Incision Activity of Human Apurinic Endonuclease (Ape) at Abasic Site Analogs in DNA

doi:10.1074/jbc.270.27.16002

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Volume 270, Number 27, Issue of July 07, pp. 16002-16007, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.

Incision Activity of Human Apurinic Endonuclease (Ape) at Abasic Site Analogs in DNA (*)

David M. Wilson III , Masaru Takeshita (1), Arthur P. Grollman (1), Bruce Demple (§)

From the (1)Harvard University School of Public Health, Department of Molecular and Cellular Toxicology, Boston, Massachusetts 02115 and the State University of New York, Department of Pharmacological Sciences, Stony Brook, New York 11794

ABSTRACT

INTRODUCTION

EXPERIMENTAL PROCEDURES

RESULTS

ABSTRACT

The major apurinic/apyrimidinic (AP) endonuclease of human cells, the Ape protein, incises DNA adjacent to abasic sites to initiate DNA repair and counteract the cytotoxic and mutagenic effects of AP sites. Here we address the determinants of Ape AP endonuclease activity using duplex DNA substrates that contain synthetic analogs of AP sites: tetrahydrofuranyl (F), propanediol (P), ethanediol (E), or 2-(aminobutyl)-1,3-propanediol (Q). The last of these, a branched abasic structure, was a poor substrate for which Ape had k > 1000-fold lower than for F. In contrast, the specificity constant (k/K) for E or P of Ape purified from HeLa cells was only 5-8-fold lower than for F. Positioning a phosphorothioate ester immediately 5` to F inhibited Ape incision activity 20-fold (Rp isomer) or > 10,000-fold (Sp isomer). Although Ape did not have detectable endonuclease activity toward single-stranded substrates or unmodified double-stranded DNA, the enzyme displayed a low level of 3`-exonuclease activity for duplex DNA (<0.03% of its AP endonuclease activity), which was influenced by the reaction conditions. The base positioned opposite F did not dramatically affect the cleavage efficiency of Ape, but an F:F arrangement was cleaved at approximately one-third of the efficiency of F:C. A 3`-mismatch diminished P and E cleavage only slightly and F not at all. A 5`-mismatch reduced the Ape cleavage rate 4-10-fold for F and 100-fold for P and E. A series of substrates with F at different positions along the oligonucleotide showed that Ape requires 4 base pairs 5` to the abasic site and 3 base pairs on the 3`-side. The implications of these results for substrate recognition by Ape are discussed.

INTRODUCTION

Apurinic/apyrimidinic (AP)()sites in DNA are formed by a variety of mechanisms that include spontaneous or chemically-induced hydrolysis of the N-glycosylic bond and enzymatic release of modified bases by DNA glycosylases (Loeb and Preston, 1986; Wallace, 1988; Lindahl, 1993; Demple and Harrison, 1994). It has been estimated that 10,000 AP sites are generated per mammalian cell/day by purine-glycosyl bond hydrolysis under normal physiological conditions (Lindahl and Nyberg, 1972); other sources of AP sites add to this burden. Abasic sites are potentially lethal and mutagenic (Loeb and Preston, 1986). To defend against these consequences, cells possess ``class II'' (hydrolytic) AP endonucleases that cleave on the 5`-side of AP sites to produce 3`-OH groups and 5`-deoxyribose 5-phosphate residues (Demple and Harrison, 1994). Release of the 5`-abasic residue and DNA repair synthesis and ligation complete the repair process (Demple and Harrison, 1994).

A characteristic form of oxidative DNA damage is free radical-initiated breaks bearing 3`-phosphates or 3`-phosphoglycolate esters (Giloni et al., 1981; Henner et al., 1983; Demple et al., 1986). These oxidative damages, along with the 3`-deoxyribose products of DNA glycosylase-associated ``class I'' AP endonuclease (-lyase) activities (Demple and Harrison, 1994), are refractory to DNA polymerases. The ``class II'' AP endonucleases also have 3`-repair diesterase activity specific for this collection of lesions (Demple and Harrison, 1994).

Two families of ``class II'' AP endonuclease/3`-repair diesterase enzymes have been identified. Endonuclease IV of Escherichia coli and Apn1 protein of the yeast Saccharomyces cerevisiae are homologous nucleases (Popoff et al., 1990) that perform demonstrable roles in correcting oxidative or alkylation damages in vivo (Cunningham et al., 1986; Ramotar et al., 1991). Yeast lacking Apn1 display a substantially increased spontaneous mutation rate (Ramotar et al., 1991; Kunz et al., 1994), which underscores the role of AP endonucleases in preventing mutagenesis due to DNA damage caused by by-products of normal metabolism.

The second family of AP endonuclease/3`-repair diesterases includes E. coli exonuclease III and the predominant human AP endonuclease called Ape protein ((Demple et al., 1991); also called Hap1 (Robson and Hickson, 1991), Apex (Seki et al., 1992), or Ref1 (Xanthoudakis et al., 1992)). Exonuclease III has a clear in vivo repair function in E. coli (Demple et al., 1986; Cunningham et al., 1986). Expression of the human AP endonuclease in E. coli can substitute for exonuclease III in repair of alkylation-induced AP sites, but evidently not efficiently for repair of oxidative damage (Demple et al., 1991; Robson and Hickson, 1991; Seki et al., 1992). This pattern is consistent with the in vitro properties of Ape, which has a relatively low 3`-repair activity (present at 1/200 of its AP endonuclease activity; Chen et al., 1991). Thus, other human enzymes may be involved in removal of 3` damages (Chen et al., 1991; Winters et al., 1992). On the other hand, Ape displays a powerful hydrolytic AP endonuclease activity about 10-fold greater than found for exonuclease III (Kane and Linn, 1981; Chen et al., 1991). A detailed understanding of the requirements of the Ape enzyme for substrate recognition and cleavage is clearly desirable. We have approached this goal by determining the activity of purified human AP endonuclease on a variety of synthetic oligonucleotide substrates containing AP site analogs.

EXPERIMENTAL PROCEDURES

Materials

The oligonucleotides employed in these studies were synthesized as described previously (Takeuchi et al., 1994) except for the oligonucleotide containing 2-(aminobutyl)-1,3-propanediol residue (Q), which was purchased from Operon Technologies, Inc. (Emeryville, CA). Structures of abasic sites are shown in Fig. 1, and the sequences of the oligodeoxynucleotides are displayed in . Bacteriophage T4 polynucleotide kinase was purchased from New England Biolabs (Beverly, MA). The [

P]ATP (specific activity, 3000 Ci/mmol) and [

H]dTTP (specific activity, 70-90 Ci/mmol) were obtained from DuPont NEN. Exonuclease III was purchased from Life Technologies, Inc. DNase I was obtained from Sigma.

Figure 1: Structures of natural and synthetic abasic sites. The equilibrium between the ring (AP, right) and open-chain (AP, left) forms of deoxyribose favors the former. The other symbols are shown to the right of each moiety.

HeLa AP Endonuclease

Human Ape protein was purified from HeLa S3 cell extracts essentially as described by Chen et al.(1991), except that the Affi-Gel blue step was omitted. Ape enzyme activity was detected during the purification by monitoring the release of radiolabeled 3`-phosphoglycolaldehyde residues from a synthetic DNA substrate (Chen et al., 1991). The purified Ape protein used here had an AP endonuclease activity of 5.3

units/mg of protein, determined using a synthetic substrate (Levin and Demple, 1990). One unit of enzyme cleaves or releases 1 pmol of damaged sites/min under standard conditions (Levin and Demple, 1991; Chen et al., 1991).

Purification of Recombinant Ape Proteins

Two plasmids were constructed to express Ape protein for purification from E. coli. pDW204 was generated by inserting a polymerase chain reaction-generated fragment containing the Ape coding region into the EcoRI and BamHI restriction sites of the pKEN2 vector (a gift from Dr. G. L. Verdine). Its expression in lacI

strains is inducible by isopropyl-1-thio-

-D-galactopyranoside under the control of a tac promoter. pGEX-Ape (graciously provided by Drs. Mark R. Kelley and James P. Carney) was constructed by inserting the Ape coding sequence into a BamHI- and EcoRI-digested pGEX-3X (Pharmacia Biotech Inc.) behind the tac promoter and in frame with the glutathione S-transferase gene.

Intact Ape protein was prepared as follows. Overnight cultures of DW5281 (xth nfo-1::kan derivative of strain XA90) containing pDW204 were diluted 1:100 into LB broth and grown for 2 h at 37 °C, and isopropyl-1-thio--D-galactopyranoside was added to a final concentration of 1 mM. After 4 h of shaking at 37 °C, the cells were harvested by centrifugation and resuspended in 50 mM Hepes-KOH, pH 7.5, 125 mM KCl, 10% glycerol. The cells were lysed by agitation with glass beads in a Mini-Bead-Beater (Biospec Products, Bartlesville, OK). After centrifugation at 12,000 g, the soluble material was applied to a column of phosphocellulose (Whatman P-11) equilibrated with 50 mM Hepes-KOH, pH 7.5, 125 mM KCl, 10% glycerol. The column was washed with several column volumes of equilibration buffer, and the bound material was then eluted with a linear gradient of KCl (125-600 mM). Fractions containing full-length Ape protein (determined by immunoblotting; Demple et al.(1991)) were pooled and concentrated using a Centriprep 30 apparatus (Amicon, Inc., Beverly, MA). The phosphocellulose pool was further fractionated on an AcA54 (Pharmacia) gel filtration column equilibrated with 50 mM Hepes-KOH, pH 7.5, 150 mM KCl, 10% glycerol. Fractions were analyzed for AP endonuclease activity and protein concentration (determined by Coomassie Blue staining), and the active fractions were pooled to yield ``recombinant Ape.''

For purification of glutathione S-transferase-Ape fusion protein, BL21 cells containing pGEX-Ape construct were induced with isopropyl-1-thio--D-galactopyranoside as described for DW5281/pDW204. After collection and resuspension, cells were lysed by sonication, and Triton X-100 was added to a final concentration of 1%. Following centrifugation for 10 min at 12,000 g, the supernatant was applied to a glutathione-Sepharose 4B (Pharmacia) column equilibrated with 50 mM Hepes-KOH, pH 7.5, 150 mM KCl, 10% glycerol, 0.1 mM dithiothreitol. The column was washed with equilibration buffer containing 1% Triton X-100, Ape-glutathione S-transferase fusion protein was eluted with 10 mM reduced glutathione (Sigma), and the active fractions were pooled. To release Ape from the hybrid protein, samples were incubated with CaCl (6 mM final concentration) and Factor Xa (0.5 units; Pierce, Rockford, IL), and the cleavage was performed overnight at 4 °C. The ``clipped'' protein was applied to a small phosphocellulose column as described above and eluted with buffer containing 400 mM KCl.

Preparation of Double-stranded DNA Substrates

Oligonucleotides containing abasic sites were 5`-end-labeled by treatment with T4 polynucleotide kinase and [

P]ATP (Sambrook et al., 1989). The DNA strand to be labeled was always in molar excess over the ATP. The kinase was heat-inactivated by incubation at 90 °C for 2 min; the reaction was placed immediately on ice, and an equimolar amount of the complementary oligonucleotide was added. This mixture was heated at 56 °C for 10 min, and the two DNA strands were annealed, first at room temperature for 1 h and then at 4 °C overnight.

Enzymatic Reactions

Reactions to measure incision activity of Ape were performed in 10 µl of 50 mM Hepes-KOH, pH 7.5, 50 mM KCl, 100 µg/ml bovine serum albumin, 10 mM MgCl

, 0.05% Triton X-100 (Chen et al., 1991) containing the indicated amounts of double-stranded DNA substrate. After incubation at 37 °C with various amounts of Ape protein for the indicated times, the reactions were stopped by placing them on ice and immediately adding 5 µl of formamide solution (Sambrook et al., 1989). The samples were then heated at 65 °C for 5 min, and 3-µl aliquots were analyzed on 20% polyacrylamide, 7 M urea gels run at 20 V/cm. After electrophoresis, the gels were autoradiographed to locate the labeled substrate and product oligodeoxynucleotides. The conversion of substrate to product was calculated by excising the substrate and product bands, counting the radioactivity in the gel slices in a Beckman LS 1801 scintillation counter, and calculating fractional conversion (fractional conversion = cpm in product/(cpm in product + cpm in substrate)).

Enzymatic reactions utilizing endonuclease IV (AP endonuclease activity of 80,000 units/mg; Levin et al.(1988)) or exonuclease III (22,000 units/mg; Life Technologies, Inc.) with MgCl were performed as described by Takeuchi et al.(1994).

Exonuclease Activity of Ape

To specifically measure exonuclease activity of Ape, two methods were employed. For the first method, the procedure described by Seki et al.(1991), using 3`-end-labeled copolymers, was implemented. These experiments were performed with various Ape protein preparations. In the second method, poly(dA-dT) duplex DNA containing a

P-radiolabeled dUMP approximately every 600 nucleotides (Levin et al., 1991) was treated with DNase I (see figure legends for concentration) for 2 min at room temperature in Ape AP endonuclease buffer (see ``Enzymatic Reactions'' of this section). DNase I was subsequently inactivated by incubating for 5 min at 65 °C, and the reaction was placed on ice. Approximately 1 pmol of the resulting DNA substrate was then incubated with recombinant Ape produced in bacteria or exonuclease III for 30 min at 37 °C. The DNA polymer was precipitated on ice for 10 min by the addition of trichloroacetic acid to a final concentration of 0.65 M. After centrifugation at 12,000

g, the free radiolabeled nucleotides in the acid-soluble supernatant were quantified by liquid scintillation counting. Immunoprecipitation of Ape protein with rabbit antiserum and protein A-Sepharose beads (Sigma) was performed essentially as described by Chen et al.(1991), except that the primary and secondary incubations were shortened to 2 and 1 h, respectively.

RESULTS

Incision Kinetics of Ape on Synthetic AP Site Analogs

Oligonucleotides containing synthetic AP sites (ethanediol (E), propanediol (P), or furanyl (F) moieties) were 5`-end-labeled and annealed to the unlabeled complementary strand with deoxycytosine positioned opposite the abasic site (sequences 1, 2, or 3 annealed to sequence 7; see ). Ape incision activity on the resulting labeled 18-bp, double-stranded DNA substrates was estimated by calculating the percent conversion of substrate (18 bp) to product (9 bp) after separation by denaturing gel electrophoresis (see ``Experimental Procedures''). Time course experiments revealed that Ape catalyzed incision of the three substrates at nearly equal rates (Fig. 2; see below). The kinetic parameters for these reactions were determined by varying substrate concentrations. These studies revealed a <2.5-fold variation in the turnover number (k

), while the specificity constants (k

) for these abasic sites varied only

8-fold (F > P > E), primarily due to >10-fold range for apparent K

(). None of these sites was cleaved in single-stranded DNA, and a 10-molar excess of unlabeled AP site containing single-stranded DNA did not alter Ape incision rates for duplex DNA substrates (data not shown).

Figure 2: Incision products of Ape on duplex oligodeoxynucleotides containing E, P, or F. Substrates (200 nM) were incubated with Ape (100 pg/ml) under standard reaction conditions for the time indicated, and the reaction products were analyzed on a 7 M urea, 20% polyacrylamide gel (see ``Experimental Procedures''). Uncleaved, single-stranded substrate migrates as an 18-mer, while the incision products migrate as 9-mers.

We also explored the action of Ape on a commercially available oligonucleotide substrate containing a branched abasic site, a 2-(aminobutyl)-1,3-propanediol residue (Q, sequence 4 in ; Fig. 1). In order to detect Ape-mediated incision at Q, a concentration of enzyme was required that was >1000-fold higher than needed for F (Fig. 3, lanes10 and 11). Similarly, concentrations of E. coli exonuclease III that converted nearly all of the F-containing substrate to product demonstrated very little endonucleolytic activity on Q, although some exonuclease activity was observed (Fig. 3, lanes5 and 6). A 10-fold higher amount of exonuclease III did not dramatically increase the amount of product generated following incubation with Q (Fig. 3, lane7), but incision of Q by E. coli endonuclease IV occurred at a rate only

8-fold slower than for F (Fig. 3, lanes3 and 4versuslane2).

Figure 3: Activity of AP endonucleases on the branched Q substrate. Duplex DNA substrates containing either F or Q (100 nM) were incubated for 5 min with endonuclease IV, exonuclease III, or Ape under standard reaction conditions in 10 µl and analyzed by gel electrophoresis (see ``Experimental Procedures''). Lane1, Q, no enzyme; lane2, F, 1 ng of endonuclease IV; lane3, Q, 1 ng of endonuclease IV; lane4, Q, 10 ng of endonuclease IV; lane5, F, 1 ng of exonuclease III; lane6, Q, 1 ng of exonuclease III; lane7, Q, 10 ng of exonuclease III; lane8, Q, 1 ng of endonuclease IV; lane9, Q with no enzyme; lane10, Q, 1 ng of Ape; lane11, Q, 10 ng of Ape. In lanes2-4 and 8 the upper bands are due to residual substrate.

Exonuclease Activity for Ape

In previous studies of Ape protein isolated from HeLa cells, we were unable to detect significant 3`-exonuclease activity using an end-labeled, blunt-ended duplex DNA substrate (Chen et al., 1991). However, it seemed possible that the exonuclease activity reported for this protein by others (Seki et al., 1991, 1992, 1993) could depend on the substrate or reaction conditions. We therefore adopted the approach of Seki et al.(1991) and investigated exonuclease activity of Ape purified from HeLa cells and of recombinant protein isolated by two different procedures from E. coli, either as intact Ape or as the Ape segment liberated from a fusion with glutathione S-transferase (see ``Experimental Procedures''). These experiments revealed a low but consistent amount of exonuclease activity for all three forms of Ape (I). This activity depended on the assay buffer, and was observed at levels 1.5-7-fold higher in the reaction buffer of Seki et al. (1991) than in our standard conditions.

The possibility that the exonuclease detected in our experiments might be due to a contaminant was addressed in immunological studies. A polyclonal rabbit antiserum generated against highly purified Ape from HeLa cells (Chen et al., 1991) immunoprecipitated the exonuclease activity associated with both HeLa-derived Ape protein and recombinant Ape isolated from E. coli (Fig. 4A). It is very unlikely that cross-reacting material other than Ape protein would be present in both of these preparations. Thus, the weak 3`-exonuclease is probably an activity of Ape itself.

Figure 4: 3`-Exonuclease activity of Ape protein. A, immunoprecipitation of Ape-specific exonuclease. Ape purified from HeLa cells or the recombinant protein isolated from E. coli (expression vector pDW204; see text) was incubated with the indicated amount of Ape-specific rabbit antiserum (Chen et al., 1991) followed by the addition of protein A-Sepharose beads, further incubation, and removal of the beads by centrifugation (see ``Experimental Procedures''). The resulting supernatants were then assayed for 3`-exonuclease activity by the method of Seki et al. (1991). Addition of >1 µl of serum was precluded by the presence of exonuclease activity in the serum itself. B, alternative exonuclease assay. Poly(dA-dT) containing one [

P]dUMP residue/

600 nucleotides was treated with 0.1 ng (upperthreebars) or 1 ng (lowerthreebars) of DNase I (see ``Experimental Procedures''). This DNA was then incubated with no protein, 3 AP endonuclease units of recombinant Ape protein, or 4 AP endonuclease units of exonuclease III. Following precipitation with trichloroacetic acid, radioactivity in the supernatant was determined by scintillation counting.

An alternative approach with a different substrate revealed no significant exonuclease activity for recombinant Ape protein under our standard reaction conditions (Fig. 4B). Consistent with the exonuclease constituting a minor activity of Ape, the expected sizes of substrate and product were observed in assays of Ape with oligonucleotide substrates (see Fig. 2, 3, and 6).

Inhibition by Phosphorothioate

Since the phosphodiester cleaved by Ape is positioned immediately 5` to the abasic site (Chen et al., 1991), we examined the effects on Ape activity of substituting this phosphate with a phosphorothioate residue (sequences 5 and 6 in ; Fig. 1). Ape actively incised a duplex with an Rp phosphorothioate at a rate of approximately one-twentieth of that seen for an F substrate with a conventional phosphodiester (Fig. 5). Cleavage of the corresponding Sp isomer was not detected (Fig. 5); the calculated cleavage rate of Ape for the Sp isomer was <10

of that found for F. Inhibition by 5`-phosphorothioates is consistent with this site as the target for hydrolysis by Ape.

Figure 5: Activity of Ape on phosphorothioate substrates. Results show the incision rates of the phosphorothioate substrates (Rp

F or Sp

F) compared with those for E, P, or F containing normal phosphodiesters (see Fig. 2). Ape was present at a concentration of 0.1 ng/ml (E, F, P) or 1 ng/ml (Rp

F, Sp

F), and the reactions were performed as described (see ``Experimental Procedures'').

Influence of the Local DNA Structure on Ape Incision Rates

The overall requirements of Ape for duplex DNA structure around the cleavage target site were explored using a series of substrates containing different bases in the opposing strand (sequences 12-18 in ) or base mismatches located in different positions (sequences 1-3 and 8-11 in ), or by varying the position of the abasic site along the duplex. The identity of the base opposite an F residue did not significantly influence the rate of cleavage by Ape (). However, a substrate containing a F:F pair was incised at only approximately one-third of the rate of substrates with a normal base in the opposite strand (). We have not determined to what extent this reduction is due to cleavage of the unlabeled strand by Ape before the enzyme interacts with the labeled strand. Preliminary experiments

(

)indicate that such cleavage might be inhibitory.

A mismatch placed immediately 5` to an F moiety reduced the rate of Ape cleavage by 4-fold compared with F in a fully base-paired context (). An adjacent 5`-mismatch had an even more pronounced effect on the E or P sites, reducing the Ape incision activity 100-fold (). In contrast, a base mismatch on the 3`-side had only marginal effects on the cleavage rate for any of the substrates. Mismatches on both the 5`- and the 3`-sides had an even greater inhibitory effect on the Ape endonuclease activity than a 5`-mismatch alone, with the relative effect of the dual mismatch particularly pronounced in the case of the F moiety (). Thus, Ape seems to have a stronger requirement for duplex DNA structure 5` to the abasic site than it does for the 3`-side.

Activity of Ape was also analyzed using a series of 18-bp duplex substrates with F placed at 1-17 bases from the 5` terminus. Incision by Ape was detected only when F was flanked by at least 4 base pairs 5` and 3 base pairs 3` to the lesion (Fig. 6). Cleavage by Ape on the substrate with F at position 15 (with 3 base pairs on the 3`-side) was significantly diminished compared with the substrate with F at position 14 (Fig. 6). Analysis of these reactions using a DNA sequencing gel (Sambrook et al., 1989) confirmed that little, if any, cleavage was occurring with F at positions 1-4 or 16 and 17 (data not shown).

Figure 6: Effect of the position of F within a duplex oligodeoxynucleotide on the incision activity of Ape. Sequences where F was placed at positions 1-17 (as indicated above each lane) throughout an 18-bp duplex DNA were incubated with Ape (100 pg/ml) under standard reaction conditions and analyzed by gel electrophoresis (see ``Experimental Procedures'').

DISCUSSION

The mode of substrate recognition by AP endonucleases has been the subject of discussion for nearly 2 decades, initially in an effort to account for the apparently diverse activities of bacterial enzymes such as E. coli exonuclease III (Weiss, 1976). Despite their limited exonuclease activity against undamaged DNA, the eukaryotic class II AP endonucleases hydrolyze a broad array of substrates ranging from intact AP sites to 3`-terminal nucleotide fragments to 3`-phosphates (Demple and Harrison, 1994). The recognition mechanism(s) of this class of enzymes must, therefore, accommodate different types of DNA damage, seemingly in all sequence contexts, while ignoring the structural fluctuations of normal DNA. The studies presented here represent the first systematic effort to define key elements of substrate recognition and incision by Ape, the main AP endonuclease of human cells.

The substrate profile for Ape was similar to that found for E. coli exonuclease III in the presence of Ca (Takeuchi et al., 1994). The relatively robust activity of these enzymes against E (ethanediol derivative) contrasts with the profile for E. coli endonuclease IV, for which E is a poor substrate (Takeshita et al., 1987; Takeuchi et al. 1994). In a more limited study, the bovine AP endonuclease homologous to exonuclease III (Robson et al., 1991) acted relatively efficiently on E (Sanderson et al., 1989). The opposite preference obtains for the branched Q substrate, which is utilized poorly by Ape and exonuclease III (Ca) but effectively by endonuclease IV. These preferences may be summarized as follows: the Ape/exonuclease III class prefers abasic sites without branches at carbon 4 (equivalent to carbon 2 in Q; Fig. 1), but will tolerate distortions of phosphodiester spacing implicit in the structure of E.

A detailed assessment of the structural requirements for Ape acting on abasic sites is warranted, since different types of abasic sites may be formed under different circumstances. In addition to AP sites formed by acid- or glycosylase-catalyzed base hydrolysis (Lindahl and Nyberg, 1974; Wallace, 1988; Demple and Harrison, 1994), free radical agents such as x-rays (von Sonntag, 1987) or bleomycin (Steighner and Povirk, 1990) form modified abasic sites containing oxidized forms of deoxyribose. Enzymes such as the Ape protein may have to cope with these lesions as the products of metabolic and environmental mutagens. Indeed, recent work has shown that exonuclease III and endonuclease IV of E. coli, which represent the two families of ``class II'' AP endonucleases (Demple and Harrison 1994), distinguish strongly between abasic sites oxidized at the 1 or the 4 position (Häring et al., 1994). It remains to be determined how Ape protein acts on these modified sites.

The duplex DNA context is of importance for Ape, as it is for exonuclease III (Takeuchi et al., 1994). As suggested by Weiss(1976), the double-helical structure on the 5`-side of the abasic site is much more important than that on the 3`-side. Thus, Ape protein requires at least four base pairs 5` to the lesion, but no more than three base pairs on the 3`-side. Ape and exonuclease III may be able to cleave duplex substrates as short as eight base pairs, although neither enzyme attacks abasic sites in single-stranded DNA.

Consistent with the evident requirement that AP endonucleases act on damages produced anywhere in the genome, the nature of the nucleotide opposite the abasic site is essentially irrelevant. An exception was the case where F was opposed by another F (abasic) site. Although we have not directly addressed the possibility that this poor activity is due to prior cleavage of the unlabeled DNA strand, the fact that the extent of substrate cleavage in these assays was always 20% argues against this interpretation. The poor activity of Ape on such a bistranded substrate may underlie the biological effectiveness of antitumor drugs such as bleomycin, which generate bistranded lesions in abundance (Steighner and Povirk, 1990).

One key point addressed here was the 3`-exonuclease activity reported for mammalian AP endonucleases (Seki et al., 1991, 1992, 1993). Several other groups (Nes, 1980; Kane and Linn, 1981; Sanderson et al., 1989; Chen et al., 1991; Winters et al., 1994) failed to detect such an activity, but employed different substrates and different reaction conditions. We made an effort here to reproduce the assay of Seki et al.(1991) and were able to detect a very low level of exonuclease activity that was stimulated when using their conditions. This activity was sensitive to the nature of the reaction buffer and was more pronounced under conditions of high osmolarity (0.25 M sucrose). It also appears that the detailed nature of the duplex DNA substrate affects this activity. Since the exonuclease was detected both for native Ape purified from HeLa cells and for recombinant protein isolated by two different procedures from bacteria, it seems unlikely to have arisen from a contaminant common to all these situations; this conclusion was bolstered by immunological experiments. The very weak intrinsic exonuclease of Ape (4 orders of magnitude lower than the AP endonuclease) may instead reflect the homology of this protein to more potent exonucleases from E. coli and Drosophila (Sander et al., 1993). Although the biological relevance of such a weak activity is not obvious, it could arise either from a small amount of modified (truncated?) Ape protein or from some minor component of the heterogeneous poly(dA-dT) substrate. A more puissant activity toward a more specialized DNA structure could be significant biologically.

The powerful effects of a 5`-phosphorothioate on cleavage by the Ape protein merit comment. The simplest interpretation of this result is that this phosphate is attacked by the enzyme during cleavage (Demple and Harrison, 1994). The inhibitory effect of such a substitution is consistent with the homology between Ape protein and exonuclease III (Eckstein, 1985). More noteworthy is the stereospecific effect of the Sp isomer, which might suggest a stereospecific attack by Ape. Structural studies of these proteins are required to address this and other fundamental issues. The recent report (Mol et al., 1995) of a crystal structure for exonuclease III suggests that such information may soon be at hand.

Table: List of oligodeoxynucleotides

Dashes represent the identical sequence as for oligonucleotide 1 (sequences 2-11), oligonucleotide 12 (sequences 13 and 14), or oligonucleotide 14 (sequences 15-18). Sequences for oligonucleotides 5 and 6 are identical to the sequence 1, except for the centrally located 5`-phosphorothioate F analogs, denoted Rp and Sp. Underlined letters indicate either the AP site analog or the base positioned opposite the AP site.

Table: Kinetic parameters of Ape for duplex DNA substrates containing synthetic AP sites

Reactions containing 8-400 nM duplex DNA substrate were performed under standard conditions (see ``Experimental Procedures'') for 5 min at 37 °C. Ape (35,500 Da) was present at a final concentration of 100 pg/ml (2.8 pM). Values of K and k were determined from Lineweaver-Burk plots.

Table: Exonuclease activities of native and recombinant Ape proteins

3`-Exonuclease activity was determined using a 3`-[H]dTMP-labeled poly (dA-dT) substrate prepared as described by Seki et al. (1991) but using either the reaction buffer of Chen et al. (1991) as standard conditions or that of Seki et al. (1991) as modified conditions. AP endonuclease activity was assayed according to Levin and Demple (1990) as modified by Chen et al. (1991). The isolation of recombinant Ape proteins is described under ``Experimental Procedures.'' AP endo, AP endonuclease activity; 3`-exo, 3`-exonuclease activity.

Table: Effect of the base positioned opposite F on endonuclease activity

Enzymatic activity was determined as described under ``Experimental Procedures'' with Ape present at final concentrations of 0.1 and 1 ng/ml and with Ape activity within the linear range. Results were calculated based on at least four independent measurements and are expressed as rates normalized to the F:C arrangement; means and standard deviations are shown.

Table: Effects of flanking mismatches on endonuclease activity

FOOTNOTES

*: This work was supported by Grants GM40000 (to B. D.) and CA17395 and CA47995 (to A. P. G.) from the National Institutes of Health and by National Research Service Award 1 F32 CA62845-01 (to D. M. W.) from the United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§: To whom correspondence should be addressed. E-mail: demple@mbcrr.harvard.edu.
: The abbreviations used are: AP, apurinic/apyrimidinic; F, tetrahydrofuranyl; P, propanediol; E, ethanediol; Q, 2-(aminobutyl)-1,3-propanediol; Sp and Rp, the phosphorothioate diastereomers; bp, base pair(s).
: D. M. Wilson III and B. Demple, unpublished data.

ACKNOWLEDGEMENTS

We thank David M. Wilson (no relation) for assistance with some of the experiments, Dr. Elena Hidalgo for helpful advice during the purification of recombinant Ape, Dr. Richard A. O. Bennett for input on the manuscript, the Cell Culture Center (Minneapolis, MN) and Dr. Lynn Harrison for providing HeLa S3 cells, Dr. Gregory Verdine (Harvard University) for pKEN2 and the XA90 strain, Drs. Mark R. Kelley and James P. Carney (Indiana University Medical School) for the pGEX-Ape construct, and Dr. Bernard Weiss (University of Michigan) for supplying the dCTP deaminase used during substrate preparation.

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