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J. Biol. Chem., Vol. 275, Issue 29, 22427-22434, July 21, 2000

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Apurinic Endonuclease Activity of Yeast Apn2 Protein^*

Ildiko Unk, Lajos Haracska, Robert E. Johnson, Satya Prakash, and Louise Prakash

From the Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1061

Received for publication, April 3, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Abasic (apurinic/apyrimidinic; AP) sites are generated in vivo through spontaneous base loss and by enzymatic removal of bases damaged by alkylating agents and reactive oxygen species. In Saccharomyces cerevisiae, the APN1 and APN2 genes function in alternate pathways of AP site removal. Apn2-like proteins have been identified in other eukaryotes including humans, and these proteins form a distinct subfamily within the exonuclease III (ExoIII)/Ape1/Apn2 family of proteins. Apn2 and other members of this subfamily contain a carboxyl-terminal extension not present in the ExoIII/Ape1-like proteins. Here, we purify the Apn2 protein from yeast and show that it is a class II AP endonuclease. Deletion of the carboxyl terminus does not affect the AP endonuclease activity of the protein, but this protein is defective in the removal of AP sites in vivo. The carboxyl terminus may enable Apn2 to complex with other proteins, and such a multiprotein assembly may be necessary for the efficient recognition and cleavage of AP sites in vivo.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Abasic (apurinic/apyrimidinic; AP)¹ sites are formed in DNA by spontaneous hydrolysis of the N-glycosylic bond and from the action of DNA glycosylases on modified bases. It has been estimated that a mammalian cell loses up to 10,000 purines per day from its genome (1). Class II AP endonucleases cleave the phosphodiester backbone on the 5'-side of the AP site and produce a 3'-OH group and a 5'-baseless deoxyribose 5'-phosphate residue. Removal of the 5'-abasic residue followed by DNA repair synthesis and ligation completes the repair process (2, 3).

Two families of class II AP endonucleases have been identified in prokaryotes and eukaryotes. In Escherichia coli, exonuclease III (ExoIII) constitutes ~90% of the total AP endonuclease activity, and endonuclease IV represents about 10% of the total AP endonuclease (2, 3). In the yeast Saccharomyces cerevisiae, Apn1 and Apn2 are the respective homologs of E. coli endonuclease IV and ExoIII (4-7). Apn1 and Apn2 represent alternate pathways for the repair of AP sites in yeast, since the apn1 apn2 double mutant strain displays a synergistic increase in sensitivity to the alkylating agent methyl methanesulfonate (MMS), and the repair of AP sites is severely impaired in this mutant (6). Consistent with the high mutagenicity of AP sites, the frequency of MMS-induced mutations is greatly elevated in the apn1 apn2 strain (6).

Ape1, the human homolog of ExoIII, also known as Hap1, Apex, or Ref-1, is a class II AP endonuclease (8, 9). The yeast Apn2 protein is unique in that it possesses a C-terminal domain that is absent from the ExoIII/Ape1 proteins (6). Recently, a sequence, Ape2, has been identified in humans that possesses the C-terminal domain present in S. cerevisiae Apn2, and Ape2 also is more closely related to Apn2 than to ExoIII or Ape1. Thus, yeast Apn2 and its human counterpart, Ape2, represent a new subfamily in the ExoIII/Ape1/Apn2 family of proteins (see Fig. 1). Here, we purify the yeast Apn2 protein and show that it has an AP endonuclease activity. We also provide evidence that the Apn2 specific C terminus is not required for the AP endonuclease activity, but, interestingly, it is indispensable for the in vivo function of the protein in the repair of AP sites.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Plasmids-- The wild type APN2 gene was obtained by gap repair from the wild type yeast strain EMY74.7. The APN2 gene was first amplified from total yeast genomic DNA by PCR from nucleotides (nt) 202 to +2306, encompassing the 1558-nt APN2 open reading frame. This PCR fragment was then cloned into the 2-µm multicopy plasmid YEplac181, generating plasmid pBJ589. pBJ589 was digested with the restriction enzymes BglII and HindIII to remove the APN2 coding region and used to transform strain EMY74.7. Plasmid DNA, which had undergone recombination and incorporated the genomic APN2 gene, was recovered from yeast and used for overexpression. To overexpress APN2, a unique BamHI restriction site was generated 10 nt upstream of the APN2 initiation codon by PCR and cloned into YCplac33. The wild type 1.37-kilobase pair APN2 internal BglII fragment from the gap-repaired APN2 gene was then used to replace the same fragment in the PCR-generated fragment, generating plasmid pBJ622. The regions of APN2 generated by PCR and not replaced by the wild type gene were sequenced and found not to contain any mutations. The APN2 gene was then cloned in frame with the glutathione S-transferase gene, which is under the control of a galactose-inducible phosphoglycerate promoter in plasmid pHQ241, generating plasmid pBJ627.

Expression and Purification of Wild Type Apn2 Protein from Yeast-- The gluthathione S-transferase (GST)-Apn2 fusion protein was overexpressed in the protease-deficient BJ5464 yeast strain, using the plasmid pBJ627. Cells were grown overnight to stationary phase in complete synthetic medium lacking leucine to select for the plasmid. The overnight culture was diluted 10-fold in fresh medium lacking dextrose and containing 2% galactose and incubated for additional 10 h at 30 °C before cells were collected by centrifugation. Cells were resuspended in cell breakage buffer containing 50 mM Tris, pH 7.0, 50 mM KCl, 10% sucrose and protease inhibitors, and disrupted by a French press. After clarification of the crude cell lysate by centrifugation (45,000 × g, 60 min), it was loaded onto a glutathione-Sepharose 4B column (Amersham Pharmacia Biotech). The column was extensively washed with high salt buffer (100 mM Tris, pH 7.5, 1 M NaCl, 0.01% Nonidet P-40, 10% glycerol), followed by low salt buffer (100 mM Tris, pH 7.5, 100 mM NaCl, 0.01% Nonidet P-40, 10% glycerol). The GST-Apn2 protein was then eluted from the column with low salt buffer containing 20 mM glutathione. Fractions containing the peak of GST-Apn2 protein were pooled, concentrated using a Centricon 30 concentrator, and dialyzed against buffer A (20 mM KH₂PO₄ pH 7.5, 100 mM KCl, 5 mM 2-mercaptoethanol, 0.01% Nonidet P-40, 10% glycerol). The dialysate was applied onto a Mini-S column and developed with a gradient of 100-1000 mM KCl in buffer A. Fractions containing nearly homogeneous GST-Apn2 protein were pooled and concentrated in the Centricon 30 concentrator.

Expression and Purification of C-terminally Deleted Apn2 Proteins-- To generate C-terminal deletions of Apn2, the BamHI fragment of pBJ627 containing the wild type APN2 gene was cloned into pGEX-3X (Amersham Pharmacia Biotech), generating plasmid pIU13. Fragments corresponding to nucleotides 972-1107 and nucleotides 972-1473 of the APN2 gene were PCR-amplified from genomic DNA and cloned into pIU13 as HindIII fragments, generating plasmids pIU14 and pIU16, respectively. From these plasmids, the 3'-truncated derivatives of the APN2 gene were cloned into the pHQ241 expression vector as BamHI fragments. The regions amplified by PCR were sequenced and found not to contain any mutations. The C-terminally deleted Apn2 proteins were overexpressed and purified as described above for the wild type protein.

Oligonucleotide Substrates-- The 75-nt synthetic oligomer, 5'-AGCTACCATGCCTGCCTCAAGAGTTCGTAAXATGCCTACACTGGAGTACCGGAGCATCGTCGTGACTGGGAAAAC-3', containing a single tetrahydrofuran, an abasic site analog at position 31 indicated by X, was purchased from Midland Co. (Midland, TX). The complementary 75-nt oligomer contained a cytosine opposite the abasic site. The oligonucleotide containing the AP site was radiolabeled at the 5'-end, using T4 polynucleotide kinase and [-³²P]ATP, and then annealed to the complementary oligonucleotide.

AP Endonuclease Assay-- Standard AP endonuclease assays (10 µl) contained 50 mM Tris, pH 8.0, 100 mM NaCl, 1 mM dithiothreitol, 100 µg of bovine serum albumin, and 10% glycerol. Routinely, 50 fmol of ³²P-labeled oligonucleotide were used in the reactions with 50 fmol of Apn2. Reactions were incubated at 30 °C for 10 min and stopped by the addition of formamide/EDTA gel loading buffer. DNA products were separated on 7 M urea, 8% polyacrylamide denaturing gels and quantified by PhosphorImager analysis and the ImageQuant Software (Molecular Dynamics, Inc., Sunnyvale, CA).

MMS Sensitivity and Mutagenesis-- Cells were grown overnight in yeast extract-peptone-dextrose (YPD) medium, sonicated to disperse clumps, and resuspended in 0.05 M KPO₄ buffer, pH 7.0, to yield a density of 3 × 10⁸ cells/ml. Appropriate dilutions of 0.5 ml of MMS at twice the desired final concentration were added to 0.5-ml cell suspensions, and cells were incubated with vigorous shaking at 30 °C for 20 min. The reaction was terminated by the addition of 1 ml of 10% sodium thiosulfate. Appropriate dilutions of cells were plated on YPD for viability determinations and on synthetic complete medium lacking arginine but containing canavanine for determining the frequency of forward mutations to canavanine resistance (can1^r) in the CAN1^S gene. Plates were incubated at 30 °C and counted after 3 and 4-5 days for viability and mutagenesis determinations, respectively.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Apn2 Is a Member of a Distinct Subfamily within the ExoIII/Ape1/Apn2 Family of AP Endonucleases-- The S. cerevisiae Apn2 and human Ape1 and Ape2 proteins all contain several highly conserved motifs found in the ExoIII family of AP endonucleases (Fig. 1A). However, the S. cerevisiae Apn2 protein exhibits a higher degree of homology to the human Ape2 protein than to Ape1. For instance, while the yeast Apn2 protein shares 15% identity and 32% similarity with the human Ape1 protein, it displays 25% identity and 43% similarity with the Ape2 protein. In addition, the S. cerevisiae Apn2 and human Ape2 proteins both contain a conserved carboxyl-terminal extension that is absent from Ape1 and ExoIII (Fig. 1A) (6). This carboxyl-terminal extension is also present in Apn2-like proteins from Schizosaccharomyces pombe and Arabidopsis thaliana (Fig. 1). X-ray structural studies of Ape1 have indicated that Glu⁹⁶, Asp²⁸³, and His³⁰⁹ are involved in metal binding and catalysis (10); these residues are invariant, and the sequences flanking them have been very highly conserved among Apn2, Ape1, and ExoIII proteins (Fig. 1A and Ref. 6). Apn2 and its counterparts in S. pombe and humans also contain a highly conserved sequence, VXAEEG(I/L)(T/S)GXL, which is present at residues 112-122 in S. cerevisiae Apn2 (Fig. 1A). This sequence is absent from Ape1 (Fig. 1A). Consistent with the high degree of similarity between ExoIII and Ape1 (6) and between yeast Apn2 and human Ape2 proteins (Fig. 1A), phylogenetic analyses support the existence of two subfamilies: ExoIII/Ape1 and Apn2/Ape2 (Fig. 1B).

View larger version (60K): [in this window] [in a new window]   Fig. 1.   Homology among Apn2-like proteins. A, sequence alignment of S. cerevisiae Apn2 with Apn2 from S. pombe and human Ape1 and Ape2 proteins. Identical and highly conserved residues are highlighted. The arrows indicate the invariant Glu, Asp, and His residues that are known to be involved in metal binding and catalysis in Ape1. The unique motif within the Apn2/Ape2 subfamily in the N terminus is indicated by a line over it. The inverted triangles indicate the positions of truncations in 369 and 491 Apn2 proteins. Sc, S. cerevisiae; Sp, S. pombe; Hs, Homo sapiens. B, phylogenetic relationships of members of the Apn2/Ape1/Ape2/ExoIII family from various organisms. Division of the ExoIII/Ape1-like and Apn2/Ape2-like subfamilies of proteins is indicated on the right. Alignments and phylogenetic analyses were done using the Macaw and Clustal N programs.

Overexpression of Apn2 Protein in Yeast and Purification of the Enzyme-- To facilitate the purification of Apn2 protein from yeast, the APN2 gene was fused in-frame downstream of the GST gene in the overexpression plasmid pBJ627, and the resulting fusion protein was purified to near homogeneity from a protease-deficient yeast strain (Fig. 2A). After the GST column step, the purity of the protein was ~90%, while after the second Mini-S chromatography step, there was no other visible protein band on the polyacrylamide gel besides GST-Apn2 (Fig. 2A). The GST-APN2 expression plasmid pBJ627 rescues the high MMS sensitivity of the apn1 apn2 yeast strain (Fig. 8A), indicating that the GST-Apn2 fusion protein functions normally in vivo.

View larger version (9K): [in this window] [in a new window]   Fig. 2.   Purity and AP endonuclease activity of GST-Apn2. A, purified GST-Apn2 fusion protein was analyzed on an 8% denaturing polyacrylamide gel and stained with Coomassie Blue. Lane 1, molecular weight standards; lane 2, 2.5 µg of purified GST-Apn2 protein. B, AP endonuclease activity of purified GST-Apn2 was assayed using a 75-nucleotide double-stranded DNA substrate, which contained a single abasic site at position 31 in one of the strands. The 5'-labeled DNA substrate (50 fmol) was incubated without (lane 2) or with (lane 3) 50 fmol of purified GST-Apn2 for 10 min at 30 °C. The reaction products were analyzed on a 7 M urea, 8% polyacrylamide gel, and the DNA bands were visualized by autoradiography. The uncleaved, single-stranded DNA containing the AP site is 75 nt long, while the incision product is 30 nt long. Lane 1 contains 5'-32P-labeled oligomers of the indicated length.

AP Endonuclease Activity of Apn2-- The AP endonuclease activity of the purified GST-Apn2 protein was assayed using a 5'-³²P-end labeled 75-nt duplex DNA substrate, in which one strand contains a single abasic site at position 31. Incubation of the 75-nt DNA substrate with the GST-Apn2 protein generated a 30-nt labeled oligomer (Fig. 2B, lane 3), indicating that Apn2 catalyzes the hydrolysis of the 5'-phosphodiester bond at the abasic site. The high degree of purity of the GST-Apn2 preparation strongly suggested that the observed AP endonuclease activity was intrinsic to the Apn2 protein. To further establish this point, we examined whether the AP endonuclease activity copurifies with the GST-Apn2 protein. As shown in Fig. 3, in different fractions from the Mini-S chromatography step, the level of AP endonuclease activity closely parallels the quantity of Apn2 protein. The strict coelution of the AP endonuclease activity with the protein indicates that this activity is intrinsic to Apn2.

View larger version (52K): [in this window] [in a new window]   Fig. 3.   Coelution of AP endonuclease activity with GST-Apn2 protein. Fractions from the Mini-S chromatography step were assayed for AP endonuclease activity. Top, 1 µl of each fraction was separated on an 8% denaturing polyacrylamide gel and stained with Coomassie Blue. Bottom, fractions were diluted 100-fold and examined for AP endonuclease activity with the 75-nt AP site-containing duplex. The positions of the intact 75-nt DNA and the 30-nt incision product are indicated on the left.

The DNA incision activity of Apn2 increases with incubation time for up to 30 min (Figs. 4, A and B), shows optimal activity in the pH range of 7.5-8.8 (Fig. 4, C and D), and the enzyme is active at relatively high salt concentrations. Maximal activity was observed at 150 mM NaCl; higher salt concentrations inhibited the enzyme activity, and at lower salt concentrations, the incision reaction was less efficient (Fig. 4, E and F).

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Optimal reaction conditions for GST-Apn2 protein. The activity of GST-Apn2 was assayed under various conditions, using the 75-nt AP site-containing DNA. A, time dependence; B, graphical representation of results in A; C, pH dependence. MES was used for pH 5 and 6, and Tris was used for pH 6.8, 7.5, 8.0, and 8.8; D, graphical representation of results in C; E, NaCl concentration dependence; F, graphical representation of results in E. np, no protein.

The AP endonuclease activity of Ape1 is strongly dependent upon magnesium (9-11). Surprisingly, Apn2 is active in the absence of any added magnesium or other metals. Furthermore, Apn2 activity is resistant to a 24-h incubation in 1 mM EDTA at 4 °C, and the addition of magnesium or other metals does not stimulate this activity (Fig. 5). This suggests that Apn2 binds the metal very tightly, and under the conditions used to purify Apn2, metal binding is not disrupted.

View larger version (65K): [in this window] [in a new window]   Fig. 5.   Effect of EDTA on Apn2 activity. GST-Apn2 was preincubated with 1 mM EDTA at 4 °C for 24 h. Incision assays were performed without () or in the presence of 1 mM of the indicated metal ions in the reaction buffer. The first lane contains no protein (np), and in the second control lane (c), the reaction was performed without preincubating GST-Apn2 with EDTA and in the absence of any metals.

Type II AP endonucleases incise the 5'-phosphodiester of an abasic site, yielding a 3'-OH and a 5'-PO₄ deoxyribose residue (2, 3). To establish the nature of the 3' terminus left after incision, we used terminal transferase to label the 3'-OH ends by [-³²P]ddATP after the incision reaction (Fig. 6A), which would effect the addition of one nucleotide to DNA fragments. The terminal transferase end-labeling reaction resulted in the appearance of two labeled incised products, a 31-nt and a 46-nt DNA fragment (Fig. 6B, lane 2). The formation of the 31-nt fragment indicates that Apn2 incision generates a 3'-OH end 5' to the AP site. To determine the nature of the 5'-end generated after incision, we phosphorylated the free 5'-OH ends of the reaction products using T4 polynucleotide kinase and [-³²P]ATP (Fig. 6A). As expected, this reaction resulted in the labeling of the 30-nt fragment and the 75-nt uncleaved DNA (Fig. 6B, lane 4). However, we observed no labeling of the 45-nt DNA fragment (Fig. 6A), indicating the presence of a PO₄ group at the 5' terminus, since under the conditions employed for labeling, T4 polynucleotide kinase will not add a PO₄ group if one is already present. The presence of the PO₄ group at the 5' terminus in the 45-nt fragment was verified by using an exchange reaction in which T4 polynucleotide kinase replaces an existing PO₄ group at the 5' terminus with the ³²P-labeled PO₄ of [-³²P]ATP. Under these conditions, the reaction resulted in the labeling of the 45-nt fragment (data not shown). Thus, Apn2 action generates a 3'-OH and a 5'-PO₄ terminus at the AP site.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Nature of the 5' and 3' termini remaining after incision at an AP site by Apn2. A, flow diagram for the reactions. , an AP site; P inside a circle, radioactively labeled nucleotides. B, the unlabeled 75-nt AP site containing duplex DNA was incubated without (lanes 1 and 3) or with (lanes 2 and 4) GST-Apn2 at 30 °C for 10 min. After ethanol precipitation, the reactions were 3'-end-labeled by terminal transferase in the presence of [-32P]ddATP (lanes 1 and 2) or 5'-end labeled by T4 polynucleotide kinase using [-32P]ATP (lanes 3 and 4). Lane 5 contains 5'-32P-labeled DNA size markers.

Proficient AP Endonuclease Activity in the C-terminally Deleted Apn2 Proteins-- To study the contribution of the C terminus to the biochemical and biological functions of Apn2, we constructed two deletion mutations: 369, which lacks the last 151 residues, and 491, which lacks only the last 29 residues of Apn2 (Fig. 1A). Thus, 369 removes almost the entire C-terminal portion specific to the Apn2 subfamily, whereas 491 deletes a highly conserved sequence present in the C terminus. In both of these mutants, however, all of the motifs involved in metal binding and catalysis remain intact (Fig. 1A).

The C-terminally deleted GST-Apn2 proteins were expressed in and purified from yeast in the same manner as the wild type proteins using glutathione-Sepharose and Mini S column chromatography. This purification resulted in almost homogeneous preparations of GST-Apn2 369 and GST-Apn2 491 protein, and both proteins exhibited the size expected for these truncations (Fig. 7A). Both mutant proteins display AP endonuclease activity comparable with wild type Apn2 (Fig. 7B). Thus, even the deletion of almost the entire carboxyl-terminal portion does not debilitate the AP endonuclease activity of Apn2 in vitro.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Purity and activity of the carboxyl-terminally deleted Apn2 proteins. A, 1 µg of the GST-Apn2 deletion mutant proteins was loaded on an 8% denaturing polyacrylamide gel and stained with Coomassie Blue. Lane 1, molecular weight standards; lane 2, GST-Apn2 369 protein; lane 3, GST-Apn2 491 protein. B, The AP endonuclease activity of the wild type GST-Apn2 (wt) and the GST-Apn2 491 and GST-Apn2 369 mutant proteins were assayed as described in the legend to Fig. 2B. The positions of the uncut (75-nt) full-length substrate and the incised product (30 nt) are indicated on the left. The first lane contains the control reaction without any Apn2 protein.

Requirement of the C Terminus for the Biological Function of Apn2-- To determine the contribution of the C terminus to Apn2 function in vivo, we tested the sensitivity of C-terminal deletions to the alkylating agent MMS. MMS alkylates bases in DNA, particularly adenine at the N-3 position (3MeA) and guanine at the N-7 position (7MeG). The alkylated bases are removed by the action of N-methylpurine DNA glycosylase, which has a broad specificity toward various base modifications, including 3MeA and 7MeG (12, 13). The AP sites resulting from this DNA glycosylase action would then be acted upon by class II AP endonucleases (2, 3).

In S. cerevisiae, deletion of APN1 confers a moderate increase in sensitivity to MMS concentrations higher than 0.1%, whereas deletion of APN2 has no effect on MMS sensitivity, even at higher concentrations (6). In the absence of both APN1 and APN2, however, yeast cells display extremely enhanced MMS sensitivity, even at low MMS concentrations (Fig. 8A). Introduction of plasmid pBJ627, which expresses the wild type GST-Apn2 protein, into the apn1 apn2 strain rescues the MMS sensitivity of this strain (Fig. 8A). Thus, the wild type GST-Apn2 protein functions normally in vivo. Surprisingly, plasmid pPM1034, which expresses the GST-369 Apn2 protein, did not complement the MMS sensitivity of the apn1 apn2 mutant strain (Fig. 8A). In fact, the MMS sensitivity of the apn1 apn2 strain was nearly the same whether or not it carried the plasmid for expressing the GST-369 Apn2 protein. By contrast, introduction of plasmid pPM1036, which expresses the GST-491 Apn2 protein, into the apn1 apn2 strain, conferred a large increase in MMS resistance (Fig. 8A).

View larger version (14K): [in this window] [in a new window]   Fig. 8.   Effect of carboxyl-terminal deletions on the in vivo function of Apn2. A, MMS sensitivity of apn1 apn2 yeast strain carrying C-terminal deletions of Apn2. Cells were grown overnight in YPD medium and treated with MMS at the concentrations indicated for a 20-min period. Appropriate dilutions were spread onto YPD plates. Each curve represents the average of two or more experiments for each strain. , EMY74.7, wild type; , YRP190, apn1; , YRP269, apn1 apn2; , YRP574, apn1 apn2 strain carrying the wild type APN2 gene on plasmid pBJ627; , YRP575, apn1 apn2 strain carrying the 369 apn2 mutation on plasmid pPM1034; , YRP577, apn1 apn2 strain carrying the 491 apn2 mutation on plasmid pPM1036. B, MMS-induced mutagenesis at the CAN1 locus. Cells were grown and treated as described above. Appropriate dilutions were spread onto YPD plates for viability determination and onto synthetic complete medium lacking arginine and containing canavanine for the determination of CAN1S to can1r forward mutation frequency. , EMY74.7, wild type; , YRP190, apn1; , YRP269, apn1 apn2; , YRP575, apn1 apn2 strain carrying the 369 apn2 mutation on plasmid pPM1034.

The lack of functional complementation by the 369 Apn2 protein may suggest a role for the Apn2 C terminus in the removal of AP sites or in a step subsequent to the incision reaction. For example, in the absence of the C terminus, the excision or repair synthesis steps may be affected. Because AP sites are noncoding, they are highly mutagenic, and accordingly, the frequency of MMS-induced mutations rises sharply in the apn1 apn2 strain (6). The apn1 apn2 strain expressing the GST-369 Apn2 protein also displays a large increase in the frequency of MMS induced can1^r mutations (Fig. 8B). The high mutagenicity suggests that AP sites are not removed in the apn1 apn2 strain expressing the 369 protein.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Yeast Apn2 and its orthologs in higher eukaryotes differ from E. coli ExoIII and human Ape1 in possessing a C terminus that is absent from the ExoIII/Ape1 subfamily. Genetic studies in yeast have indicated that Apn1 and Apn2 constitute alternative pathways of AP site repair. The Apn1 protein has previously been shown to be an AP endonuclease (14). Here, we overproduce and purify the Apn2 protein from yeast and show that it has an AP endonuclease activity that cleaves the phosphodiester bond 5' to the AP site and generates a 3'-OH and 5'-PO₄ terminus. Thus, Apn2 is a class II AP endonuclease.

To examine the role of the Apn2-specific C terminus in the biochemical and biological functions of the protein, we purified the 369 and 491 Apn2 proteins, which lack almost the entire C terminus and the last 29 residues of Apn2, respectively. Consistent with the presence of the motifs involved in metal binding and catalysis in the amino terminus, both C-terminally truncated proteins display nearly normal AP endonuclease activity. Interestingly, however, the C terminus is indispensable for the biological function of the protein, since expression of 369 protein does not elevate the repair efficiency of the apn1 apn2 strain. The carboxyl-terminal 29 residues that include the highly conserved sequence NXGRKFWXCXRXXG, however, are not needed for biological function, since expression of 491 protein complements the MMS sensitivity of the apn1 apn2 strain.

How might the carboxyl terminus contribute to Apn2 function in vivo? Although the C-terminally deleted Apn2 protein has nearly normal AP endonuclease activity in vitro, it is possible that the C terminus modulates Apn2 activity in vivo. Because of histone and nonhistone proteins bound to DNA, and because of high compaction of DNA, the recognition of lesions could be a formidable task in vivo. Thus, in contrast to the ability of Apn2 to act upon AP sites in naked DNA in vitro, the highly compacted chromatin in vivo may necessitate that Apn2 be a part of the multiprotein complex for efficient recognition and cleavage of AP sites to occur. The Apn2 C terminus may be crucial for complex formation with other proteins, and in its absence, such a complex may not form. Such a possibility is supported from studies of nucleotide excision repair in yeast, where the repair of UV damage from the nontranscribed regions of the genome is strictly dependent upon the RAD7 and RAD16 genes (15, 16). In vitro, however, the incision reaction can be reconstituted on UV-damaged DNA in the absence of Rad7 and Rad16 proteins (17), although the addition of Rad7-Rad16 complex to the purified reconstituted system stimulates the incision reaction (18). Thus, the absolute need for the Rad7 and Rad16 proteins is manifested in vivo but not in vitro. The Rad7-Rad16 protein complex has affinity for UV-damaged DNA, and it has been proposed that the Rad7-Rad16 complex functions in locating the damage in nontranscribed regions of DNA (18, 19). Although the damage can be recognized and incised in vitro on naked DNA without the Rad7 and Rad16 proteins, in the absence of these proteins, the search and demarcation of DNA damage may become quite inefficient in vivo.

The identification of AP endonuclease activity in the yeast Apn2 protein and the demonstration that although the C terminus of the protein is not required for this activity it still is necessary for the in vivo function of the protein strongly suggest that the human Ape2 protein would also have such an activity and that the C terminus of the human protein also will be indispensable for its in vivo function.

By contrast to nucleotide excision repair, which involves the concerted action of several distinct multiprotein complexes (17-21), base excision repair has been thought to rely upon the sequential action of several independent enzymatic activities. For example, following the recognition and removal of the damaged base by a DNA glycosylase, an AP endonuclease is thought to cleave at the AP site (2, 3). However, the possibility that base excision repair involves a multiprotein complex is suggested by the observation that a glycosylase and an AP endonuclease may be present together in a complex with DNA polymerase  and DNA ligase I (22). Our studies raise the possibility that the recognition and cleavage of AP sites by Apn2 in vivo is accomplished by a multiprotein complex.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant CA41261 and Department of Energy Grant DE-FG03-00ER62910.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Sealy Center for Molecular Science, University of Texas Medical Branch, 6.104 Medical Research Bldg., 11th and Mechanic St., Galveston, TX 77555-1061. Tel.: 409-747-8601; Fax: 409-747-8608; E-mail: lprakash@scms.utmb.edu.

Published, JBC Papers in Press, May 5, 2000, DOI 10.1074/jbc.M002845200

	ABBREVIATIONS

The abbreviations used are: AP, apurinic/apyrimidinic; ExoIII, exonuclease III; MMS, methyl methanesulfonate; PCR, polymerase chain reaction; GST, glutathione S-transferase; YPD, yeast extract-peptone-dextrose; nt, nucleotide(s); MES, 4-morpholineethanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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