AP endonuclease and poly(ADP-ribose) polymerase-1 interact with the same base excision repair intermediate

DNA Repair 3 (2004) 581–591

AP endonuclease and poly(ADP-ribose) polymerase-1 interact with the same base excision repair intermediate Cheryl Cistulli a , Olga I. Lavrik a,b , Rajendra Prasad a , Esther Hou a , Samuel H. Wilson a,∗ a

Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, 11 T.W. Alexander Drive, Research Triangle Park, NC 27709, USA b Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, Prospect Laventiev 8, 630090 Novosibirsk, Russia Accepted 2 September 2003

Abstract Base excision repair (BER) is a defense system that protects cells from deleterious effects secondary to modified or missing DNA bases. BER is known to involve apurinic/apyrimidinic endonuclease (APE) and DNA polymerase ß (ß-pol) among other enzymes, and recent studies have suggested that poly(ADP-ribose) polymerase-1 (PARP-1) also plays a role by virtue of its binding to BER intermediates. The main role of APE is cleavage of the DNA backbone at abasic sites, and the enzyme also can catalyze 3 - to 5 -exonuclease activity at the cleaved abasic site. Photocross-linking studies with mouse embryonic fibroblast (MEF) cell extracts described here indicated that APE and PARP-1 interact with the same APE-cleaved abasic site BER intermediate. The model BER intermediate used includes a synthetic abasic site sugar, i.e. tetrahydrofuran (THF), in place of the natural deoxyribose. APE cross-linked efficiently with this intermediate, but not with a molecule lacking the 5 -THF phosphate group, and the same property was demonstrated for PARP-1. The addition of purified APE to the MEF extract reduced the amount of PARP-1 cross-linked to the BER intermediate, suggesting that APE can compete with PARP-1. APE and PARP-1 were antagonists of each other in in vitro BER related reactions on this model BER intermediate. These results suggest that PARP-1 and APE can interact with the same BER intermediate and that competition between these two proteins may influence their respective BER related functions. © 2004 Elsevier B.V. All rights reserved. Keywords: Base excision repair; AP endonuclease; Poly(ADP-ribose) polymerase-1; FEN1

1. Introduction Biological survival requires a delicate balance between genomic stability and genetic variation. The former prevents deleterious mutations and the latter provides a source of variants for natural selection. DNA repair proteins maintain genomic stability by detecting, removing, and repairing lesions [1–3]. The main mammalian cell DNA repair pathway removing a damaged base is base excision repair (BER) [4,5], Abbreviations: APE, apurinic/apyrimidinic endonuclease; ß-pol, DNA polymerase ß; BER, base excision repair; dRP, deoxyribose phosphate; DTT, dithiothreitol; FAB-dCTP, exo-N-[ß-(p-azidotetrafluorobenzamido)ethyl] deoxycytidine 5 -triphosphate; FEN1, flap endonuclease 1; MEF, mouse embryo fibroblast; PARP-1, poly(ADP-ribose) polymerase-1; THF, tetrahydrofuran; UDG, uracil-DNA glycosylase; XRCC1, X-ray repair cross complementing group 1 ∗ Corresponding author. Tel.: +1-919-541-3267; fax: +1-919-541-3592. E-mail address: [email protected] (S.H. Wilson). 1568-7864/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.dnarep.2003.09.012

which occurs in several forms in the cell depending on the activity of the enzymes involved and the efficiency of processing the DNA intermediate during each step of the pathway. One form of BER is initiated when the damaged or inappropriate base is removed by a DNA glycosylase or by spontaneous chemical hydrolysis of the glycosidic bond, resulting in the abasic site in double-stranded DNA [2,6]. The abasic or apurinic/apyrimidinic (AP) site is cleaved by a class II AP endonuclease (such as apurinic/apyrimidinic endonuclease (APE)), which incises the phosphodiester backbone 5 to the AP site, leaving 3 -hydroxyl and 5 -deoxyribose phosphate (dRP) termini [7]. Repair then proceeds through either the single-nucleotide or long patch BER (2–10 nucleotide patch) subpathway [8–10]. However, subpathway choice in BER appears to be influenced by the rate-limiting step after the repair process has been initiated, i.e. removal of the 5 -dRP group by the dRP lyase activity of DNA polymerase ß (ß-pol) [11–14]. For example, when the 5 -dRP group

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is not efficiently removed, continued DNA synthesis is favored either by ß-pol or another polymerase leading to long patch BER; on the other hand, when the dRP group is efficiently removed, DNA ligase can seal the nick in the BER intermediate completing the single-nucleotide BER process. Our previous studies involving photoaffinity labeling of mouse embryonic fibroblast (MEF) extract proteins had identified poly(ADP-ribose) polymerase-1 (PARP-1) as a major BER intermediate binding factor along with ß-pol and flap endonuclease 1 (FEN1) [15]. The BER intermediate used in this work was an oligonucleotide model representing the product of AP endonuclease cleavage of the AP site. The natural BER intermediate at this step contains the 5 -dRP group, which is removed by the ß-pol dRP lyase activity in normal single-nucleotide BER. It is known that accumulation of this BER intermediate, by virtue of a defect in the ß-pol dRP lyase activity, makes MEF cells hypersensitive to DNA alkylating agents, triggering apoptosis or necrotic cell death depending on the amount of the alkylating agent used and the status of cellular PARP-1 activity [14]. PARP-1 activity is required to protect wild-type MEF cells from apoptosis, even after very low doses of alkylating agent exposure [15]. In our biochemical studies, we have used 5 -tetrahydrofuran (THF) phosphate as the cleaved AP site sugar phosphate instead of the natural 5 -deoxyribose phosphate. The photoaffinity labeling of PARP-1 by this model BER intermediate was much less when an alternate intermediate was used that did not contain the 5 -THF phosphate group [15]. All of this information points to the biological importance of an interaction between PARP-1 and the 5 -dRP-containing BER intermediate. The PARP-1 and 5 -dRP-containing BER intermediate interaction is involved in some way in apoptosis signaling, but the interaction also may be involved in BER itself. Dantzer et al. [16] found a deficiency in in vitro BER mediated by PARP-1 null mouse cell extracts, and PARP-1 has been found to stimulate gap-filling DNA synthesis by ß-pol in an in vitro long patch BER reaction; this long patch BER stimulation by PARP-1 required FEN1 and the 5 -dRP group in the BER intermediate [17]. In the current study, we extended information using the photoaffinity labeling approach by showing that another extract protein interacting with the 5 -THF-containing BER intermediate was APE. Recent biochemical and genetic studies indicate that in single-nucleotide BER, ß-pol is the major DNA polymerase, recognizing the gap, incorporating one nucleotide, and then removing the dRP group [8,9,14,18]. While ß-pol is considered a moderately accurate polymerase that lacks an intrinsic exonuclease activity, APE has 3 - to 5 -exonuclease activity [19] that could be important in BER. The 3 -exonuclease of APE shows a significant preference for removal of mismatched versus matched nucleotides from the 3 -terminus of nicked DNA [20–22]. Therefore, APE could serve as a proofreading enzyme, excising nucleotides misincorporated by polymerases during BER [23]. It is unknown whether other proteins involved in BER might contribute

such 3 -exonuclease proofreading activity. Other exonucleases including Trex1 and WRN are also mismatch-selective [21,24,25], but it is not yet clear whether these enzymes can contribute a proofreading function in BER. Recently, Matsuda et al. [26] reported that the error rate in a reconstituted single-nucleotide BER system with purified enzymes (APE, ß-pol, and DNA ligase I) was lower than the error rate of single-nucleotide gap filling DNA synthesis by purified ß-pol. This suggested that the BER process through protein–protein interactions or the proofreading activity of APE might be responsible for enhanced fidelity of BER. The present study was undertaken to determine if APE is one of the MEF extract proteins labeled with the photoaffinity probe representing the 5 -dRP-containing BER intermediate. We found that APE was selectively photocross-linked by the intermediate. APE, PARP-1, and FEN1 appeared to share specificity for the 5 -dRP group in the BER intermediate, whereas ß-pol did not. Yet, we were surprised to observe that addition of purified APE could compete with PARP-1, but not with ß-pol, for cross-linking. We examined implications of this for BER related activities of PARP-1 and APE and discuss the possibility that APE may play a regulatory function in BER by binding to the product of its AP endonuclease reaction and regulating activities of PARP-1 that require binding to the same 5 -dRP-containing BER intermediate.

2. Materials and methods 2.1. Materials Synthetic oligodeoxyribonucleotides were from Oligos Etc Inc. (Wilsonville, OR). [␥-32 P]dATP and [␣-32 P]dCTP (3000 Ci/mmol) were from Amersham Biosciences (Piscataway, NJ). Polynucleotide kinase was from Promega (Madison, WI). Human APE, ß-pol, uracil-DNA glycosylase (UDG), FEN1, PCNA, and PARP-1 were purified as described previously [15,17,27–29]. Mutant Y171A of APE was a gift from Phyllis Strauss (Northeastern University, Boston, MA). Antiserum specific for APE was raised by immunization of rabbit as described [30]. 2.2. Mouse embryonic fibroblast cell extracts ß-Pol null mouse embryonic fibroblasts expressing a FLAG epitope-tagged ß-pol have been described previously [15]. Cells were routinely grown at 34 ◦ C in a 10% CO2 incubator in Dulbecco’s modified Eagle’s medium supplemented with GlutaMAX-1 (Invitrogen, Carlsbad, CA) and 10% fetal bovine serum (HyClone, Logan, UT). Cells were routinely tested and found to be free of mycoplasma contamination. Extracts were prepared as previously described [31]. Cells (5 × 106 ) were resuspended in 100 ␮l Buffer I (10 mM Tris–HCl, pH 7.8, 200 mM KCl), and an equal volume of

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Buffer II (10 mM Tris–Cl, pH 7.8, 200 mM KCl, 2 mM EDTA, 40% glycerol, 0.2% NP-40, 2 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride, 10 mg/ml apoprotinin, 5 mg/ml leupeptin, 1 mg/ml pepstatin A) was added. Cell suspension was then rotated at 4 ◦ C for 1 h and centrifuged at 14,000 rpm for 10 min.

incubated for 1 h at 4 ◦ C, and then the beads were collected by centrifugation and washed three times with Buffer II. After a final wash, the beads were resuspended in 50 ␮l SDS-PAGE sample buffer, boiled for 5 min, and the soluble proteins were separated on a 10% SDS-PAGE gel. The gel was dried and subjected to autoradiography.

2.3. DNA substrates and 5 -end labeling

2.7. Base excision repair assay

Preparation of DNA substrates and 5 -end labeling were carried out essentially as described previously [17]. The sequence of the uracil (U) or tetrahydrofuran (also referred to as F)-containing DNA was as follows: 5 -CTGCAGCTGATGCGCU/FGTACGGATCCCCGGGTAC-3 . Oligodeoxynucleotides were 5 -32 P-phosphorylated with T4 polynucleotide kinase [32]. Unreacted [␥-32 P]ATP was removed by a MicroSpinTM G-25 column (Amersham Biosciences) using the manufacturer’s protocol.

A 34-bp DNA substrate containing uracil or F/THF at position 16 was pre-treated with 10 nM UDG in 50 mM Hepes, pH 7.5, 20 mM KCl, and 2 mM DTT. The reaction was incubated at 37 ◦ C for 20 min. The repair reaction (10 ␮l) was assembled on ice that contained 50 mM Hepes, pH 7.5, 20 mM KCl, 2 mM DTT, 10 mM MgCl2 , 4 mM ATP, 20 ␮M each dATP, dGTP, dTTP, and 2.3 ␮M [␣-32 P]dCTP (specific activity: 1 × 106 dpm/pmol) and 100 nM DNA. The reaction was initiated by adding APE (50 nM), ß-pol (2.5 nM), FEN1 (20 nM) or PARP-1 (50 nM), as indicated in the figure legends. Note that all reaction mixtures contained UDG at a final concentration of 10 nM. Incubation was at 37 ◦ C for 25 min. DNA products were analyzed as described previously [17].

2.4. Primer-template annealing Lyophilized oligodeoxynucleotides were resuspended in H2 O and the optical density was measured. Complementary oligodeoxynucleotides were mixed in equimolar concentration and annealed in 10 mM Tris–HCl, pH 7.4, and 1 mM EDTA by heating the solution to 90 ◦ C for 3 min, followed by slow cooling to room temperature. 2.5. Photoaffinity labeling Photoaffinity labeling was carried out as previously described [15]. Briefly, the reaction mixture (10 ␮l) contained 50 mM Tris–HCl, pH 7.8, 10 mM MgCl2 , 0.64 mg/ml MEF extract, 0.4 ␮M 32 P-labeled DNA containing either an abasic site, a nicked abasic site, or uracil (see Table 1), and 20 ␮M exo-N-[ß-(p-azidotetrafluorobenzamido)-ethyl]deoxycytidine 5 -triphosphate (FAB-dCTP). The reaction mixtures were incubated at 25 ◦ C for 30 min to allow incorporation of photoreactive probe exo-N-[ß-(p-azidotetrafluorobenzamido)ethyl]deoxycytidine 5 -monophosphate (FAB-dCMP). The reaction mixtures were spotted onto Parafilm, placed on ice, and irradiated with near UV light (312 nm) with a UV Stratalinker (Stratagene, La Jolla, CA). Photoaffinity-labeled proteins were separated by 10% SDS-PAGE. Gels were dried and autoradiographed. 2.6. Co-immunoprecipitation Co-immunoprecipitaion of APE from a photoaffinity cross-linked reaction mixture was performed essentially as described previously [33]. Briefly, 10 ␮l anti-APE rabbit polyclonal immune or preimmune serum was added to photoaffinity cross-linked reaction mixture (300 ␮l) and incubated for 4 h at 4 ◦ C. Immunocomplex was adsorbed onto protein A Sepharose beads (20 ␮l) that were pre-equilibrated with protein extraction Buffer II. The reaction mixture was

2.8. DNA substrates for exonuclease assay OL1: [32 P]5 -CTGCAGCTGATGCGCC OL2: [32 P]5 -CTGCAGCTGATGCGCT OL3: 5 -PUGTACGGATCCCCGGGTAC-3 OL4: 5 -PFGTACGGATCCCCGGGTAC-3 OL5: 5 -PGTACGGATCCCCGGGTAC-3 OL6: 3 -GACGTCGACTACGCGGCATGCCTAGGGGCCCATG-5 where P is phosphate, U uracil and F is tetrahydrofuran. Substrate

Match (C–G)

Mismatch (T–G)

dRP THF Nick

OL1 + OL3 OL1 + OL4 OL1 + OL5

OL2 + OL3 OL2 + OL4 OL2 + OL5

Equimolar amounts of purified oligonucleotide with T or C (OL1/OL2, 16 mer) at the 3 -terminus, the downstream oligonucleotide (OL3/OL4/OL5, 19 mer) and the 34 mer template (OL6) were mixed and annealed to form a nicked double-stranded DNA. Then, the uracil or F-containing DNA substrate (1 ␮M) was pre-treated with 10 nM UDG in 50 mM Hepes, pH 7.5, 20 mM KCl, and 2 mM DTT. The reaction mixture was incubated at 37 ◦ C for 20 min. The DNA substrate, thus prepared, contained a nick at position 16 and phosphate (P), deoxyribose phosphate, or tetrahydrofuran (F or THF) phosphate at the 5 -terminus in the nick. 2.9. APE exonuclease activity assay The standard exonuclease reaction was assembled on ice in a reaction mixture (10 ␮l) that contained 200 nM

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Table 1 Nucleotide sequence and structure of DNA molecules used in photoaffinity cross-linking Nucleotide sequence

Designation

DNA1

DNA2

DNA3

DNA4

Abbreviations: C∗ , FAB-dCMP; F, THF; P, phosphate; dC∗ TP, FAB-dCTP.

32 P-labeled

DNA substrate, 50 mM Hepes, pH 7.5, 20 mM KCl, 2 mM DTT, 10 mM MgCl2 , and 4 mM ATP. Cleavage was initiated by adding APE (50–100 nM) for 0.5–60 min at 37 ◦ C. The reactions were stopped by adding 10 ␮l of DNA gel-loading solution (95% formamide, 20 mM EDTA, 0.02% xylene cyanole, and 0.02% bromphenol blue), heated for 3 min at 80 ◦ C and analyzed by a 15% polyacrylamide gel containing 8 M urea. The gel was dried under vacuum and scanned by a PhosphorImager, Model 450 (Molecular Dynamics). Data were analyzed using ImageQuant software. The turnover rate of APE was calculated for each DNA substrate from the initial velocity. In the situations where the velocity decreased with time, the data were fit to

an exponential equation and the initial velocity determined at t = 0 (i.e. initial slope).

3. Results 3.1. Identification of APE and BER intermediate interaction in the MEF extract The experiments described below were carried out with a photoreactive DNA substrate that resembles a BER intermediate. The BER intermediate was constructed from a photoaffinity labeling probe, FAB-dCTP (indicated as dC*TP in

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mobility as proteins identified in a previous study [15], and these species are indicated in Fig. 1. Low molecular weight proteins were also photoaffinity-labeled; these proteins are designated “unknown proteins” (Fig. 1, lane 3) and were not further evaluated. However, the previous work did not identify the 42 kDa cross-linked protein as APE. The possibility that this protein was APE was tested here. Anti-APE polyclonal antibody or non-immune IgG was used in immunoprecipitation experiments in which the precipitated labeled protein was analyzed by SDS-PAGE. The anti-APE antibody precipitated the 42 kDa product (Fig. 1, lane 1), whereas non-immune IgG did not precipitate any labeled proteins (Fig. 1, lane 2). We concluded from these experiments that the 42 kDa cross-linked product was APE. 3.2. A 5 -sugar phosphate is required for efficient cross-linking of PARP-1 and APE

Fig. 1. Photoaffinity cross-linking and immunoprecipitation. 32 P-labeled DNA (0.4 ␮M) was pre-incubated with the a MEF extract expressing a FLAG epitope-tagged ß-pol for 30 min at 25 ◦ C in the presence of 20 ␮M FAB-dCTP (lane 3). The reaction mixture was irradiated with near UV light (λmax = 312 nm). Photoaffinity labeled proteins were immunoprecipitated with anti-APE IgG (lane 1) or preimmune IgG (lane 2). UV cross-linked proteins were separated by SDS-PAGE and visualized by autoradiography. The electrophoretic mobility of the BER proteins and protein markers are indicated. Lane 3 depicts 1/10th of the input of lanes 1 and 2. Incorporation of photoreactive FAB-dCMP moiety into DNA1 is shown schematically in the diagram below the photograph of the gel. The positions of APE and PARP-1 are indicated.

Fig. 1), and a 32 P-labeled 34-base pair oligonucleotide with a synthetic abasic site sugar (THF) (DNA1 ) (Table 1). Low energy UV light (312 nm) activates an arylazido moiety in the photoreactive group. When these reagents are preincubated with MEF extract, APE cleaves the abasic site and FAB-dCMP is incorporated onto the 3 -terminal hydroxyl by a DNA polymerase, generating a nicked molecule with THF phosphate at the 5 -side and FAB-dCMP at the 3 -side of the nick [15]. Note that the ß-pol dRP lyase activity is unable to cleave the 5 -THF phosphate group. Several proteins are labeled when the 32 P-labeled DNA photoaffinity-labeling molecule is incubated with MEF extract and then exposed to near-UV light. Fig. 1 (lane 3) shows that six proteins in the extract were selectively labeled in these current experiments. Three of the six proteins (PARP-1, FEN1, and ß-pol) had a similar electrophoretic

Several photoaffinity DNA probes were used to examine the DNA interaction specificity of APE. Photoreactive probes carried a nick with a 5 -THF phosphate group (DNA1,2 , Fig. 2B) or a 5 -phosphate group (DNA3,4 , Fig. 2B). PARP-1 and APE cross-linked strongly to the THF phosphate-containing molecules (Fig. 2A, lanes 2 and 4). DNA3 and DNA4 , which are identical chemically, but constructed differently, did not cross-link to APE (Fig. 2A, lanes 6 and 7), although PARP-1 cross-linked to DNA3 and DNA4 weakly (Fig. 2A, lanes 6 and 7). These results indicate that APE and PARP-1 interact preferentially with BER intermediates carrying the 5 -THF phosphate group. We noted also that FEN1 labeling was modestly reduced when the 5 -THF phosphate group was removed from the photoreactive probe molecule; this observation was not further pursued here. 3.3. APE competes with PARP-1 for a 5 -sugar phosphate in the BER Intermediate The following experiments examined whether APE and PARP-1 compete for binding to the same BER intermediate. In this case, a photoaffinity probe was formed containing the 5 -THF phosphate group at a nick, and an excess of purified APE was added to the reaction mixture before irradiation with UV light. The addition of APE reduced the amount of PARP-1 and FEN1 cross-linked to the BER intermediate, but not the amount of ß-pol cross-linked (Fig. 3, compare lanes 1 and 2). In contrast, preincubation and irradiation in the presence of an excess of a reference protein, PCNA, did not alter PARP-1 or APE cross-linking to the labeled probe (Fig. 3, lane 3). These data suggest that PARP-1 and APE can compete for the THF phosphate-containing BER intermediate and that these proteins recognize the same features in the BER intermediate. These results with APE and PARP-1 were extended using an in vitro BER related assays (see below), whereas the results regarding FEN1 were not further pursued here.

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Fig. 3. APE competes with PARP-1 for 5 -THF phosphate-containing DNA. 32 P-labeled DNA1 (0.4 ␮M) was pre-incubated with MEF extract for 30 min at 25 ◦ C. Additions were made as follows prior to UV irradiation: buffer (lane 1), APE (1 ␮M; lane 2) or PCNA (1 ␮M; lane 3). The electrophoretic mobility of BER proteins and protein markers are indicated.

Fig. 2. A 5 -sugar phosphate is required for PARP-1 and APE cross-linking. A photograph of an autoradiogram (A) and schematic diagrams of different DNAs and incorporation of FAB-dCTP (dC*TP) (B) are shown. 32 P-labeled DNA1,2,3, or 4 (0.4 ␮M) was pre-incubated with MEF extract for 30 min at 25 ◦ C with (lanes 2, 4, 6, and 8) or without (lanes 1, 3, 5, and 7) FAB-dCTP. The reaction mixtures were UV-irradiated at 312 nm and the photoaffinity labeled products were analyzed as described in Section 2. The electrophoretic mobility of BER proteins and protein markers are indicated.

3.4. 5 -Sugar phosphate and APE regulate the BER subpathways A long patch BER DNA synthesis reaction was analyzed in vitro using a 34-base pair substrate with a cleaved AP site at position 16 (Fig. 4). The reaction mixtures included ß-pol alone, ß-pol and PARP-1/FEN1, or all four enzymes, as indicated in Fig. 4. The reaction products are a 16-nucleotide (16-mer) molecule with [␣-32 P]dCMP incorporated at position 16 (a single-nucleotide BER intermediate) and longer products resulting from strand displacement synthesis (long patch BER intermediates) [17]. Fig. 4 (lane 1), shows that ß-pol alone, at the level used in these experiments, synthesized primarily the 16-mer, i.e. the single nucleotide addition product. As observed previously [17], the combination of PARP-1 and FEN1 (Fig. 4, lane 2) stimulated strand dis-

Fig. 4. APE inhibits PARP-1/FEN1-mediated stimulation of strand displacement DNA synthesis by ß-pol. A 34-base pair duplex DNA containing tetrahydrofuran phosphate (FP) (lanes 1–3) at position 16 was pre-treated with UDG (10 nM) at 37 ◦ C for 20 min and incubated with ß-pol (2.5 nM) with (+) or without (−) PARP-1 (50 nM)/FEN1 (20 nM) and/or APE (50 nM), as indicated. Incubation was for 25 min at 37 ◦ C. A schematic representation of the substrate DNA is shown at the top of the photograph of the gel. LP and SN refer to long patch and single-nucleotide BER, respectively.

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Table 2 Rate of APE exonuclease activity (expressed as nmol/min)a,b Group at 5 side of nick DNA substratec

Match (C–G)

Mismatch (T–G)

Phosphate Deoxyribose phosphate THF phosphate

0.05 0.03 0.003

0.9 0.5 0.01

a The turnover rate of APE was calculated for each DNA substrate from the initial velocity as described in Section 2. b Data represents the average of three independent experiments. c DNA substrates were prepared by annealing upstream (16-mer) and downstream (18 or 19-mer) oligonucleotides to the template (34-mer) and the duplex DNA was then pre-treated with uracil, as described in Section 2. The DNA substrate, thus prepared, contained a matched (C–G) or mismatched (T–G) and a phosphate, deoxyribose phosphate, or THF phosphate at the 3 -, and 5 -ends in the nick, respectively.

placement DNA synthesis on this substrate. However, in the presence of APE strand displacement synthesis was partially inhibited (Fig. 4, lane 3). For example the 18-mer (three dNMP additions) and 19-mer (four dNMP additions) products were reduced approximately two-fold and much more accumulation of the single-nucleotide addition product was observed. These results indicate that APE, under some circumstances, can interfere with the PARP-1/FEN1 mediated stimulation of long patch BER. It is also important to note that APE alone did not stimulate BER (not shown). 3.5. 5 -Sugar phosphate flap and APE exonuclease activity In light of the results above suggesting that APE could be a negative regulator of PARP-1’s role in strand displacement DNA synthesis in long patch BER, the interaction between APE and the 5 -THF phosphate group on a BER intermediate was examined further. Recently, several studies revealed that gapped and nicked substrates with a 5 -THF group are not used efficiently by the APE 3 -exonuclease activity [19,34,35]. Here, this effect was examined by kinetic analyses of the APE exonuclease using various DNA substrates. Nicked DNA substrates for the 3 -exonuclease carried 5 -phosphate or a 5 -sugar phosphate along with either matched (C–G) or mismatched (T–G) 3 -termini at the nick. Time courses of 3 -exonuclease activity were measured (Fig. 5), and the rates of activity for six DNA substrates are summarized in Table 2. These data confirmed that the activity was higher with the T–G mismatched than with the C–G matched substrates. The 5 -deoxyribose phosphate substrates supported less activity than the 5 -phosphate substrates. Yet, the 5 -THF-phosphate-containing molecules supported even less activity than the 5 −deoxyribose substrates. Our interpretation of these results is that after strand cleavage of the AP site-containing BER intermediate, APE can dissociate and then re-bind to the 5 -sugar phosphate-containing product molecule, resulting in a form of product inhibition. Even though 5 -THF was used here only as a model for the natural sugar, it appears that binding

Fig. 5. Kinetic analysis of the 3 -exonuclease activity of APE. DNA substrates were constructed as described in Section 2. DNA substrate (200 nM) (A, dRP; or C, nick) was incubated with 50 nM APE at 37 ◦ C for 0, 0.5, 1, 3, 5, or 10 min. Incubation for the THF phosphate-containing substrate (B, THF) was with 100 nM APE at 37 ◦ C for 0, 0.5, 1, 2, 5, 10, 20, or 60 min. Reaction products were separated by 15% PAGE gel containing 8 M urea (inset). Product formation by APE exonuclease was determined as a function of incubation time. The rates of exonuclease of APE were calculated from the initial velocities. For the situations where the velocities decreased with time, the data were fit to an exponential equation and the initial velocity was determined at t = 0 (i.e. initial slope). Data for the graphs were taken from a representative experiment. (䊉) Mismatched and (䊏) matched substrates.

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to this synthetic analogue was stronger than binding to the natural deoxyribose. For the mismatched substrates, there was a large difference in exonuclease activity of APE between the 5 -THF phosphate-containing substrate versus the 5 -phosphate-containing substrate. In other experiments (not shown), the intrinsic nature of the APE exonuclease activity was confirmed using the APE Y171A mutant, an alteration in the 3 -exonuclease active site domain; we found that this mutant did not have activity on these nicked substrates. 3.6. PARP-1 can activate of APE exonuclease activity on the 5 -THF-containing substrate PARP-1 and APE interact with the 5 -THF phosphatecontaining BER intermediate substrate, and in the case of the APE, this can lead to product inhibition of the APE endonuclease activity [29]. This product binding may be related to the weak 3 -exonuclease activity observed with the 5 -THF phosphate-containing substrate (Table 2). The

ability of PARP-1 to stimulate APE 3 -exonuclease activity on this substrate was examined. In these experiments (Fig. 6), the APE 3 -exonuclease activity on the mismatched substrate was weak, as shown above. However, when APE and PARP-1 were co-incubated with the substrate, the APE 3 -exonuclease activity was higher (Fig. 6A and B). These results were not due to exonuclease contamination of PARP-1, since PARP-1 did not significantly degrade the substrate in the absence of APE. These data are consistent with the hypothesis that PARP-1 can compete with APE for binding at the 5 -THF site, and as a result, more APE is released from the 5 -THF site and is available for 3 -exonuclease activity.

4. Discussion In our photoaffinity cross-linking experiments with model BER intermediates, we found that both PARP-1 and APE showed a preference for interacting with the 5 -THF

Fig. 6. PARP-1 stimulates the APE 3 -exonuclease activity. (A) A 34-base pair duplex DNA containing a T–G mismatch at position 16 and a tetrahydrofuran phosphate (FP) at the 5 -end of the nick was prepared as described in Section 2. The DNA substrate (100 nM) was incubated with (+) or without (−) APE (100 nM) and/or PARP-1 (100 nM), as indicated. Reactions were incubated at 37 ◦ C for 5, 10, 20, and 30 min. Lanes 1, 5, 9, and 13 are minus protein controls (no enzymes added). The cleaved product (**) migrates faster than the 16-mer substrate. A schematic representation of the substrate is shown at the top of the photograph of the gel. (B) The rate of exonuclease activity of APE with (䊏) or without (䊉) PARP-1 was determined as a function of incubation time. APE exonuclease activity was calculated from the initial velocities, as in Fig. 5.

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phosphate-containing intermediate over the 5 -phosphate containing intermediate. The synthetic THF sugar phosphate group was used here, and in other studies, for convenience because it is resistant to spontaneous and dRP lyase mediated removal. Although THF itself is not biologically relevant, we consider the binding specificity observed for this sugar analogue as reflective of binding to the natural deoxyribose phosphate-containing BER intermediate. The results presented here support the idea that PARP-1 and APE interact with the BER intermediate containing the 5 -deoxyribose phosphate terminus and under certain circumstances can compete for interaction at this site. This interplay between PARP-1 and APE could have important implications for BER. We examined the substrate specificity of APE 3 exonuclease using matched and mismatched nicked DNA oligonucleotide substrates with 5 -phosphate, 5 -dRP or 5 -THF phosphate (see Materials and methods). The results confirmed recent findings by several groups that APE 3 -exonuclease is lower on substrates with the 5 -THF phosphate group at nicks (Fig. 5B and C; references [19,20,34,35]). These results suggest that after endonucleolytic cleavage and release of the product, APE can bind to the 5 -THF phosphate flap-containing structures and, hence, is not able to participate as effectively in the 3 -exonuclease reaction. This picture of product binding by APE has interesting implications. From in vitro studies of BER with purified enzymes, it is known that removal of the natural 5 -deoxyribose phosphate group by ß-pol is slower than the single-nucleotide gap filing DNA synthesis step. Therefore, after ß-pol has incorporated a dNMP residue, APE may bind to the 5 -sugar phosphate flap-containing structure. The product recognition feature of APE, allowing it to bind the BER intermediate after ß-pol has inserted a nucleotide, would mean that APE is in proximity to both proofread and facilitate the dRP lyase activity of ß-pol [36]. Competition between PARP-1 and APE for binding to the BER intermediate may depend on the amount of each protein present in the nucleus and factors affecting both the location and binding ability of the proteins. The ratio of APE and PARP-1 is not constant in cells. For example Ramana et al. [37] showed that HeLa cells treated with doses of hypochlorite displayed an increase in APE mRNA and protein. Increased expression of APE has also been reported in cervical cancers [38], germ cell tumors [39] and prostate cancer [40]. On the other hand, upregulation of PARP-1 expression has not been reported, either associated with exposure to genotoxic agents or in cancer to our knowledge. Tartier et al. [41] used a microbeam to locally irradiate mammalian cells and showed that poly(ADP-ribose) synthesis increased, but PARP-1 protein was not upregulated. PARP-1 is considered to be involved in DNA strand break damage recognition [42–44]. PARP-1 binds DNA single-strand breaks and synthesizes (ADP-ribose) polymers from NAD+ ; this results in auto-poly(ADP-ribosyl)ation of PARP-1 and poly(ADP-ribosyl)ation of many other pro-

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teins involved in DNA metabolism (i.e. DNA polymerases and histones); poly(ADP-ribosyl)ated PARP-1 dissociates from DNA and loses its catalytic activity until the automodification has been removed and it binds again to DNA. PARP-1-null mice are hypersensitive to alkylating agents and [45], among other possibilities, this is consistent with the idea that PARP-1 may play a role in BER. A physical interaction between PARP-1, X-ray repair cross complementing group 1 (XRCC1) and ß−pol has been observed, and this too is consistent with a role for PARP-1 in BER. Recently, PARP-1 was found to interact with 5 -dRP-containing BER intermediates [15,46] and to stimulate strand displacement DNA synthesis by ß-pol and 5 -flap cleavage by FEN1 [15,47]. Further, repair of AP sites was impaired in cell-free extracts of PARP-1 null MEFs [16], and Le Page et al. [48] showed that the poly(ADP-ribosyl)ation activity of PARP-1 is essential for repair of 8-oxoG in a non-transcribed plasmid sequence in mouse cells. In summary, it is likely that BER proteins act in a coordinated manner, yet mechanisms that facilitate such coordination beyond protein–protein interactions remain to be identified. Results presented here illustrate how specificity for recognition of the BER intermediates themselves could enable APE and PARP-1 coordination. When APE binds to the 5 -dRP-containing BER intermediate, it may prevent PARP-1 binding. Hence, APE may conduct both endonuclease and accessory protein functions in BER.

Acknowledgements We thank Jennifer Myers for editorial assistance, Miriam Sander for assistance with preparation of the manuscript, and Dr. Phyllis Strauss for her generous gift of APE Y171A. We also thank Drs. Thomas Kunkel, Kataryzyna Bebenek, William Beard, and Julie Horton for critical reading of the manuscript. Olga I. Lavrik was supported, in part, by a grant from the Russian foundation for Basic Research (02-04-48404) and by a Collaborative Linkage grant from NATO-Ref. 978233.

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