Involvement of Flap Endonuclease 1 in Base Excision DNA Repair*

Kyung Kim, Siham Biade, and Yoshihiro MatsumotoDagger

From the Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Base excision repair can proceed in either one of two alternative pathways: a DNA polymerase beta -dependent pathway and a proliferating cell nuclear antigen (PCNA)-dependent pathway. Excision of an apurinic/apyrimidinic (AP) site by cutting the phosphate backbone on its 3' side following incision at its 5' side by AP endonuclease is a prerequisite to completion of these repair pathways. Using a reconstituted system with the proteins derived from Xenopus laevis, we found that flap endonuclease 1 (FEN1) was a factor responsible for the excision of a 5'-incised AP site in the PCNA-dependent pathway. In this pathway, DNA synthesis was not required for the action of FEN1 in the presence of PCNA and a replication factor C-containing fraction. The polymerase beta -dependent pathway could also use FEN1 for excision of the synthetic AP sites, which were not susceptible to beta -elimination. In this pathway, FEN1 was functional without PCNA and replication factor C but required the DNA synthesis, which led to a flap structure formation.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Base excision repair is a major mechanism for repair of damaged bases in DNA (1). Major lesions to be repaired by this mechanism are bases with a relatively small modification or apurinic/apyrimidinic (AP)1 sites. These types of damage are induced by ionizing radiation and exposure to various chemicals, such as alkylating agents, as well as by attack from reactive species generated during normal cellular metabolism. To protect their DNA from such persistent damage formation, most living organisms are equipped with vital mechanisms for base excision repair. In a present model for base excision repair (2), an altered base is removed by a specific DNA-N-glycosylase to leave an AP site, which is accordingly a common intermediate. The next step in this mechanism is the incision of the phosphate backbone immediately 5' to the AP site by AP endonuclease, generating a 3'-hydroxyl terminus and a 5'-terminus with a deoxyribose phosphate (dRP) group. The 5'-dRP residue is then excised, and a DNA polymerase fills in the gap. Finally, the DNA strand is sealed by the action of a DNA ligase.

Recent studies with in vitro repair systems derived from vertebrates indicate that AP site repair in higher eukaryotes may proceed by either one of two alternative pathways: a DNA polymerase beta  (pol beta )-dependent pathway and a proliferating cell nuclear antigen (PCNA)-dependent pathway (3, 4). The pol beta -dependent pathway requires a minimum of three proteins for AP site repair: AP endonuclease, pol beta , and DNA ligase. In this pathway, pol beta  catalyzes not only DNA synthesis but also excision of a dRP residue (5). This dRP excision is via beta -elimination catalyzed by the amino-terminal 8-kDa domain of pol beta . Consequently, the pol beta -dependent pathway can repair an unmodified natural AP site efficiently but not a reduced AP site or a synthetic AP site analog, 3-hydroxy-2-hydroxymethyltetrahydrofuran (tetrahydrofuran), neither of which is susceptible to beta -elimination. In contrast, the PCNA-dependent pathway is able to repair the synthetic AP site analog as efficiently as the natural AP site (3). Therefore, the excision of dRP residues during PCNA-dependent repair is not via beta -elimination but seems to be via hydrolysis. PCNA-dependent AP site repair has been reconstituted in vitro with proteins from Xenopus laevis ovaries consisting of three homogeneously purified proteins, AP endonuclease, PCNA, and DNA polymerase delta , and two protein fractions, BE-1B and BE-2 (3). Although BE-1B and BE-2 have not been purified as homogeneous proteins, the requirement of these fractions for DNA synthesis, excision of AP sites, and ligation steps suggests that replication factor C (RF-C), a 5'right-arrow3' exonuclease, and a DNA ligase are included in one or the other of these fractions.

Flap endonuclease 1 (FEN1) was originally isolated as a DNA structure-specific endonuclease that cleaves a flap strand of branched DNA with a 5' single-stranded terminus at the position near its junction to the double-stranded structure (6). Subsequently, it was found to be identical or homologous to previously isolated proteins DNaseIV (7), pL (8), 5'right-arrow3' exonuclease (9), and MF1 (10). Thus FEN1 carries several distinct nuclease activities on specific structured DNA substrates. It works as an endonuclease on 5'-flap structured DNA, as a 5'right-arrow3' exonuclease on nicked or gapped dsDNA (9, 11), and as a ribonuclease on RNA-primed Okazaki fragments generated during discontinuous DNA replication (10, 12). In addition, FEN1 has an activity for removing 5'-incised AP sites (13, 14). Furthermore, recent studies demonstrate that PCNA directly binds to FEN1 and stimulates its activity (15, 16). Therefore, FEN1 is a candidate for the factor responsible for AP site excision in the PCNA-dependent repair pathway.

In this study, we cloned a cDNA coding for X. laevis FEN1 (X-FEN1). Using the highly purified protein expressed from this cDNA, we show here that FEN1 is essential for PCNA-dependent AP site repair and is found in the BE-2 fraction from X. laevis ovaries. FEN1 can also assist the pol beta -dependent pathway in the absence of PCNA to repair the synthetic AP site analog, which cannot be removed via beta -elimination. The mode of its function in the pol beta -dependent pathway is, however, distinct from that in the PCNA-dependent pathway.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Repair Factors-- PCNA, AP endonuclease, and the BE-2 fraction from X. laevis ovaries were prepared as described previously (3). The BE-1 fraction was prepared by the procedures described (3) except that S150 extract (17) was used as a starting material instead of the F3 fraction.

Cloning of a cDNA Coding for X-FEN1-- A part of the X-FEN1 coding cDNA was obtained by PCR amplification of total X. laevis oocyte cDNA with degenerative primers designed for the conserved amino acid sequences between mouse and human FEN1 (see Fig. 1). This 240-kilo-base pair partial cDNA fragment was used as a probe for screening an X. laevis oocyte cDNA library in lambda  ZAP vector (18). Positive phage clones were isolated and converted to plasmids according to the manufacturer's instructions (Stratagene). The clone carrying the longest cDNA insert (1.9 kilobases) was chosen for further characterization. This cDNA fragment was digested with EcoRI and either KpnI or SalI, and the resultant, smaller fragments were subcloned into pBS- (Stratagene). Each subclone was sequenced with a reverse primer and a T7 primer with an automated sequencer (Applied Biosystems).

Overproduction and Purification of X-FEN1-- The coding region of the cloned X-FEN1 cDNA was transferred into a pRSET vector for overexpression in bacteria (19). The X-FEN1 protein was overproduced from the pRSET carrying the X-FEN1 cDNA in a bacterial strain, BL21(lambda DE3)(pLysS) (Ref. 20; obtained from Novagen), by induction with 1 mM isopropyl-1-thio-beta -D-galactopyranoside for 3 h at 25 °C. The bacterial cells from a 250-ml culture were harvested by centrifugation and lysed by sonication in Buffer A (50 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 0.1 mM EGTA, 20% glycerol, 1 mM dithiothreitol, 2 mM benzamidine-HCl, 0.2 mM phenylmethylsulfonyl fluoride) plus 500 mM NaCl. After centrifugation at 18,000 × g for 20 min, the supernatant was diluted with 1.5 volumes of Buffer A without NaCl and loaded onto a 10-ml column of DEAE-Sepharose FF (Amersham Pharmacia Biotech) that had been preequilibrated with Buffer A plus 200 mM NaCl. The flow-through fraction from the DEAE column was diluted with an equal volume of Buffer A without NaCl and loaded onto a 1-ml Hitrap SP column (Amersham Pharmacia Biotech) that had been preequilibrated with Buffer A plus 100 mM NaCl. Elution was performed with a linear gradient of 100-500 mM NaCl in Buffer A. The fractions containing the FEN1 protein were pooled, diluted with Buffer A without NaCl to give a conductivity equivalent to 70 mM NaCl, and loaded onto a 1-ml column of single-stranded DNA cellulose (Sigma). FEN1 was recovered by a stepwise elution with Buffer A plus 500 mM NaCl.

The two site-directed mutants of X-FEN1, D86A and D181A, were constructed by a PCR-based method (21). These mutant proteins were overproduced and purified in the same manner as the wild-type FEN1 protein.

Anti-X-FEN1 Antibody-- Polyclonal antibodies against X-FEN1 were raised in a rabbit by three injections with the purified X-FEN1 protein by the standard procedures (22). Polyclonal antiserum from the rabbit was purified by using a 1-ml column of X-FEN1-affinity resin that had been prepared using Affi-Gel 10 (Bio-Rad) and the purified X-FEN1 protein (1.2 mg) according to manufacturer's instructions. Rabbit serum was diluted 10-fold in 10 mM Tris-HCl (pH 7.5) and passed 10 times through the X-FEN1-affinity column. The column was then washed with 20 ml of 10 mM Tris-HCl (pH 7.5) followed by another wash with 20 ml of 500 mM NaCl, 10 mM Tris-HCl (pH 7.5). The anti-X-FEN1 antibody was then eluted with 100 mM glycine (pH 2.5) and neutralized with 100 mM Tris-HCl (pH 8.0).

Immunoblotting of X-FEN1-- Protein samples were subjected to electrophoresis in an SDS-containing 10% polyacrylamide gel as described by Laemmli (23) and electrotransferred to an Immobilon P membrane (Millipore) in transfer buffer (10 mM 3-cyclohexylamino-1-propane sulfonic acid (pH 11.0), 10% methanol) at 100 V for 1 h in a cold room as described by LeGendre et al. (24). The blot was incubated with 1:4000 dilution of anti-X-FEN1 antibody in PBS plus 0.5% Tween 20 overnight. The immunoreactive proteins were visualized by colorimetric development after incubation with alkaline phosphatase-conjugated anti-rabbit IgG secondary antibody.

AP Site Repair Assay-- 32P-Prelabeled covalently closed circular DNA substrates containing a synthetic AP site analog, 3-hydroxy-2-hydroxymethyltetrahydrofuran (tetrahydrofuran) residue, were prepared as described previously (3). Among these substrates, the 5'-labeled DNA has a [32P]phosphate at the position 5 nucleotides away from the lesion toward 5', whereas the 3'-labeled DNA has a 32P atom at the position 10 nucleotides away from the lesion toward 3'. Repair assay with these covalently closed circular DNA substrates was conducted as described previously (3). Briefly, the indicated proteins were incubated with either the 5'- or 3'-labeled DNA containing a tetrahydrofuran site at 25 °C for 30 min. In a standard reaction, 7 ng of X. laevis AP endonuclease, 20 ng of X. laevis PCNA, 30 ng of X-FEN1, and 0.4 µg of protein of the BE-1 fraction were employed. For the pol beta -dependent repair with three purified proteins, 7 ng of AP endonuclease, 50 units of X. laevis pol beta , and 0.1 unit of T4 DNA ligase (Life Technologies, Inc.) were used. After the reaction was stopped by the addition of SDS, DNA was recovered by phenol/chloroform extraction and ethanol precipitation, digested with either PvuII or HinfI and subjected to electrophoresis in a denaturing 6% (for the PvuII digests) or 20% (for the HinfI digests) polyacrylamide gel. Subsequently, the gel was subjected to autoradiography with an x-ray film and was quantitatively analyzed with a Fuji BAS1000 phosphorimage analyzer.

AP Site Excision Assay-- Two oligonucleotides complementary to each other (see Fig. 3; the 3'-terminal 32P-adenosine in the upper strand was added afterward) were prepared by an automated DNA synthesizer in the DNA synthesis facility of Fox Chase Cancer Center. A tetrahydrofuran site in the upper strand was introduced by using its phosphoramidite derivative, dSpacer (Glen Research Corp.). The prelabeled oligonucleotide substrate was prepared by annealing the two oligonucleotides and end-filling at the 3' end of the lesion-containing strand with [alpha -32P]dATP and reverse transcriptase. In the AP site excision assay, this oligonucleotide substrate (40 fmol) was incubated with 100 ng of undamaged plasmid DNA, 7 ng of X. laevis AP endonuclease, and indicated proteins in 10 µl of the reaction buffer containing 20 mM HEPES-KOH (pH 7.5), 100 mM KCl, 10 mM MgCl2, 1 mM dithiothreitol, 0.1 mg/ml bovine serum albumin. After incubation at 25 °C for 30 min, the reaction mixtures were mixed with 10 µl of sequencing gel loading solution (95% formamide, 0.5% SDS, 0.025% bromphenol blue, 0.025% xylene cyanol FF), boiled for 1 min, and subjected to electrophoresis on a 20% polyacrylamide gel containing 8 M urea (40 cm long) at 2400 V for 7 h. Subsequently, the gel was subjected to autoradiography with an x-ray film and also analyzed with a Fuji BAS1000 phosphorimage analyzer.

Detection of the PCNA/X-FEN1 Interaction-- The indicated amounts of PCNA and FEN1 were mixed in a 10 µl of buffer containing 20 mM HEPES-KOH (pH 7.5), 50 mM KCl, 0.1 mM EDTA, 20% glycerol, 1 mM dithiothreitol, 0.01% Triton X-100, and 0.1 mg/ml bovine serum albumin and incubated on ice for 20 min. Subsequently, the samples were subjected to electrophoresis in a 5% polyacrylamide gel (ratio of acrylamide to bis, 38:1) in TBE buffer (45 mM Tris borate, 1 mM EDTA). The proteins were electrotransferred to an Immobilon-P membrane and detected by immunoblotting with a monoclonal anti-PCNA antibody, 19F4 (a generous gift from Bruce Stillman), or the affinity-purified anti-FEN1 antibody as described above.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Isolation of an X. laevis FEN1 Homolog-- To test whether FEN1 can serve for excision of a dRP residue during AP site repair by the reconstituted system with X. laevis proteins, we first isolated a cDNA coding for X. laevis FEN1. Because the amino acid sequences of human and mouse FEN1 are 96% identical (25, 26), we designed three degenerative primers from their conserved sequences and used them for nested PCR amplification on X. laevis oocyte cDNA. One of the PCR products thus obtained coded an amino acid sequence that was highly homologous to mammalian FEN1. This cDNA fragment was used as a probe for screening a cDNA library derived from X. laevis oocytes. One of the clones we isolated from the cDNA library contained an open reading frame coding for a protein, the amino acid sequence of which was more than 80% identical to mammalian FEN1 (Fig. 1). For further characterization, the protein coded by this cDNA, named X-FEN1, was overproduced in bacteria and purified as a more than 95% homogeneous protein as described under "Experimental Procedures."


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Fig. 1.   Amino acid sequence of X-FEN1. The X-FEN1 sequence is aligned with those of mouse FEN1 (M-FEN1; Ref. 25) and human FEN1 (H-FEN1; Ref. 26), in which - and . represent a residue that is the same as in X-FEN1 and a deletion, respectively. Among these sequences, three regions from which degenerative primers were designed for PCR amplification are indicated with a thick underline.

Identity of X-FEN1 as a Factor in the BE-2 Fraction Essential for AP Site Excision-- Previous biochemical characterization of X. laevis repair factors suggested that the BE-2 fraction may contain a 5'right-arrow3' exonuclease, the size of which should be between 29 and 66 kDa (3). To examine whether the BE-2 fraction contains the X-FEN1 protein, we prepared a polyclonal antibody against recombinant X-FEN1 and used it for immunoblotting. This anti-X-FEN1 antibody recognized a protein in BE-2 that had the same mobility as that of the recombinant X-FEN1 in SDS-containing polyacrylamide gel electrophoresis (Fig. 2A). The same result was obtained with another anti-X-FEN1 antibody prepared independently by D. Carroll and colleagues (data not shown). Comparison of the signal obtained from BE-2 with those from various amounts of purified X-FEN1 revealed that the BE-2 fraction included approximately 30 ng of X-FEN1 per µl (Fig. 2A).


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Fig. 2.   Comparison of the recombinant X-FEN1 and the BE-2 fraction. A, immunoblotting of X-FEN1. One µl of the BE-2 fraction (lane 1) and 30 (lane 2), 3 (lane 3), and 0.3 (lane 4) ng of the purified protein of the recombinant X-FEN1 were subjected to electrophoresis in an SDS-containing 10% polyacrylamide gel and immunoblotted with an affinity-purified anti-X-FEN1 antibody as described under "Experimental Procedures." B, repair complementation with X-FEN1. Repair of the synthetic AP site (tetrahydrofuran) was conducted as described under "Experimental Procedures" with AP endonuclease, PCNA, the BE-1 fraction, and one of the following proteins: no additional repair factor (lane 1); 0.5 µl of the BE-2 fraction (lane 2); 1 (lane 3), 3 (lane 4), 10 (lane 5), and 30 (lane 6) ng of the wild-type X-FEN1; 1 (lane 7), 3 (lane 8), 10 (lane 9), and 30 (lane 10) ng of the X-FEN1-D86A mutant; 1 (lane 11), 3 (lane 12), 10 (lane 13), and 30 (lane 14) ng of the X-FEN1-D181A mutant. After the reaction, DNA was digested with PvuII and subjected to electrophoresis in a denaturing 6% polyacrylamide gel. The percentage of repaired DNA in each reaction, which was calculated after scanning the gel with a phosphorimage analyzer, is shown in the bottom panel.

We tested the ability of the recombinant X-FEN1 protein to replace the BE-2 fraction in a reconstituted system for AP site repair. The DNA substrate used in this system contains a tetrahydrofuran site, a lesion that is repaired 4-fold more efficiently by the PCNA-dependent pathway than by the pol beta -dependent pathway (3). In these assays, we employed X. laevis AP endonuclease, PCNA, and the BE-1 fraction that supplied DNA polymerase delta , RF-C, and DNA ligase (3). As expected, the BE-2 fraction was essential for complete AP site repair (Fig. 2B, lanes 1 and 2). In addition, the recombinant X-FEN1 protein also supported the repair activity when the BE-2 fraction was omitted from the assay (Fig. 2B, lanes 3-6). Ten ng of purified X-FEN1 supported a similar level of AP site repair to that of 0.5 µl of BE-2. This relative activity of the BE-2 fraction compared with the purified X-FEN1 is in good agreement with the relative quantity of X-FEN1 in the same fraction estimated by immunoblotting. Furthermore, we tested two site-directed mutants of X-FEN1, D86A and D181A. Human FEN1 proteins carrying the corresponding mutations have been reported to lose flap endonuclease activity (21). The X-FEN1 mutant proteins also lost the activity for supporting AP site repair (Fig. 2B, lanes 7-14), ruling out the possibility that contaminated bacterial proteins, rather than X-FEN1, might complement the repair activity. Taken together, these results are consistent with the hypothesis that X-FEN1 is the essential factor for the activity of the BE-2 fraction in AP site repair.

Damage Excision by X-FEN1-- The recombinant X-FEN1 was tested for the activity for removing a dRP residue from the 5'-incised AP site. In this experiment, we employed a double-stranded oligonucleotide substrate carrying a tetrahydrofuran residue in the middle of one strand (Fig. 3A). In the presence of AP endonuclease, which cut the phosphate backbone at the 5' side of AP sites, X-FEN1 converted the incised product to shorter fragments (Fig. 3B, lane 3). Compared with the molecular size marker corresponding to the product that lost only the 5'-terminal tetrahydrofuran residue (Fig. 3B, lane 1), these short products appeared to result from excision of the tetrahydrofuran together with one or more 3'-adjacent nucleotides from the incised AP site. We could not detect an intermediate resulting from excision of only the sugar phosphate residue. This is consistent with the previous report that FEN1 cannot remove the 5'-terminal dRP residue as a free form but only as a part of an oligonucleotide (27).


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Fig. 3.   Excision of the 5'-incised AP site. A, structure of the double-stranded oligonucleotide substrate used for excision assay. A tetrahydrofuran residue and a 32P label are designated by Delta  and an asterisk, respectively. The fragment to be produced by AP endonuclease incision (incised) and the fragment to result from excision of only the AP site from the incised product (excised) are also indicated. B, excision by X-FEN1. Excision reactions were conducted as described under "Experimental Procedures" with AP endonuclease and the following repair proteins: no additional repair protein (lane 2); 10 ng of X-FEN1 (lane 3); 10 ng of X-FEN1 and 20 ng of PCNA (lane 4); 10 ng of X-FEN1 and 0.4 µg of BE-1 protein (lane 5); 10 ng of X-FEN1, 20 ng of PCNA, and 0.4 µg of BE-1 protein (lane 6). As a molecular size marker, the DNA fragment corresponding to the one resulting from excision of only the AP site from the incised fragment was loaded in lane 1.

Mammalian FEN1 is known to be bound to and stimulated by PCNA (15, 16). To examine whether X-FEN1 can bind to PCNA, we subjected mixtures of PCNA and X-FEN1 to native polyacrylamide gel electrophoresis followed by immunoblotting. In this gel electrophoresis system, the free X. laevis PCNA protein ran toward the anode (Fig. 4A, lane 1), whereas the free X-FEN1 protein barely entered the gel (Fig. 4B, lane 3). The difference in their mobilities seems to result from their isoelectric points (4.3 for X. laevis PCNA; 7.9 for X-FEN1). When mixed with X-FEN1, X. laevis PCNA exhibited a slower mobility in gel electrophoresis (Fig. 4A, lane 2). The band detected by an anti-PCNA antibody was also recognized by the anti-X-FEN1 antibody (Fig. 4B, lane 2), indicating that it should contain a complex of X-FEN1 and PCNA. The experiment with increasing amounts of the X-FEN1 protein revealed that the molar ratio of the two proteins in the complex was approximately 1 to 1 (Fig. 4C). We also examined the effect of X. laevis PCNA and RF-C on the X-FEN1 activity. In the excision assay with the oligonucleotide substrate, however, neither the purified PCNA protein nor the BE-1 fraction containing the RF-C activity increased the efficiency of removal of the 5'-terminal tetrahydrofuran residues by X-FEN1 (Fig. 3B, lanes 4-6). A possible explanation for the failure of PCNA to stimulate X-FEN1 in this experiment is that the linear oligonucleotide used in the excision assay may be a poor substrate for the PCNA-dependent reaction. Podust et al. (28) reported that PCNA was much less stably loaded on linear DNA than on circular DNA. We also observed that the PCNA-dependent pathway was able to efficiently repair AP sites on circular DNA but not on linear DNA (29). Thus, all of the subsequent experiments were conducted with circular DNA as a repair assay substrate.


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Fig. 4.   Interaction between X-FEN1 and PCNA. Native 5% polyacrylamide gel electrophoresis with X-FEN1 and PCNA was conducted as described under "Experimental Procedures." These proteins were detected by immunoblotting with anti-PCNA antibody (A and C) or anti-X-FEN1 antibody (B). In A and B, the following proteins were subjected to gel electrophoresis after a 20-min incubation on ice: 2 µg of PCNA (lane 1); 2 µg of PCNA and 2 µg of X-FEN1 (lane 2); 2 µg of X-FEN1 (lane 3). In C, 1 µg of PCNA (35 pmol of monomer) and the following amounts of X-FEN1 were mixed and subjected to gel mobility shift assay: no X-FEN1 (lane 1); 0.038 µg of X-FEN1 (0.89 pmol) (lane 2); 0.076 µg of X-FEN1 (1.8 pmol) (lane 3); 0.15 µg of X-FEN1 (3.6 pmol) (lane 4); 0.38 µg of X-FEN1 (8.9 pmol) (lane 6); 0.76 µg of X-FEN1 (18 pmol) (lane 6); 1.5 µg of X-FEN1 (36 pmol) (lane 7). Under this electrophoresis condition with TBE buffer (pH 8.3), most of the free X-FEN1 molecules remained in the sample well.

Analysis of Intermediate Products during FEN1-associated Repair-- To further characterize the action of FEN1 in base excision repair, we analyzed intermediate products in the repair reaction in which either the ligation or the DNA synthesis step was blocked. Ethidium bromide is known to strongly inhibit the activity of most of DNA ligases. We tested whether this DNA intercalating agent could specifically block the ligation step during AP site repair. Among the four steps in AP site repair, the incision by AP endonuclease turned out to be strongly inhibited by ethidium bromide (data not shown). When the DNA substrates had been treated with AP endonuclease prior to the addition of ethidium bromide, neither DNA synthesis nor dRP excision was significantly suppressed by the addition of 10 µg/ml ethidium bromide (Fig. 5A). However, this concentration of ethidium bromide blocked ligation and, therefore, more than 80% of the repair of tetrahydrofuran sites. Therefore, we examined the dRP excision by X-FEN1 under conditions in which the ligation step was blocked by 10 µg/ml ethidium bromide. As shown in Fig. 5B, excision of dRP residues by X-FEN1 was observed only when both PCNA and the BE-1 fraction were added together to the reaction, suggesting that X-FEN1 required PCNA and RF-C for its action on circular DNA.


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Fig. 5.   Analysis of intermediate products in the repair reaction blocked by ethidium bromide. A, titration of ethidium bromide. The indicated concentrations of ethidium bromide (EtBr) were added to the repair reaction of either the 5'- or 3'-labeled AP site-containing DNA that had been predigested with AP endonuclease. The repair reactions were conducted as described under "Experimental Procedures" with PCNA, X-FEN1, and the BE-1 fraction. After the repair reaction, DNA was recovered, digested with HinfI, and analyzed by electrophoresis in a denaturing 20% polyacrylamide gel. B, stimulation of the excision activity of X-FEN1 by PCNA and BE-1. Repair reactions were conducted in the presence of 10 µg/ml ethidium bromide (except for lane 1, in which ethidium bromide was omitted from the reaction) with the indicated proteins and either the 3'- or the 5'-labeled DNA that had been pretreated with AP endonuclease.

Because the BE-1 fraction also contains pol delta , it is still possible that DNA synthesis might be involved in stimulating the dRP excision carried out by the flap endonuclease activity of X-FEN1. To test this possibility, we examined the repair reaction in which DNA synthesis was suppressed by omitting dNTPs from the reaction mixture (Fig. 6). As a result, the AP site repair was almost completely hindered, whereas X-FEN1 still efficiently excised the AP site residues in the presence of PCNA and the BE-1 fraction (Fig. 6A, lane 8). The products resulting from excision by FEN1 lost the AP site residue and at least one 3'-adjacent nucleotide (Fig. 6A, compare lanes M1, M2, and 8). In the absence of either PCNA or the BE-1 fraction, however, X-FEN1 scarcely catalyzed the excision reaction (lanes 2, 6, and 7). This result indicates that X-FEN1, along with PCNA and RF-C, can efficiently excise a dRP residue even from the 5'-terminus that does not form a stable flap structure.


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Fig. 6.   Analysis of intermediate products in the repair reaction without dNTPs. A, repair of the synthetic AP site with various combinations of protein factors. Repair reactions on the 3'-labeled DNA were conducted as described under "Experimental Procedures" in the absence or presence of 20 µM dNTPs with AP endonuclease and the indicated proteins. After the repair reaction, DNA was digested with HinfI and subjected to electrophoresis in a denaturing 20% polyacrylamide gel. Positions of the repaired product (I), the ligation intermediate (II), the fragment just after incision by AP endonuclease (III), and two molecular weight markers (IV and V) are indicated by arrows. The ligation intermediate is a product resulting from addition of AMP to the 5'-terminus of the incised fragment by DNA ligase. The molecular weight makers, IV and V (loaded on lanes M1 and M2), were prepared by digestion of the unmodified DNA with RsaI and KpnI, respectively. B, structures of repair products, intermediates, and molecular weight markers. A synthetic AP site analog and a 32P label are designated by Delta  and an asterisk, respectively.

Supplementation of the Pol beta -dependent Repair with X-FEN1-- As previously reported, the reconstituted system consisting of AP endonuclease, pol beta , and DNA ligase cannot repair the tetrahydrofuran site because this lesion is not susceptible to beta -elimination (5). However, the study of the reconstituted system with X. laevis proteins (3) demonstrates that the pol beta -dependent pathway can repair the tetrahydrofuran site at a low level in the presence of the BE-2 fraction, which now turns out to contain X-FEN1. Our recent observation also indicated that a mouse cell extract repaired the tetrahydrofuran site by the pol beta -dependent pathway (29), although not efficiently. In these repair reactions, FEN1 may play a role in the pol beta -dependent pathway to excise the tetrahydrofuran residue. Therefore, we tested whether X-FEN1 can assist the pol beta -dependent pathway to repair the synthetic AP site analog. When the 5'-labeled covalently closed circular DNA containing a tetrahydrofuran residue was incubated with AP endonuclease, pol beta , and DNA ligase, repaired products were not detected, but some intermediate products, resulting from an elongation by DNA synthesis of several nucleotides, were observed (Fig. 7A, lane 2). When X-FEN1 was added to this repair system, however, the synthetic AP site was efficiently repaired (Fig. 7A, lane 3). Because this repair system did not include either PCNA or RF-C, the mechanism for the FEN1 action may be different from that in the PCNA-dependent reaction. To elucidate the PCNA-independent mechanism of FEN1 excision, we further characterized the X-FEN1 action during the pol beta -dependent repair reaction with the 3'-labeled DNA (Fig. 7B). Unlike PCNA and BE-1, which together stimulated excision by X-FEN1 without DNA synthesis (Fig. 7B, lane 10), pol beta  by itself did not stimulate the X-FEN1 activity for excision (lane 4). However, the addition of dNTPs with pol beta  to the reaction significantly induced the X-FEN1 activity (lane 8), suggesting that the flap structure formation at the AP site by the strand-displacing DNA synthesis was a prerequisite to its excision by X-FEN1 in this situation. This is in stark contrast to the FEN1 action in the PCNA-dependent pathway, in which the flap structure was not essential for the excision.


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Fig. 7.   Synthetic AP site repair by the pol beta -dependent pathway supplemented with X-FEN1. A, repair of the synthetic AP site with purified factors. Repair reactions on the 5'-labeled DNA were conducted as described under "Experimental Procedures" with AP endonuclease, T4 DNA ligase, and the indicated proteins. B, stimulation of the excision activity of X-FEN1 by pol beta  or PCNA and BE-1. Repair reactions on the 3'-labeled DNA were conducted as described under "Experimental Procedures" in the absence or presence of 20 µM dNTPs with AP endonuclease and the indicated proteins.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The results presented here demonstrate that FEN1 is a factor involved in the PCNA-dependent pathway for base excision repair. Excision of AP sites is a prerequisite to completion of base excision repair. Typically, it proceeds via an incision of the phosphate backbone at the 5' side of the AP site followed by another incision at its 3' side. Whereas the 5' incision is mostly carried out through hydrolysis by AP endonuclease, the 3' incision of AP sites can proceed via either beta -elimination or hydrolysis. In higher eukaryotes, pol beta  has an activity for excision of a dRP residue from 5'-incised AP site through beta -elimination (5). Thus, the pol beta -dependent pathway is able to complete repair of natural AP sites without additional factors for 3' incision. On the other hand, FEN1 is able to excise a dRP residue from the 5'-incised AP site via hydrolysis (13, 14). In this reaction, the 5'-terminal dRP residue is released together with its 3'-adjacent nucleotide(s) as a part of an oligonucleotide (27). The hydrolytic excision by FEN1 can be applied not only to unmodified natural AP sites but also to modified or synthetic AP sites that are refractory to beta -elimination. These characteristics of FEN1 confer two significant properties on the PCNA-dependent pathway for base excision repair. First, this pathway can repair modified or synthetic AP sites as efficiently as unmodified natural AP sites. Second, this pathway replaces at least two nucleotides during the repair reaction. These properties are distinct from those of the pol beta -dependent pathway, which repairs preferentially natural AP sites through the replacement of a single nucleotide (30, 31).

FEN1 can physically bind to PCNA with a stoichiometry of three FEN1 molecules to one PCNA trimer (16, 32). The result of our gel mobility shift of PCNA with X-FEN1 provides additional evidence for their strong interaction. Li et al. (15) reported that the flap endonuclease activity of FEN1 on oligonucleotide substrates was stimulated when a highly excess amount of PCNA was added. They also observed a similar stimulation of FEN1 exonuclease activity by PCNA on circular DNA but not on oligonucleotide substrates. We here demonstrated that PCNA with the RF-C-containing fraction induced the X-FEN1 activity for AP site excision on circular DNA but not on oligonucleotides. The stimulation of FEN1 by PCNA on circular DNA was detected with a reasonable molar ratio for their stoichiometric interaction. The simplest model provided from our result and the established function of PCNA and RF-C is that FEN1 bound to PCNA is recruited to the nicked site by RF-C. Because we employed the BE-1 fraction instead of the purified RF-C, it is still possible that other factors in BE-1, such as DNA polymerase delta  or DNA ligase, may also participate in the stimulation of X-FEN1. In previous studies with an S150 extract from X. laevis oocytes, we observed that omission of dNTPs from the repair reaction blocked not only DNA synthesis but also excision of the 5'-terminal AP site (33). This result appears to contradict the result described here. One possible explanation to resolve this contradiction is that DNA polymerase delta  (or epsilon ) may block the nuclease activity of FEN1 in the absence of DNA synthesis in the S150 extract, whereas an excess amount of FEN1 in the reconstituted system may overcome the blocking by DNA polymerase. It remains to be determined whether PCNA can interact with DNA polymerase delta /epsilon and FEN1 simultaneously and, if so, whether the 5'-terminus of the nicked site can be accessible to FEN1 in this complex.

The activity for AP site excision by X-FEN1 was also stimulated by pol beta . Because it was observed only when DNA synthesis was allowed, the stimulation by pol beta  seems to target the flap endonuclease activity. Li et al. (15) and Wu et al. (16) reported that the flap endonuclease of FEN1 can be stimulated by PCNA. In our experiments, however, the efficiency of AP site excision by X-FEN1 with pol beta  was comparable to that by X-FEN1 with PCNA and the BE-1 fraction, suggesting that PCNA is not essential for the FEN1 activity on the flap-structured DNA formed by pol beta . These observations may be explained by the possibility that a flap-structured DNA can be a better substrate for FEN1 than a nicked DNA and accordingly can be processed at an apparently efficient rate without PCNA. Indeed, it has been reported that FEN1 has a 2-4-fold higher affinity for the flap structure than the nicked site (11). Another possibility is that pol beta  may play the role of PCNA in stimulation of FEN1 on a flap-structured DNA. However, this possibility was ruled out by our observation that once DNA synthesis displaced several nucleotides of the downstream strand carrying a 5'-incised AP site, X-FEN1 was equally active for excision in either the presence or the absence of pol beta  (data not shown). Thus, the generation of a 5'-flap structure is sufficient to explain the stimulation of FEN1 by pol beta .

The efficient excision of AP sites by FEN1 with pol beta  allowed the pol beta -dependent pathway to repair tetrahydrofuran sites that cannot be excised by dRP lyase activity of pol beta . Recently, Klungland and Lindahl (34) reported that FEN1 is involved in base excision repair for the AP sites reduced with sodium borohydride and the oxidized AP sites generated by ionizing radiation. These lesions are similar to the tetrahydrofuran sites used in this study for their lack of susceptibility to beta -elimination. However, the results described here do not support their model, in which the pol beta -dependent repair assisted by PCNA-stimulated FEN1 may be a major contributor to the PCNA-dependent pathway in base excision repair. Furthermore, it was observed that a pol beta -knockout cell extract repaired the tetrahydrofuran site as efficiently as a pol beta -proficient isogenic cell extract (29). This observation clearly indicates the participation of a DNA polymerase other than pol beta , most likely pol delta  or epsilon , in the repair of modified AP sites.

Eukaryotes have other structure-specific nucleases and 5'right-arrow3' exonucleases in addition to FEN1. These enzymes may also serve for excision of altered AP sites. XPG is a protein coded by the gene responsible for the xeroderma pigmentosum G phenotype and is required for nucleotide excision repair. It is also homologous to FEN1 in three domains and has a structure-specific endonuclease activity that incises single-stranded DNA at its 3' border to double-stranded DNA (25, 35). When it is substituted for FEN1, however, XPG cannot support repair of altered AP sites (34).2 However, the possibility is not ruled out that some other factor(s) that can interact with XPG may be required for excision of AP sites by XPG. DNaseV has both 5'right-arrow3' and 3'right-arrow5' exonuclease activities and forms a complex with pol beta  (36). It is reported that DNaseV enhances the repair synthesis by pol beta  at the nicked site generated by AP endonuclease (37). Although this enzyme has not been well characterized yet, DNaseV may be a nuclease to excise altered AP sites in the pol beta -dependent pathway. It has not been determined whether DNaseV can interact with PCNA. Thus, it remains to be examined whether FEN1 is the only factor responsible for the excision step in the PCNA-dependent base excision repair.

Yeast FEN1 homologs (the RAD27 gene product in Saccharomyces cerevisiae and the rad2 gene product in Schizosaccharomyces pombe) have been characterized for their cellular functions. The yeast cells lacking the functional RAD27 or rad2 gene exhibit various phenotypes, such as moderate sensitivity to UV radiation, hypersensitivity to methylmethane sulfonate, deficient chromosome segregation, conditional lethality, and accumulation of S phase cells (26, 38-40). These phenotypes suggest involvement of FEN1 homologs in DNA replication, repair, and cell cycle control. Among these phenotypes, the hypersensitivity to the alkylating agent particularly supports our conclusion that FEN1 is one of the factors employed in base excision repair. Recently, Tishkoff et al. (41) reported that a mutation in RAD27 resulted in a strong mutator phenotype that caused duplication of sequences ranging from 5 to 108 base pairs flanked by direct repeats of 3-12 base pairs. These authors proposed that this mutagenic process may be initiated at DNA replication on the lagging strand, where the RAD27 gene product is required for legitimate joining of Okazaki fragments. In the RAD27-deficient cells, a displaced 5'-flap may not be removed efficiently, leading to a high incidence of duplication between distant short direct repeats (Fig. 8A). Our results provide an additional model for generation of the same type of mutations, which can be initiated during base excision repair in RAD27-deficient cells (Fig. 8B). It is likely that the duplications generated during base excision repair may be relatively short compared with those arising from DNA replication because, unlike the replication machinery, the base excision repair complex may lack some factors, such as a helicase and a single-strand DNA-binding protein, that are required for displacing a long range of the downstream DNA strand. This model can be tested by examining whether treatment with the alkylating agent can increase the specific type of mutations in these cells.


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Fig. 8.   Schematic models of duplications generated in FEN1-deficient cells. A, model for DNA replication. B, model for base excision repair. A 5'-incised AP site is designated by Delta .

    ACKNOWLEDGEMENTS

We thank Gordon Chan for providing the X. laevis total cDNA; M. Bibikova and D. Carroll for providing their anti-X. laevis FEN1 antibody; Bruce Stillman for the anti-PCNA antibody; R. D. Wood for the purified XPG protein; and A. Bellacosa, S. W. Johnson, R. A. Katz, and A. T. Yeung for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants CA63154 and CA06927 and by an appropriation from the Commonwealth of Pennsylvania.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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF036327.

Dagger To whom correspondence should be addressed: Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111. Tel.: 215-728-5272; Fax: 215-728-4333; E-mail: y_matsumoto{at}fccc.edu.

1 The abbreviations used are: AP, apurinic/apyrimidinic; FEN1, flap endonuclease 1; PCNA, proliferating cell nuclear antigen; RF-C, replication factor C; pol, DNA polymerase; dRP, deoxyribose phosphate; PCR, polymerase chain reaction.

2 K. Kim and Y. Matsumoto, unpublished data.

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Top
Abstract
Introduction
Procedures
Results
Discussion
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