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J. Biol. Chem., Vol. 278, Issue 38, 36242-36249, September 19, 2003

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Modulation of the 3'5'-Exonuclease Activity of Human Apurinic Endonuclease (Ape1) by Its 5'-incised Abasic DNA Product^*

Donny Wong , Michael S. DeMott  and Bruce Demple 

From the Department of Cancer Cell Biology, Harvard School of Public Health, Boston, Massachusetts 02115

Received for publication, June 9, 2003

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The major abasic endonuclease of human cells, Ape1 protein, is a multifunctional enzyme with critical roles in base excision repair (BER) of DNA. In addition to its primary activity as an apurinic/apyrimidinic endonuclease in BER, Ape1 also possesses 3'-phosphodiesterase, 3'-phosphatase, and 3'5'-exonuclease functions specific for the 3' termini of internal nicks and gaps in DNA. The exonuclease activity is enhanced at 3' mismatches, which suggests a possible role in BER for Ape1 as a proofreading activity for the relatively inaccurate DNA polymerase . To elucidate this role more precisely, we investigated the ability of Ape1 to degrade DNA substrates that mimic BER intermediates. We found that the Ape1 exonuclease is active at both mismatched and correctly matched 3' termini, with preference for mismatches. In our hands, the exonuclease activity of Ape1 was more active at one-nucleotide gaps than at nicks in DNA, even though the latter should represent the product of repair synthesis by polymerase . However, the exonuclease activity was inhibited by the presence of nearby 5'-incised abasic residues, which result from the apurinic/apyrimidinic endonuclease activity of Ape1. The same was true for the recently described exonuclease activity of Escherichia coli endonuclease IV. Exonuclease III, the E. coli homolog of Ape1, did not discriminate among the different substrates. Removal of the 5' abasic residue by polymerase alleviated the inhibition of the Ape1 exonuclease activity. These results suggest roles for the Ape1 exonuclease during BER after both DNA repair synthesis and excision of the abasic deoxyribose-5-phosphate by polymerase .

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The formation of apurinic/apyrimidinic (AP)¹ sites in DNA is the most common consequence of exposure of cells to DNA-damaging agents of both endogenous and environmental origin (1). AP sites are formed as repair intermediates by DNA glycosylases, which remove certain mismatched bases or base lesions formed by reactive oxygen species, alkylating agents, or other environmental insults. AP sites can also form spontaneously via acid-catalyzed hydrolysis of the N-glycosylic bonds linking the bases to the sugar-phosphate backbone of DNA. Such spontaneous depurination forms an estimated 10,000 AP sites per day in each human cell (2), and the activity of DNA glycosylases would certainly add to this burden. Indeed, the steady-state level of AP lesions is estimated in some studies to be much higher, approaching 50,000 or more per cell depending on its age and tissue source (3, 4).

AP sites in mammalian cells are repaired by the DNA base excision repair (BER) pathway. The major human AP endonuclease, Ape1 (also called Apex, HAP1, or Ref-1), initiates BER by hydrolyzing the 5'-phosphodiester bond of the AP site to create a DNA repair intermediate that has a single strand break bracketed by 3'-hydroxyl and 5'-deoxyribose-5-phosphate (dRP) termini. Ape1 interacts with DNA polymerase (pol) during BER to recruit the polymerase to the incised AP site (5), where the polymerase catalyzes both nucleotide insertion and dRP excision. The sequence of these latter two activities is still unclear, but all the combined enzymatic activities of Ape1 and pol acting at an AP site would yield a nick that can be sealed by DNA ligase to complete the repair (6, 7).

Ape1 is a multifunctional protein with proposed diverse roles in the cell. In addition to its DNA repair activities, Ape1 can activate transcription factors via a redox mechanism (8), form a transcriptional repressor complex for the negative calcium responsive element (9–11), and act as an important target for the granzyme A-mediated cell death pathway (12). In addition to its major role as an AP endonuclease during BER, Ape1 also possesses a weak 3'-phosphatase activity and a 3'-phosphodiesterase activity against abasic residues or fragments (13). These activities are required for the removal of 3'-blocking groups created by ionizing radiation, oxygen free radicals, radiomimetic anti-tumor drugs, and the 3'-AP lyase activities of bifunctional DNA glycosylases (1, 14). Ape1 also has a 3'5'-exonuclease that excises undamaged DNA nucleotides (see below). Knocking out both alleles coding for Ape1 in mice (the APEX gene) results in early embryonic lethality (15–17), which point to critical roles for Ape1. However, it has not yet been established which Ape1 activities are required for survival. Although Ape1 protein levels have been dramatically reduced for short periods in cells in culture using small interfering RNA (12), stable cell lines lacking the protein have not been reported.

The modest 3'5'-exonuclease activity of the mouse and human Ape1 proteins has been known for some time (18, 19), but the biological role of the exonuclease remained obscure. In contrast, the robust AP endonuclease activity of Ape1 is related to the demonstrated roles in BER of exonuclease III in Escherichia coli or Apn1 in yeast (1, 13). Recently, it was shown that Ape1 acting as an exonuclease can remove therapeutic anti-tumor nucleoside analogs incorporated at the 3' ends of DNA oligonucleotides, which suggested that Ape1 contributes to cellular resistance to these drugs (20). This work was further extended to show that the exonuclease activity of Ape1 is enhanced on DNA mispairs at the 3' termini of nicks and gaps (21). Because the primary mammalian BER polymerase, pol , has relatively low fidelity and lacks an associated proofreading exonuclease (22, 23), the Ape1 exonuclease could provide the "missing" proofreading activity during BER (24). To explore a possible role for the exonuclease activity of Ape1 during BER, we investigated the ability of the enzyme to excise nucleotides at the 3' termini of different BER intermediates.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Reagents and Enzyme—Urea was purchased from American Bioanalytical Corp. (Natick, MA). An acrylamide-bisacrylamide solution (40%, 29:1 ratio) was purchased from Bio-Rad, Inc. (Hercules, CA). E. coli exonuclease III, uracil DNA glycosylase, and T4 polynucleotide kinase were obtained from New England Biolabs (Beverly, MA). One unit of exonuclease III is defined by the manufacturer as the amount of enzyme required to release 1 nmol of nucleotides in 30 min. E. coli endonuclease IV was purified as previously described (25). Recombinant wild-type human Ape1 and the D283A/D308A mutant form were purified as previously described (26). The R177A mutant form of Ape1 was kindly provided by Drs. Tadahide Izumi and Sankar Mitra of the University of Texas Medical Branch (Galveston, TX). Recombinant wild-type human pol was a generous gift from Drs. Rajendra Prasad and Sam Wilson, NIEHS, National Institutes of Health (Research Triangle Park, NC).

DNA Substrates—Oligonucleotides were synthesized and high-performance liquid chromatography-purified by Operon Technologies, Inc. (Alameda, CA) or Midland Certified Reagent Co., Inc. (Midland, TX). The sequences of DNA oligonucleotides used in this study are shown in Table I. Upstream strands were 5'-end-labeled by T4 polynucleotide kinase using a molar excess of [-³²P]ATP (3000 Ci/mmol, PerkinElmer Life Sciences, Boston, MA). Double-stranded DNA substrates were made by heating the radioactively labeled oligonucleotides with the appropriate template and downstream oligonucleotides to 90 °C and then annealing by slow cooling to room temperature. Unincorporated [-³²P]ATP was removed using Micro Biospin P-30 columns (Bio-Rad) following the manufacturer's protocols. DNA substrate purity was monitored by autoradiography following electrophoresis on 8% non-denaturing gels and 12% denaturing polyacrylamide gels containing 7 M urea. The different substrates used in this study are listed in Table II along with the structural components of each substrate. Substrates S2, S4, S6, and S8 were incised with a catalytic amount of Ape1 just prior to use in exonuclease assays. Substrates S22 and S23 were sequentially treated with catalytic amounts of uracil DNA glycosylase and Ape1 to create nicked and gapped DNA with 5'-dRP termini. These substrates were also used immediately following pretreatment due to the labile nature of the 5'-dRP.

Exonuclease Assays—Exonuclease reactions were performed in BER buffer (27). Briefly, standard reactions contained 50 mM Hepes-KOH (pH 7.5), 8 mM MgCl₂, 5% (v/v) glycerol, 0.5 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, 10 nM DNA substrate, and enzyme concentrations as indicated in the figure legends. After incubation at 30 °C, the reactions were terminated at the indicated times by the addition of formamide loading buffer (90% formamide, 10 mM EDTA, bromphenol blue, and xylene cyanol) and heating at 100 °C for 2 min. The DNA products were then resolved by electrophoresis on acrylamide (14%) gels containing 7 M urea. After drying, the gels were analyzed using a Molecular Imager System (Model GS-525, Bio-Rad), and the results were quantified using Molecular Analyst software (Bio-Rad). The fraction of substrate degraded by the Ape1 exonuclease was calculated as previously described (28). Briefly, the percent exonuclease activity of each reaction was calculated using Equation 1,

(Eq. 1)

where N represents the amount of the 21-mer DNA substrate, N - 1 is the amount of the 20-mer exonuclease product, N - 2 is the amount of the 19-mer exonuclease product, and so on.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Comparison between Human Ape1 and E. coli Exonuclease III for Excision of 3' Mismatches on BER Intermediates—Previous reports on the exonuclease activity of Ape1 focused on its activity at 3' mismatched nucleotides at gaps and nicks in DNA (21, 29, 30), but the activity of Ape1 on BER intermediates with incised abasic residues was not investigated. We used DNA containing the synthetic AP analog tetrahydrofuran (F) to create a set of DNA substrates that simulate these base excision repair intermediates (Fig. 1A). Tetrahydrofuran residues are resistant to cleavage by -elimination reactions (31) but are cleaved by Ape1 with the same catalytic efficiency as regular AP sites (19). After 5' incision by Ape1, F residues serve as stable structural analogs of the 5'-dRP moiety that is produced following 5' incision of natural AP sites. We found that the Ape1 exonuclease is most active on 3' A/G mismatches adjacent to a single-nucleotide gap. However, the exonuclease was significantly less active at mismatches positioned at nicks or at gaps bearing 5'-incised F residues (Fig. 1, B and D).

View larger version (50K): [in this window] [in a new window]   FIG. 1. Substrate specificity of Ape1 exonuclease compared with E. coli exonuclease III. A, schematic diagram of oligonucleotide substrates (see Table II for detailed sequences). Asterisks and carets denote the locations of the 32P label and internal 3'-OH termini, respectively. B, Ape1 exonuclease activity. Standard enzyme reactions contained 10 nM DNA with increasing concentrations of Ape1 are denoted by the wedges (no enzyme, 0.5, 1.5, 5, 15, or 50 nM). The enzyme incubations were 10 min. The intact DNA substrates are indicated by an arrow. C, exonuclease III activity on various structures. The reactions contained increasing amounts of exonuclease III, as denoted by the wedges (no enzyme, 0.0001, 0.001, 0.01, 0.1, or 1 unit). The arrow indicates the intact DNA substrates. D, quantification of Ape1 exonuclease activity. Experiments as shown in panel B were quantitated in a PhosphorImager. The bars indicate the average fraction of each DNA substrate degraded in 10 min. Error bars indicate the S.E. (n = 3).

Exonuclease III is the E. coli homolog of Ape1 and shares many of the same DNA repair activities (13). In addition to being an AP endonuclease, exonuclease III also possesses potent 3'-phosphodiesterase and 3'-phosphatase activities and a well characterized and robust 3'5'-exonuclease (32, 33). Exonuclease III shares structural homology with Ape1 reflected in 28% amino acid sequence identity, including highly conserved catalytic residues (29, 34). To determine if exonuclease III activity was inhibited similarly to the Ape1 exonuclease by the presence of 5'-incised F residues at nicks and gaps, we assayed the ability of exonuclease III to degrade these same four DNA substrates across a range of enzyme concentrations. Exonuclease III exhibited only a small difference in activity between gaps and nicks (Fig. 1C, panels 1 and 3). The bacterial enzyme was not inhibited at all by the presence of the incised dRP analog (Fig. 1C, panels 2 and 4). Thus, the inhibition of the 3'5'-exonuclease activity of Ape1 is a feature specific to the mammalian enzyme.

Ape1 Exonuclease Activity for Correctly Matched 3' Termini Is Inhibited by the Presence of Incised Abasic Residue—Previous experiments indicated that Ape1 is generally inefficient at excising non-mismatched nucleotides (19, 21, 35) but that the enzyme may also be more active at excising some properly paired nucleotides than certain mismatched pairs (29). The difference in these reports may depend on the overall sequence context of the DNA substrate, which can affect the exonuclease activity by up to two orders of magnitude (29). Our experiments utilized a well characterized 51-mer oligonucleotide substrate that has long been used by several groups for routine BER assays (26, 36). In the sequences used here (Fig. 2A), kinetic analysis showed that Ape1 was able to digest a properly matched A/T pair (Fig. 2B), albeit at a reduced efficiency compared with an A/G mismatch. On the 3' mismatch, the same enzyme concentration resulted in digestion of most of the mismatched substrate even at the earliest time point (Fig. 2C). A comparison of estimated initial rates from exonuclease assays using a reduced amount of Ape1 indicated that the exonuclease activity is at least 3-fold more active on an A/G mismatch than on an A/T pair in this sequence context. Whereas previous studies found no difference in the exonuclease activity at nicks or gaps (21, 29), we found that Ape1 was at least 3-fold more active at gaps than at nicks in the sequence context used here, whether or not a 3' mismatch was present (Fig. 2). Furthermore, these assays showed that Ape1 was inhibited at least 3-fold by the presence of incised F residues at both nicks and single-nucleotide gaps, consistent with the data shown in Fig. 1.

View larger version (20K): [in this window] [in a new window]   FIG. 2. Inhibition of Ape1 exonuclease activity by a 5'-terminal abasic site analog, tetrahydrofuran. A, schematic diagrams of fully base-paired (S5, S6, S7, and S8) and 3'-A/G mismatched (S1, S2, S3, and S4) substrates (see Table II for details). The asterisks and carets denote the locations of the 32P label and internal 3'-OH termini, respectively. B, exonuclease activity of Ape1 on fully base-paired DNA substrates. The reactions contained 10 nM DNA and 25 nM Ape1. The fraction of total substrate converted to product at each time point is shown. Error bars indicate the S.E. for each data point (n = 3). C, Ape1 exonuclease activity on substrates with internal 3'-A/G mismatches. The reactions were performed and analyzed as described for panel B, except that some reactions with substrate S1 contained only 5 nM Ape1 (open squares).

Separate Inhibitory Effects of an Abasic Residue and a 5'-Phosphate on Ape1 Exonuclease Activity—We next wanted to explore in greater detail the role of different structural elements at the 5'-end of nicks and gaps that might affect the Ape1 3'5'-exonuclease activity. Panel A and the tops of each box of panel C in Fig. 3 illustrate schematically the various DNA structures that were analyzed. The presence of a 5'-phosphate at a single-nucleotide gap did not affect the exonuclease activity (Fig. 3B). However, the presence of a 5'-phosphate at a nick inhibited the exonuclease activity 8-fold compared with a 5'-hydroxyl (Fig. 3B).

View larger version (36K): [in this window] [in a new window]   FIG. 3. Inhibition of Ape1 exonuclease by 5'-phosphate at nicked DNA. A, schematic diagram of oligonucleotide substrates (see Table II for details). The asterisks and carets denote the locations of 32P label and internal 3'-OH termini, respectively. The p denotes the location of an unlabeled 5'-phosphate. B, effect of downstream 5'-phosphate on Ape1 exonuclease activity at single-nucleotide gaps and nicks. The reactions contained 10 nM DNA and 25 nM Ape1. Error bars indicate the S.E. for each data point (n = 3). C, Ape1 exonuclease activity on nicked and single-nucleotide-gapped substrates. A diagram of each substrate is shown above its respective gel image (see Table II for details). The asterisks and carets denote the locations of the 32P label and internal 3'-OH termini, respectively. The p denotes the location of an unlabeled 5'-phosphate. The arrows indicate the migration of the intact DNA substrate. The reactions were performed as described for panels B and C. The wedges indicate a time course (0, 5, 10, or 15 min).

We also assayed the Ape1 exonuclease activity on substrates with termini bearing 5'-unphosphorylated deoxyribose moieties or 5'-mismatched nucleotides, as well as substrates lacking a downstream strand to determine the relative inhibitory contribution made by the structural elements at the 5' terminus of each substrate (Fig. 3C). In the sequence context of our DNA substrate, differences between single-nucleotide gaps and nicks were observed only when the 5'-phosphates were present. Consistent with data shown in Figs. 1 and 2, the Ape1 exonuclease was relatively inefficient at both nicks and single-nucleotide gaps when an abasic residue containing an adjacent phosphate group was present at the 5' terminus (Fig. 3C, panels a and e). The Ape1 exonuclease activity increased only slightly when the 5'-phosphate was removed to yield a 5'-terminal abasic residue (Fig. 3C, panels b and f). However, substitution of the abasic residue with a guanine nucleotide to create a 5' G/G mismatch increased the 3'5'-exonuclease activity of Ape1 (Fig. 3C, panels c and g). DNA lacking a downstream double-stranded structure was a very poor substrate for the Ape1 exonuclease activity (Fig. 3C, panel d). This observation is consistent with structural studies, DNase footprinting analysis, and enzymatic data demonstrating that Ape1 requires at least three base pairs of duplex DNA both upstream and downstream of its active site for maximal AP endonuclease activity (37–39).

Ape1 and E. coli Endonuclease IV Are Both Inhibited by 5'-Incised Abasic DNA at Nicks and Gaps—To verify that the inhibitory effect of 5'-dRP on the 3'5'-exonuclease activity of Ape1 is a general effect rather than a sequence-specific feature of our DNA substrates, we assayed the Ape1 exonuclease activity on substrates whose sequences resemble some other previously characterized substrates (tops of each panel in Fig. 4A (29)). In this sequence context, which contained a 3'-A/C mismatch at nicks and gaps, Ape1 was slightly more active at the nicks than at the single-nucleotide gaps (Fig. 4A, compare panels a–c with d–f; Fig. 4B, compare panel a to b). Ape1 continued to be inhibited at nicks and gaps bearing 5'-incised F residues (Fig. 4A, panels c and f; Fig. 4B, panels a and b), although to a slightly lesser extent than previously observed (3-fold instead of 4-fold as shown in Fig. 2). Consistent with experiments shown in Fig. 3, we observed that Ape1 exonuclease was more active at nicked DNA when the 5'-phosphate was removed (Fig. 4B, panel b). However, we also observed the same increase in exonuclease activity at gapped substrates lacking the 5'-phosphate (Fig. 4B, panel a).

View larger version (60K): [in this window] [in a new window]   FIG. 4. Comparison of Ape1 and E. coli endonuclease IV exonuclease activities at A/C mismatches in different DNA structures. A, schematic diagrams of the substrates used in this experiment are shown in each panel (see Table II for details). The asterisks and carets denote the locations of the 32P label and internal 3'-OH termini, respectively. The reactions contained 10 nM DNA substrate and either 0.2, 1, or 5 nM Ape1 (open wedges) or 0.2, 1, or 5 nM E. coli endonuclease IV (filled wedges). Control lanes without added enzyme are indicated (solid lines). B, quantification of enzyme titrations. Panel a, activity of Ape1 on gapped substrates. Panel b, activity of Ape1 on nick substrates. Panel c, activity of endonuclease IV on gap substrates. Panel d, activity of endonuclease IV on nick substrates.

A second AP endonuclease from E. coli, endonuclease IV, was also recently demonstrated to possess an intrinsic 3'5'-exonuclease activity (40). Although structurally unrelated to Ape1, endonuclease IV also has AP endonuclease, 3'-phosphodiesterase, and 3'-phosphatase activities. Endonuclease IV is inducible as part of the oxidative stress-activated SoxRS regulon of E. coli (41), and the enzyme plays a specialized role in repairing oxidatively damaged DNA (42). To determine whether endonuclease IV is also inhibited by the presence of a 5' abasic residue, which can arise from oxidatively damaged AP sites (1), we assessed the ability of the endonuclease IV exonuclease to degrade these same substrates bearing 5'-phosphates or F residues. Endonuclease IV was nearly 2-fold more active than Ape1 as an exonuclease on gapped substrates lacking a 5'-phosphate or bearing 5' F (Fig. 4A, compare filled wedges representing endonuclease IV to open wedges representing Ape1; Fig. 4B, compare panels a and b to panels c and d). Furthermore, endonuclease IV was also inhibited at nicks and gaps bearing 5' F residues, relative to the same substrates lacking F. This effect was smaller than that observed for Ape1. Interestingly, endonuclease IV was somewhat more active on nicks and gaps when the 5'-phosphate was present than when it was removed (Fig. 4B, panels c and d), the reverse of the effect seen for Ape1.

Excision of 5'-dRP by pol Activates Ape1 Exonuclease at an Incised Natural AP Site—Using substrates with the same DNA sequence as those characterized in Fig. 4, we next wanted to assess the Ape1 exonuclease activity at a hydrolytic AP site following incision by Ape1, and to determine whether the addition of pol could stimulate the exonuclease activity. Substrates bearing a single-nucleotide gap or a nick, each containing a 5'-dRP adjacent to a 3' A/C mismatch, were generated by treatment of DNA containing a single dUMP residue with uracil-DNA glycosylase to create an AP site. This treatment was followed by incision with a catalytic amount of Ape1 (Ape1: DNA ratio 1:40). Addition of pol to the Ape1 exonuclease reactions resulted in stimulation of the exonuclease activity on nicks with 5'-dRP, but not at single-nucleotide gaps with 5'-dRP (Fig. 5). Because dRP excision by pol has been proposed to be the rate-limiting step of BER (43), deoxyribonucleoside monophosphate misincorporation by pol prior to dRP excision would result in a mismatch adjacent to a nick, displacing the dRP by one nucleotide. The stimulation of the Ape1 exonuclease activity is likely due to the dRP lyase activity of pol and not protein-protein interactions, because pol was unable to stimulate Ape1 on substrates containing preincised F residues, which are resistant to -elimination by pol (data not shown). The excision of 5'-dRP by pol on DNA containing mismatches has not been reported, but our results suggest that pol favors excision of the displaced 5'-dRP when a 3'-mismatch is present.

View larger version (25K): [in this window] [in a new window]   FIG. 5. A, activation of Ape1 exonuclease activity by pol dRP lyase. Each 25-µl reaction contained 20 nM DNA substrate, 5 nM Ape1, and where indicated, 10 nM pol . B, the fraction of total substrate converted to product was plotted for each time point. Error bars indicate the S.E. for each data point (n = 3).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The 3'5'-exonuclease activity of the murine (18) and human (19) Ape1 proteins was identified some time ago, but only recently characterized in detail. The apparent specificity of this exonuclease activity for 3'-mismatched DNA at internal nicks and gaps suggested a role for Ape1 as a proofreading exonuclease for DNA polymerase (21), which lacks its own intrinsic 3'5'-exonuclease activity (23). In E. coli, the primary repair polymerase is DNA polymerase I, which possesses its own 3'5'-exonuclease activity and thus does not need to rely on other proteins for proofreading of polymerization errors. Although other proteins with 3'5'-exonuclease activity are present in mammalian cells (44), the role of Ape1 in initiating the repair of abasic sites, demonstrated physical interaction with pol , and ability to stimulate 5'-dRP excision by pol (5) strongly implicate Ape1 in proofreading for pol during BER.

The specific activity of the Ape1 exonuclease varies rather widely among different studies (21, 29, 35). Some variation is expected due to differences in the activity or purity of recombinant enzymes prepared by each group. The Ape1 exonuclease activity can also differ by as much as two orders of magnitude depending on the sequence of the DNA substrate (21, 29). However, exonuclease III is expressed at high levels in E. coli and may constitute a highly active exonuclease contaminant that co-purifies with Ape1. To verify that the 3'5'-exonuclease activity is intrinsic to Ape1 and not due to the contaminating activity of another nuclease, we assayed two independently purified preparations of histidine-tagged Ape1 (12, 45) in addition to the untagged wild-type Ape1 used in this study. The two histidine-tagged Ape1 proteins were found to possess similar levels of exonuclease activity as the wild-type enzyme (data not shown), and like the wild-type Ape1, the exonuclease activities of both histidine-tagged Ape1 proteins were also inhibited on DNA containing a 5'-incised abasic residue (data not shown). This inhibition of Ape1 exonuclease contrasts with the lack of such inhibition for purified exonuclease III. Furthermore, the AP endonuclease-defective D283A/D308A mutant form of Ape1, which was purified in the same manner as the wild-type Ape1 used in this study (26), did not possess measurable exonuclease activity (data not shown), a further indication that contaminating bacterial exonucleases are not responsible for the observed activity. Finally, the pattern of exonuclease products formed by Ape1 in our assays is distinct from that produced by exonuclease III. Exonuclease III is much more active than Ape1 as a 3'5'-exonuclease and even small concentrations of exonuclease III can sequentially degrade DNA to produce multiple products (Fig. 1C). Consistent with results published by Hadi et al. (29), the Ape1 used in this study excised primarily one or two nucleotides. Digestion of DNA with high concentrations of Ape1 or over long periods of incubation did not result in the excision of more than two nucleotides from the original single-nucleotide gap or nick substrates.

The relationship between the exonuclease function of Ape1 and the product of its AP endonuclease activity, 5'-dRP, is intriguing in view of the high affinity of Ape1 for incised AP sites (26). Mammalian DNA BER is coordinated by a series of protein-protein and protein-DNA interactions that facilitate hand-off of DNA repair intermediates from one protein to the next (46). Tight binding of BER proteins to their products may protect the repair intermediates from the cellular milieu to limit DNA degradation or the activation of deleterious recombination pathways.

We demonstrate here that the exonuclease activity of Ape1 is inhibited by 5'-dRP at nicks and gaps and enhanced when the 5'-dRP is removed by pol . While this report was in preparation, another report (58) independently demonstrated the inhibitory effect of a 5'-incised tetrahydrofuran residue. This suggests a possible regulatory mechanism whereby excision of 5'-dRP coordinates the exonuclease activity of Ape1 during BER. Inhibition of the exonuclease activity of Ape1 by the 5'-dRP residues (that result from incision at AP sites) would protect the undamaged DNA 5' of the AP site from degradation prior to nucleotide incorporation by pol . This protective mechanism may be biologically significant given that the cellular levels of Ape1 can far surpass the concentration of pol (47, 48). The report, that the majority of AP sites detected in cells is already 5'-incised, supports this concept (4). Furthermore, if dRP excision is rate-limiting during BER (43), then deoxyribonucleoside monophosphate incorporation by pol would usually precede dRP excision. Temporally placing the Ape1 exonuclease activity after dRP excision may then ensure that the proofreading exonuclease is activated only after DNA repair synthesis has occurred. The intrinsic preference of the exonuclease for mismatches would further limit the role of Ape1 to error correction.

Crystal structures of Ape1 in complex with duplex DNA containing the abasic site analog tetrahydrofuran indicate a contact between the side chain of the arginine 177 and the 5'-phosphate at the abasic incision site (38). Arg-177 is absent from exonuclease III and appears to be conserved only in the Ape1 homologs of higher eukaryotes. Because the Ape1 exonuclease, but not exonuclease III, exhibited specificity for different DNA substrates, we hypothesized that a protein-DNA contact involving Arg-177 might be responsible for the observed inhibition of the Ape1 exonuclease on DNA containing 5'-incised AP sites. We already observed that disruption of this putative contact by removal of the 5'-phosphate at nicks increased the exonuclease activity. Thus, elimination of this putative contact at the protein level, by an alanine substitution at Arg-177 in Ape1, might increase the exonuclease activity on nicked DNA containing 5'-phosphates. Instead, we found the R177A mutant of Ape1 to exhibit an overall 60–80% reduction in exonuclease activity, with no change in its substrate specificity (data not shown). Noting that both 5'-phosphorylated and 5'-unphosphorylated F residues inhibit Ape1 (Fig. 3), we conclude that contacts to the 5'-phosphate specifically do not constitute a major determinant of the exonuclease inhibition. Compared with the wild-type protein, the R177A mutant also exhibited a similar reduction in its ability to excise 3'-phosphoglycolate esters (data now shown). Because the R177A protein exhibited wild-type AP endonuclease activity, some protein residues clearly modulate the 3'-phosphodiesterase and 3'5'-exonuclease activities selectively, even though all of the nuclease activities of Ape1 probably share a single active site carrying out hydrolysis (38).

The finding that the exonuclease activities of both Ape1 and E. coli endonuclease IV are inhibited by the presence of 5'-incised abasic residues at nicks and gaps suggests an evolutionarily defined role for this mode of inhibition. Ape1 and endonuclease IV are unrelated at the amino acid sequence and protein structural levels. Endonuclease IV utilizes three tightly bound zinc metal ions for catalysis (49), whereas Ape1 uses magnesium (38). Furthermore, the mammalian and bacterial proteins bind DNA in very different ways. Ape1 makes contact mostly with the strand containing the flipped out abasic residue and induces a 35° bend in the DNA (38). Endonuclease IV makes significant contacts with both the damaged and undamaged strands to flip out both the abasic DNA and the unpaired nucleotide, which produces an 85° kink in the DNA (49). Despite these differences, both proteins have combined AP endonuclease, 3'-phosphodiesterase, and 3'5'-exonuclease, and 3'-phosphatase activities, albeit at different relative levels (50). The biological roles in BER of the 3'5'-exonuclease activities of both endonuclease IV and exonuclease III from E. coli are still unclear. However, the modulation of the endonuclease IV exonuclease activity by the 5'-incised abasic DNA suggests a specific regulatory mechanism in BER for endonuclease IV but not for exonuclease III.

How the exonuclease activity of Ape1 is coordinated during BER remains an intriguing question. As demonstrated by DNase footprinting and structural data, Ape1 occludes at least 3 bp of double-stranded DNA upstream and downstream of AP sites when bound as an AP endonuclease (37–39). However, it is unclear whether Ape1 remains at the AP site, whereas pol inserts a nucleotide and excises the 5'-dRP residue. It is possible that recruitment of pol to the repair site results in displacement of Ape1, which would have to be recruited back to the repair site following DNA synthesis. Yet another possibility is that Ape1 and pol are both part of a multiprotein repair complex that coordinates BER. Although a protein complex containing enzymatic activities proficient for the complete repair of uracil has been isolated from bovine testis nuclear extracts, only pol and DNA ligase I were identified as individual components of this complex (51). Both Ape1 and pol interact physically with Xrcc1 (52–54), p53 (55), and proliferating cell nuclear antigen (56, 57), but no stable complex containing all these proteins has yet been identified. It seems likely that the dynamic processes of BER require transient interactions to allow the processing of large numbers of lesions on a daily basis in mammalian cells.

	FOOTNOTES

 
^* This work was supported in part by National Institutes of Health Grants GM40000 and CA71993 (to B. D.) and NIEHS Grant ES00002 to the Kresge Center for Environmental Health Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported in part by NIEHS Training Grant T32CA009078.

To whom correspondence should be addressed: Dept. of Cancer Cell Biology, Harvard School of Public Health, 665 Huntington Ave, Boston, MA 02115. Tel.: 617-432-3462; Fax: 617-432-0377; E-mail: bdemple{at}hsph.harvard.edu.

¹ The abbreviations used are: AP, apurinic/apyrimidinic; Ape1, AP endonuclease 1; pol , DNA polymerase ; dRP, deoxyribose-5-phosphate; BER, base excision repair; F, tetrahydrofuran.

	ACKNOWLEDGMENTS

 
We thank members of the Demple laboratory for useful discussions, and Drs. Tadahide Izumi, Sankar Mitra, Rajendra Prasad, and Sam Wilson for generous gifts of enzymes. We thank Tom Ellenberger for helpful comments on the manuscript and David Wilson for sharing results prior to publication.

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