Specific Binding of a Designed Pyrrolidine Abasic Site Analog to Multiple DNA Glycosylases*

Orlando D. SchärerDagger , Huw M. Nash, Josef Jiricny§, Jacques Laval, and Gregory L. Verdinepar

From the Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, the § Institute for Medical Radiobiology, August-Forel-Strasse 7, 8029 Zurich, Switzerland, and the  Groupe Réparation de l'ADN, URA 147 Centre National de la Recherche, Institut Gustave Roussy, PRII, 39, rue Camille Desmoulins, 94805 Villejuif Cedex, France

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

In the base excision DNA repair pathway, DNA glycosylases recognize damaged bases in DNA and catalyze their excision through hydrolysis of the N-glycosidic bond. Attempts to understand the structural basis for DNA damage recognition by DNA glycosylases have been hampered by the short-lived association of these enzymes with their DNA substrates. To overcome this problem, we have employed an approach involving the design and synthesis of inhibitors that form stable complexes with DNA glycosylases, which can then be studied biochemically and structurally. We have previously reported that double-stranded DNA containing a pyrrolidine abasic site analog (PYR) forms an extremely stable complex with the DNA glycosylase AlkA and potently inhibits the reaction catalyzed by the enzyme (Schärer, O. D., Ortholand, J.-Y., Ganesan, A., Ezaz-Nikpay, K., and Verdine, G. L. (1995) J. Am. Chem. Soc. 117, 6623-6624). Here we investigate the interaction of this inhibitor with a variety of additional DNA glycosylases. With the exception of uracil DNA glycosylase all the glycosylases tested bind specifically to PYR-containing oligonucleotides. By comparing the interaction of DNA glycosylases with PYR and the structurally related tetrahydrofuran abasic site analog, we assess the importance of the positively charged ammonium group of the pyrrolidine in binding to the active site of these enzymes. Such a general inhibitor of DNA glycosyases provides a valuable tool to study stable complexes of these enzymes bound to substrate-like molecules.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The preservation of genetic information encoded by DNA is of crucial importance for the survival of all organisms. Genomic DNA is continuously subjected to damage arising from a variety of sources, including spontaneous hydrolysis, oxidation, deamination, alkylation, and errors in replication (1). To counter the toxic and mutagenic effects of the lesions thus produced, DNA repair pathways exist in all known organisms that identify damaged sites in the genome, remove the lesions, and reconstitute the original DNA sequence (2). One such pathway is the base excision repair pathway, in which modified bases are excised through glycosidic bond hydrolysis, the resulting abasic sites are removed, and the correct nucleotide sequence is reinstalled by repair synthesis (2, 3). The pivotal enzymes in this pathway are DNA glycosylases, which recognize damaged bases in DNA and catalyze their removal through hydrolysis of the N-glycosidic bond linking the damaged base to its sugar (Fig. 1A). Two classes of DNA glycosylases are known: monofunctional DNA glycosylases only catalyze glycosidic bond hydrolysis and generate abasic sites as products. Bifunctional DNA glycosylases have an associated AP1 lyase activity, which enables them to catalyze the cleavage of the 3' C-O bond through a beta -elimination mechansim; some such enzymes are also able to cleave the 5' C-O bond through beta -elimination, thereby generating a one-nucleoside gap (3, 4). The remaining enzymes of the pathway remove the abasic sites (or the 3'-phosphate group in the case of glycosylase/lyases) and reinstall the original base through repair synthesis (3, 5, 6).


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Fig. 1.   A, the base excision repair pathway: various DNA damaging agents produce modified bases in DNA. DNA glycosylases recognize the damaged bases and remove them through N-glycosidic bond hydrolysis. The nucleophile (Nu) replacing the damaged base is either an activated water molecule (for monofunctional glycosylases) or an amino group of a side chain or the N terminus of the enzyme (for bifunctional glycosylase/lyases). The abasic site generated is excised by additional enzymes in the pathway. B, structural analogy between the proposed transition state structure (1) for glycosidic bond cleavage and the pyrrolidine abasic site analog (2) intended to mimic the positive charge of the transition state. The binding of 2 to DNA glycosylases is compared with the binding to the uncharged tetrahydrofuran (3) to assess the importance of the positive charge in 2 in the interaction with DNA glycosylases.

Known DNA glycosylases act on base adducts in DNA resulting from four types of chemical reactions: 1) alkylation, which takes place mainly at N7 guanine, N3 of adenine, and at O2 of thymine and cytidine (3); 2) oxidation, principally at the 8 position of purines, the 5,6 double bond of pyrimidines, and the exocyclic methyl group of thymine (7); 3) hydrolytic deamination of the exocyclic amine groups of cytosine and adenine (1); and 4) photocross-linking of neighboring pyrimidines (8).

The recognition problem faced by DNA glycosylases is a formidable one, requiring these enzymes to recognize subtle alterations in DNA bases in the context of their native counterparts, which are present in vast excess. Progress on this front requires high resolution structural information to reveal in detail the interactions between these enzymes and their substrates. However, because DNA glycosylases associate with their substrate only for the brief period of time during which the excision reaction takes place, it has been virtually impossible to gain direct structural insight into catalytically competent complexes. One approach to circumvent this problem has been to mutate essential catalytic groups in the enzyme so as to abrogate turnover, but preserve substrate recognition (9). For example, mutant versions of the bacteriophage T4 endonuclease V and the human uracil DNA glycosylase have been found to form tight complexes with a substrate thymidine dimer and a product abasic site and free uracil, respectively (10, 11).

We have developed a complementary substrate-modification approach for the study of damage recognition by DNA glycosylases, which relies on the design and synthesis of substrate-like molecules that are recognized but not cleaved by base excision repair enzymes (12-15). Our design of inhibitors of DNA glycosylases is based on a model for the transition state structure of the reaction they catalyze. Although the reaction mechanism employed by DNA glycosylases has not been studied in great detail, a mechanistic model can be proposed based on studies of enzymes that catalyze similar glycosyl transfer reactions such as glucosidases (16, 17) or nucleoside hydrolase (18). According to this model, the transition state is likely to resemble 1 in Fig. 1B, in which substantial positive charge is accumulated at O-1' and C-1'. This model applies for both monofunctional glycosylases and bifunctional glycosylase/lyases; the only difference between the two classes of enzymes is the nature of the nucleophile (Nu in 1), an enzyme activated water in the case of monofunctional glycosylases or an amino group of the enzyme for glycosylase/lyases (4, 19).

Previously, we have used two strategies to design inhibitors based on this transition state structure. We introduced electron-withdrawing fluorine groups in the deoxyribose moiety, which electronically destabilize the positive charge and thereby lower the reaction rates. This approach yielded potent inhibitors of the mammalian 3-methyladenine glycosylase ANPG and the mismatch-specific thymine DNA glycosylase TDG (12, 13). In a second approach, we used transition state mimicry to inhibit DNA glycosylases. Oligonucleotides containing a pyrrolidine abasic site analog (PYR, 2) have been synthesized to mimic the positive charge of the transition state and were shown to interact specifically with Escherichia coli 3-methyladenine DNA glycosylase AlkA and T4 endonuclease V (14, 15). Here, we extend our studies using transition state mimicry and show that duplex oligonucleotides containing a PYR residue (2) serve as inhibitors for a wide variety of DNA glycosylases. We found that the DNA glycosylases AlkA, ANPG, Tag, Fpg, MutY, endonuclease III, and TDG bind specifically to PYR-containing oligonucleotides. Uracil DNA glycosylase was the only enzyme tested that did not interact specifically with this transition state mimic. Such a general inhibitor of DNA glycosylases should provide a valuable tool to study the interaction of DNA glycosylases with their target DNA using biochemical and structural methods.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Enzymes, General Methods, and Materials-- Oligonucleotides were synthesized on a Applied Biosystems 392 DNA/RNA synthesizer and purified by denaturing gel electrophoresis. AlkA (14), ANPG (20), Tag (21), Fpg (22), endonuclease III (23), and TDG (24) were overexpressed in E. coli and purified as described previously. Uracil DNA glycosylase was a gift from Dr. Dale Mosbaugh, and MutY was a gift from Dr. Stephen Lloyd. The exact enzyme concentration in our preparations was determined using gel-shift titrations (see below). [3H]Dimethyl sulfate-treated calf thymus DNA (specific activity: 785 cpm/pmol methylated nucleotide) was prepared as described previously (25). T4 polynucleotide kinase was obtained from New England Biolabs. [gamma -32P]ATP was obtained from NEN Life Science Products. A FUJIX BAS 200 phosphorimager was used for radioactivity quantification.

Synthesis, 5'-32P-End Labeling and Annealing of Oligonucleotides-- Oligonucleotides containing the pyrrolidine (14) and tetrahydrofuran (26, 27) residues were synthesized as described previously. The 8oxoguanine-containing oligonucleotide was a gift of Dr. Tomohiko Kawate. The sequences of the duplex oligonucleotides used in this study are shown in Table I. For gel-shift assays, 1 pmol of a modified 25-mer oligonucleotide (5'-GGA TAG TGT CCA X GTT ACT CGA AGC -3', X = PYR or THF) was 5'-32P-end-labeled using with T4 polynucleotide kinase and [gamma -32P]ATP and annealed to a 10-fold excess of a complementary 25-mer (5'-GCT TCG AGT AAC Y TGG ACA CTA TCC-3', Y = T, G, or 8-oxoguanine) in 10 mM Tris·HCl, pH 8, 1 mM EDTA, 100 mM NaCl by heating to 90 °C for 5 min and slow cooling to room temperature. For competition and inhibition assays, 10 pmol of PYR- or THF-containing or unmodified oligonucleotides were annealed to 10 pmol of unmodified complementary DNA.

                              
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Table I
Duplex oligonucleotides used in this study

Gel-shift Assays

General Procedures-- The standard binding reaction mixture (20 µl) contained 25 mM Hepes·KOH, pH 7.8, 0.5 mM EDTA, 0.5 mM dithiothreitol, 5% glycerol, 150 mM NaCl (for AlkA and ANPG) or 100 mM NaCl (for Tag, Endo III, Fpg, MutY, Ung, and TDG) and various amounts of protein and 32P-labeled oligonucleotide. The mixture was incubated for 30 min at room temperature and an aliquot analyzed by electrophoresis on a 10% nondenaturing polyacrylamide gel (37.5:1 acrylamide/bisacrylamide) using 1/2 × TBE buffer (0.045 M Tris borate, pH 8, 1 mM EDTA) at 150 V constant voltage for 2 h at room temperature. After drying, the gels were exposed to film and to a Fuji image plate for quantification of the bands on a phosphorimager.

Determination of Amount of Active Protein Molecules in Our Preparations-- The exact concentration of active enzyme molecules was determined by titrating the protein with the analog-containing DNA under stochiometric binding conditions ([DNA] = 10 nM >>  Kd).

Competition Assays-- For competition assays 10 fmol of 32P-labeled oligonucleotide were used together with 1000 fmol of unlabeled PYR-T, PYR-G, THF-T, THF-G, or A-T duplex (Table I). The protein (50 fmol) was added to the mixture last.

Determination of the Thermodynamic Binding Constant Kd-- Under conditions in which [DNA] < Kd, the concentration of the protein that affords 50% formation of a protein-DNA complex is approximately equal to Kd. The Kd = [protein][DNA]/[protein-DNA] was measured as the concentration of the protein at which half of the target DNA is bound. To keep the DNA concentration below Kd, 0.1 fmol of labeled oligonucleotide was used in a total reaction volume of 50 µl with varying amounts of protein (oligonucleotide concentration = 2 pM). The data from three to six titration gels were averaged to obtain the reported Kd values.

Assay of AlkA/Tag/ANPG Activity-- The reaction mixture (50 µl) contained 5 µg of [3H]dimethyl sulfate-treated calf thymus DNA, 25 mM Hepes·KOH, pH 7.8, 0.5 mM dithiothreitol, 0.5 mM EDTA, and various amounts of protein and the PYR-T or A-T duplex. The reaction mixture was incubated at 37 °C for 30 min and quenched by addition of 50 µl of a stop solution containing 1 mg/ml salmon sperm DNA, 0.2 M NaCl, and 1 mg/ml bovine serum albumin. The DNA was precipitated with 100% ethanol (200 µl) at -70 °C for 30 min and centrifuged at 4 °C for 30 min. 200 µl of the supernatant were mixed with 5 ml of scintillation fluid and the amount of released alkylated bases measured by liquid scintillation counting.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Experimental Design-- The interaction of DNA glycosylases with the PYR- and THF-containing duplexes was studied using the electrophoretic mobility shift assay (EMSA). The specificity of the interaction of the PYR- and THF-containing duplexes and the DNA glycosylases was determined by competition with nonspecific and specific competitors; the dissociation constants (Kd) were determined by titrating under equilibrium conditions ([DNA] <<  Kd). In some cases, we additionally used inhibition assays to determine the ability of PYR-containing duplexes to inhibit the reaction normally catalyzed by DNA glycosylases. The "Results" section is divided into three parts, according to types of damage specificities (repair of alkylated, oxidized, or deaminated bases) of the DNA glycosylases tested. The interaction between the inhibitors used in this study with a fourth class of DNA glycosylases acting on UV damage has been reported separately (15).

DNA Glycosylases That Act on Alkylated DNA-- We examined the interaction of three DNA glycosylases that act on alkylated DNA, Tag, and AlkA from E. coli and the human enzyme ANPG, with the pyrrolidine-containing oligonucleotides. All three of these enzymes are monofunctional DNA glycosylases, but differ in their substrate specificities and share no significant amino acid sequence similarity. Tag has a restricted substrate specificity, acting efficiently only on 3-methyladenine (28). AlkA, by contrast, has a relatively broad substrate specificity for adducts containing aberrantly placed methyl groups in both the major groove (7-meG) and the minor groove (3-meA, 3-meG, O2-meT, O2-meC) of DNA (29, 30). Additional substrates that are repaired at much lower rate include 5-formyluracil and 5-hydoxymethyluracil (31), N2,3-ethenoguanine (32), 1,N6-ethenoadenine (21), and hypoxanthine (33). The substrates for the mammalian DNA glycosylase ANPG include 3-methyladenine (34), 7-methylguanine (34), 1,N6-ethenoadenine and other cyclic base adducts (21, 35), hypoxanthine (33), and 8-oxoguanine (36). In contrast to AlkA, ANPG repairs uncharged lesions such as hypoxanthine and 1,N6-ethenoadenine with similar efficiency as positively charged alkylation adducts.

The interaction of the pyrrolidine-containing DNA with AlkA, ANPG, and Tag was analyzed using the EMSA (37), which detects the difference in mobility between protein-bound and unbound DNA. Titration of a 5'-32P-end-labeled duplex having PYR opposite T on the complementary strand (PYR-T) with increasing concentrations of AlkA and ANPG resulted in the appearance of a band with a reduced mobility characteristic of a protein-DNA complex (Fig. 2, lanes 2 and 6). This band was completely abolished in the presence of a 100-fold excess of unlabeled PYR-T duplex (Fig. 2, lanes 3 and 7), but was resistant to competition by a 100-fold excess of an unmodified duplex 25-mer (Fig. 2, lanes 4 and 8). These results demonstrate that the two proteins specifically recognize the presence of the pyrrolidine moiety in DNA. Further EMSA experiments under Kd conditions ([DNA] <<  Kd) revealed that the dissociation constants for AlkA and ANPG with the PYR-T duplex were 16 pM (±3 pM) and 23 pM (±4 pM), respectively (Table II). In contrast to the specific interaction between the PYR-T duplex with AlkA and ANPG, the complex formed between Tag and the PYR-T duplex was not stable under EMSA conditions. We did observe a band indicative of a protein-DNA complex, but were not able to observe more than 30% of the probe bound to the protein (data not shown). Variation of salt concentrations, pH, and addition of bovine serum albumin did not stabilize this interaction.


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Fig. 2.   EMSA assay to detect specific binding of pyrrolidine-containing DNA to AlkA and ANPG (lanes 1-4 and 5-8, respectively). Concentration of 32P-end-labeled PYR-T duplex: 0.2 nM; protein concentrations: lanes 1 and 5, no protein; lanes 2-4 and 6-8, 0.6 nM; concentration of unlabeled competitor in lanes 3 and 7 (PYR-T duplex) and 4 and 8 (A-T duplex): 20 nM (100-fold molar excess over labeled substrate).

                              
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Table II
Summary of dissociation constants (Kd) of DNA glycosylases bound to oligonucleotides containing pyrrolidine and tetrahydrofuran residues

As a test for our design strategy, we analyzed in parallel the binding of these DNA glycosylases to a singly modified 25-mer duplex containing a (uncharged) tetrahydrofuran analog (THF-T duplex) in the place of the pyrrolidine. In contrast to the exceedingly tight binding of AlkA to the charged pyrrolidine, the uncharged tetrahydrofuran exhibited little specific binding to AlkA (Fig. 3A); the Kd was measured to be 45 nM (±6 nM) (Table II), showing roughly a 3000-fold decrease in Kd (equivalent to 4.7 kcal/mol). Thus, AlkA exhibits exquisite selectivity for the presence of a positively charged NH2 group in the inhibitor. ANPG, on the other hand, shows less than 10-fold greater affinity for PYR-T as compared with THF-T (Fig. 3B, Table II).


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Fig. 3.   A, qualitative EMSA assay to detect binding of pyrrolidine-containing DNA (lanes 1-6) and tetrahydrofuran-containing DNA (lanes 7-12) to various concentrations of AlkA. Oligonucleotide concentration: 0.1 nM. AlkA concentrations are indicated. B, EMSA assay to detect binding of pyrrolidine-containing DNA (lanes 1-6) and tetrahydrofuran-containing DNA (lanes 7-12) to various concentrations of ANPG. Oligonucleotide concentration: 0.05 nM; ANPG concentrations are indicated.

The ability of PYR-containing DNA to inhibit glycosidic bond cleavage of normal substrates by AlkA, ANPG, and Tag was tested by measuring the enzyme-catalyzed release of methylated bases from [3H]dimethyl sulfate-treated calf thymus DNA in the presence of PYR-T and A-T duplex. Addition of increasing amounts of the PYR-T duplex resulted in almost complete inhibition the release of 3H-methylated bases from DNA by all three enzymes (Fig. 4). Addition of the same amount of the A-T duplex had little or no effect on the enzyme catalyzed reaction. Thus, duplex DNA containing a single pyrrolidine residue clearly functions as a specific inhibitor of AlkA, ANPG, and Tag. These results demonstrate PYR-containing DNA interacts specifically with Tag, despite the fact that the complex formed between these two molecules is unstable to gel electrophoresis conditions.


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Fig. 4.   Inhibition of the catalytic activity of AlkA (A), ANPG (B), and Tag (C) by pyrrolidine-containing DNA (PYR-T duplex) and unmodified DNA (A-T duplex). The activity is measured as the release of methylated bases from [3H]dimethyl sulfate-treated calf thymus DNA (specific activity: 785 cpm/pmol). Protein concentration: AlkA, 200 nM; Tag, 190 nM; ANPG, 160 nM. Inhibitor concentration: 0-500 nM.

Studies on DNA Glycosylases That Act on Oxidized Bases-- We next studied the interaction of three DNA glycosylases from E. coli that act on oxidized bases (endonuclease III, Fpg, and MutY) with PYR- and THF-containing DNA. Endo III and Fpg are bifunctional DNA glycosylases/AP lyases, while MutY appears to function primarily as a monofunctional glycosylase (4, 38). The alkylation-repair enzymes examined above are all monofunctional glycosylases, and it was of interest to see whether there exists any consistent difference in the interaction of mono- and bifuncitonal DNA glycosylases with the PYR- and THF-containing duplexes. Endo III excises a wide variety of oxidized pyrimidines (23). Fpg (also called MutM) and MutY from part of an intricate system in E. coli to prevent the mutagenic effect of 8-oxoguanine (oG) residues in DNA (39). Fpg efficiently removes oG from DNA, if it is paired to C, T, or G, but not if it is paired to A (40, 41). MutY excises A when mispaired with oG or G. Biochemical studies indicating that MutY favors oG·A over G·A pairs (42, 43) are consistent with genetic evidence that the oG·A pair is the more relevant MutY substrate in vivo (44).

Taking into account the substrate specificities of the enzymes, we used the PYR-T and THF-T duplexes to study the interaction with Fpg and the PYR-G and THF-G duplexes to study the interaction with Endo III and MutY. All three enzymes bound readily to the PYR- and THF-containing duplexes. This interaction was resistant to competition by a 100-fold excess of unlabeled unmodified 25-mer duplex, but susceptible to competition of 100-fold excess of unlabeled PYR-T or PYR-G duplex (Fig. 5). These results demonstrate that the interaction of all three proteins is specific for the presence of the pyrrolidine in DNA. We carried out further EMSA experiments under Kd conditions to determine the binding constants of Fpg, Endo III, and MutY to PYR- and THF-containing oligonucleotides (Table II). In addition, for MutY we determined the binding constant of an 25-mer duplex oligonucleotide containing a PYR or a THF residue opposite an oG (PYR-oG duplex and THF-oG duplex, respectively). Fpg bound the PYR-T about 8-fold more tightly than the THF-T duplex (Kd = 350 and 2600 pM, respectively). For Endo III, on the other hand, the difference in Kd of the PYR- and THF-containing DNA is 200 fold (Kd = 42 pM and 7800 pM, respectively). This high specificity for the positive charge in the pyrrolidine is reminiscent of the specificity of inhibitor binding observed for AlkA.


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Fig. 5.   EMSA assay to detect specific binding of pyrrolidine-containing duplex DNA to Fpg (lanes 1-4), MutY (lanes 5-8), and endonuclease III (lanes 9-12). Concentration of 32P-end-labeled PYR-T duplex: 0.2 nM; protein concentrations: lane 1, 5, and 9, no protein; lanes 2-4, 6-8, and 10-12, 0.6 nM; concentration of unlabeled competitor in lanes 3, 7, 11 (PYR-T duplex) and 4, 8, 12 (A-T duplex): 20 nM (100-fold molar excess over labeled substrate). The mobility difference observed in the gels for the three enzymes is reflective of their molecular weight (MutY, 39 kDa; Fpg, 31 kDa; Endo III, 27 kDa).

Endo III and AlkA are structurally homologous (19, 45, 46), and the similarity of inhibitor binding of the two proteins further supports the similarity of the active sites of the two proteins. MutY is also homologous to endo III, and one might therefore expect that MutY would interact with pyrrolidine- and tetrahydrofuran-containing DNA in a similar way as Endo III. The dissociation constants determined for MutY and are 90 pM for the PYR-G duplex, 320 pM for the THF-G duplex, 65 pM for the PYR-oG duplex, and 45 pM for THF-oG duplex. Thus, unlike Endo III, MutY does not discriminate strongly for the presence of the positive charge in the pyrrolidine. The lower dissociation constants for the PYR and THF opposite oG are consistent with the notion that the principal role of MutY is to excise adenine residues that are mispaired to 8-oxoguanine (42-44).

Studies on DNA Glycosylases That Act on Deaminated Bases-- We also investigated the interaction of pyrrolidine- and tetrahydrofuran-containing oligonucleotides with two DNA glycosylases that act on deaminated bases. Uracil DNA glycosylase is conserved throughout evolution and excises uracil bases from both double-stranded and single-stranded DNA (47). The mammalian mismatch-specific thymidine DNA glycosylase (TDG) catalyzes the removal of T and U bases from DNA if they are mispaired with G residues (48, 49). Both enzymes are monofunctional DNA glycosylases.

Under the conditions used, we failed to observe any interaction between uracil DNA glycosylase and both the PYR-G and THF-G duplex, even at enzyme concentrations at higher than 1 µM (data not shown). By contrast, TDG stably bound both the PYR-G and THF-G duplex in an EMSA assay (Fig. 6). The interaction of both duplexes with TDG was abolished by competition with a 100-fold excess of unlabeled PYR-G and THF-G duplex, respectively, but was resistant to competition with a 100-fold excess of (A-T) duplex. The Kd of the PYR-G to TDG was determined to be 16 pM, the Kd of the THF-G duplex to TDG 23 pM (Table II). These data indicate TDG does not exhibit any significant specificity for the presence of the positive charge in the pyrrolidine.


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Fig. 6.   EMSA assay to detect specific binding of pyrrolidine- and tetrahydrofuran-containing DNA to TDG (lanes 1-4 and 5-8, respectively). Concentration of 32P-end-labeled PYR-T and THF-T duplex: 0.2 nM; protein concentrations: lanes 1 and 5, no protein; lanes 2-4 and 6-8, 0.6 nM; concentration of unlabeled competitor in lanes 3 and 7 (PYR-T duplex) and 4 and 8 (A-T duplex): 20 nM (100-fold molar excess over labeled substrate).

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

An important objective of the DNA repair field is to understand how DNA repair enzymes achieve selectivity in recognition and processing of aberrant bases amid the vast excess of normal genetic material. An especially powerful way to gain insight into these issues involves high resolution structural analysis of DNA repair proteins bound to their substrates. However, the short lifetime of such enzyme-substrate complexes ordinarily renders them inaccessible to structural analysis. It has proven possible to overcome this problem through modification of either the enzyme (9-11) or the substrate (12-15, 43, 50, 51) so as to stall the normal reaction process and thereby generate long-lived enzyme-DNA complexes. In the present study, we have used the pyrrolidine (PYR) moiety as a mimic for the positive charge developed in the transition state of the glycosidic bond cleavage reaction catalyzed by DNA glycosylases (18). We probed the interactions of this PYR-containing DNA with the DNA glycosylases AlkA, ANPG, Tag, Fpg, MutY, Endo III, Ung, and TDG, and previously, with endonuclease V (15). We found that DNA containing a PYR residue interacts specifically with all the DNA glycosylases tested, with the exception of uracil DNA glycosylase. These results thus establish the pyrrolidine as a general inhibitor for DNA glycosylases and demonstrate the potential utility of the substrate modification approach for future x-ray crystallographic studies. Indeed, diffraction quality crystals of AlkA and ANPG bound to pyrrolidine-based inhibitors have been obtained.2 High resolution structural studies of DNA glycosylase-inhibitor complexes are certain to provide valuable insights into the molecular basis for substrate recognition and catalysis by these essential enzymes.

The studies described here have shown that some DNA glycosylases (AlkA, Endo III) show exquisite specificity for the positively charged pyrrolidine, while others (ANPG, Fpg, MutY, TDG, and Endo V) bind to the uncharged tetrahydrofuran moiety with almost equal affinity. In the case of AlkA, biochemical and x-ray crystallographic studies have provided insight into the structural basis for specificity in the interaction of the enzyme with the PYR moiety in DNA. Namely, mutation of the negatively charged Asp238 located next to the substrate binding pocket to the neutral Asn (D238N) resulted in a loss of the glycosylase activity of AlkA (19, 46). Moreover, whereas the wild-type enzyme was found to discriminate strongly for the positively charged PYR inhibitor over the neutral THF, the D238N AlkA mutant enzyme bound PYR- and THF-containing DNA with almost equal affinity. These data strongly suggest that the positively charged PYR nitrogen interacts directly with Asp238 in the enzyme active site. It has since been recognized that the residue corresponding to Asp238 is absolutely conserved in a superfamily of DNA glycosylases and indeed is the C-terminal residue of a helix-hairpin-helix-Gly/Pro-rich loop-Asp (HhH-GPD) motif, which is the defining characteristic of superfamily members (22). Given these findings, it is curious that other members of the HhH-GPD superfamily (such as Endo III, MutY, and perhaps even ANPG), all of which possess an active site residue corresponding to Asp238 in AlkA, do not show the same degree of discrimination as AlkA for the positively charged PYR over the neutral THF moiety in DNA.

Inhibitors of DNA glycosylases have many additional uses aside from structural studies. First, affinity chromatography on a column containing double-stranded DNA with a reduced abasic site has been used in the recent isolation and cloning of the Saccharomyces cerevisiae 8-oxoguanine DNA glycosylase OGG1 (22). Similarly, affinity purification or expression screening using DNA containing a pyrrolidine could be used for the same purpose. Second, several lesions can be repaired by multiple DNA repair pathways (2, 52, 53). A general and specific inhibitor of a given DNA repair pathway could therefore be used to investigate the relative contribution of a pathway in the processing of any given lesion. Finally, DNA glycosylases are able to excise DNA lesions generated by various antitumor agents such as the N-nitrosoureas and ionizing radiation, thereby counteracting the action of these agents (54-56). Inhibitors of DNA glycosylases might therefore be used to increase the cell-killing efficiency of certain antitumor therapeutics. The development and employment of more specific and efficient inhibitors of DNA glycosylases are ongoing in our laboratories (57).

    ACKNOWLEDGEMENTS

We thank Dr. Stephen Lloyd and Dr. Dale Mosbough for generously providing the MutY and Ung enzymes, respectively. We acknowledge Dr. Tom Ellenberger for helpful discussions and Dr. Roland Kanaar for a critical reading of the manuscript.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant 51330 (to G. L. V.) and grants from the Association pour la Recherche sur le Cancer and European Economic Community (to J. L.).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.

Dagger Present address: Dept. of Cell Biology and Genetics, Erasmus University, P. O. Box 1738, 3000 DR Rotterdam, The Netherlands.

par To whom correspondence should be addressed. Tel.: 617-495-5333; Fax: 617-495-8755; E-mail: verdine{at}chemistry.harvard.edu.

1 The abbreviations used are: AP, abasic site; PYR, pyrrolidine residue in DNA; THF, tetrahydrofuran residue in DNA; Endo III, endonuclease III; oG, 8-oxoguanine; EMSA, electrophoretic mobility shift assay; ANPG, alkyl N-purine glycosylase; TDG, thymine DNA glycosylase.

2 A. Lau and T. Ellenberger, personal communication.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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