lyases. The description of a DNA glycosylase/AP lyase activity on ssDNA is unique, because UDGs, which are the only described DNA glycosylases with in vitro activity on ssDNA, do not have an associated lyase activity (21). As was the case for several other DNA glycosylases (33), the associated AP lyase activity that nicks DNA at the site of base removal led to the description of RIPs as "endonucleases." In our investigation, the lyase activity was evident when full-length, single-stranded pUC18 (Fig. 1) or an 800-base fragment (Fig. 2) were used as substrates. The ability to detect intermediates containing abasic sites but no nicks (Figs. 1 and 2) suggested that, similar to the pyrimidine dimer-glycosylase/AP lyase T4 endonuclease V (34), the AP lyase activity was weak. ODNs were substrates for adenine-DNA glycosylase activity but were not cleaved (Fig. 3). To our knowledge, this is the first report of the absence of AP lyase activity of a native DNA glycosylase/AP lyase on an unmodified ODN. Chemical attachment of a fluorine atom at the 2' position of the 5' component of a thymine dimer site has been shown to inhibit the strand cleavage, but not the glycosylase activity, by T4 endonuclease V (31). Our results suggest that the classification of enzymes as simple DNA glycosylases or DNA glycosylase/AP lyases should not rely solely on assays using oligonucleotides as substrates nor the NaBH4 trapping procedure.
The proposed mechanism for the DNA glycosylase/AP lyase pathway (24) predicts that the fate of the covalent imino intermediate is to be hydrolyzed, either before or after the DNA undergoes aThe Physiological Function of RIPs and the Consequences of DNA Glycosylase Activity on Normal Bases in Mammalian Cells-- The proteins used in this study are from plants. They are known as ribosome-inactivating proteins, but their physiological function is not clear (1). A possible role of RIPs could be in adenine metabolism, because enzymes that catalyze cleavage of the N-glycosidic bond in nucleotides, nucleosides, or related compounds are central to salvage pathways. The observation of their induction upon stress or senescence (39) suggests that they could be involved in macromolecular turnover. Viral infection of sugar beets has been shown to induce the expression of RIPs (40). Although DNA glycosylases can be seen as defenses against potentially injurious modifications of DNA, RIPs now described as adenine-ssDNA glycosylase/AP lyases could have a protective function by damaging the genetic material of invading pathogens.
In addition to uses in agribiology (41), RIPs are being evaluated for their anti-cancer (42) or anti-viral efficacy in humans (43). Some reports have suggested that their effects may not be (only) because of inhibition of protein synthesis (3-6). The observations of selective inhibition of viral DNA synthesis by PAP (4) or elimination of the parasite 6-kb extrachromosomal (mitochondrial) DNA of P. falciparum infected erythrocytes by gelonin (3) are consistent with a DNA damaging activity of RIPs. The characterization of the activities on DNA of proteins such as the C-terminal deletion mutant of PAP that inhibits viral infection but does not depurinate host ribosomes (6) might give a better insight into the origin of their anti-viral effect. DNA glycosylases are classically described as enzymes of the DNA repair pathway (44). In the last few years, it has become clear that defects in genes that maintain the integrity of the genome may be causes of inherited predispositions to cancer (45). The expression of the UDG engineered mutants that release normal pyrimidines in Escherichia coli has been shown to cause mutations and cytotoxicity (38). The fact that three of the four mutant pyrimidine-DNA glycosylases were produced by single nucleotide substitutions led to the suggestion of the possible natural existence of such enzymes (38). Mutations in DNA glycosylase genes that lead to expression of proteins, which, similar to RIPs, are able to remove normal bases, could be linked to certain (human) diseases.A New Name for RIPs?-- Plant RIPs are a family of enzymes whose physiological function is unknown and whose pharmacological activities do not appear to be solely because of the "classical" property of inhibitors of protein synthesis after which they were named. On the basis of activities observed on various substrates in vitro, it had been proposed to reclassify them as polynucleotide:adenosine nucleosidases (15) or polynucleotide:adenosine glycosidases (14, 16). These terminologies appear to be inappropriate. We propose to postpone the renaming of these proteins until their physiological role and cytotoxic pathways are better characterized.
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ACKNOWLEDGEMENTS |
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We thank Dr. Mary-Ann Bjornsti for valuable discussions and Jolanta Fertala for technical advice in the Maxam- Gilbert sequencing procedure.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants AI-27247 and AI-41761.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Pathology,
Anatomy and Cell Biology, Jefferson Medical College, 1020 Locust St.,
Rm. 229, Philadelphia, PA 19107. Tel.: 215-503-5020; Fax: 215-923-2218;
E-mail: Theodore.Taraschi{at}mail.tju.edu.
1 The 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.
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REFERENCES |
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