Proliferating Cell Nuclear Antigen Facilitates Excision in Long-patch Base Excision Repair*

Ronald GaryDagger §, Kyung Kim, Helen L. CorneliusDagger , Min S. ParkDagger , and Yoshihiro Matsumoto

From the Dagger  Life Sciences Division, M888, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 and the  Department of Radiation Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111

    ABSTRACT
Top
Abstract
Introduction
References

There are two distinct pathways for the removal of modified DNA bases through base excision repair (BER) in vertebrates. Following 5' incision by AP endonuclease, the pathways diverge as two different excision mechanisms are possible. In short-patch repair, DNA polymerase beta  accounts for both excision activity and single nucleotide repair synthesis. In long-patch repair, the damage-containing strand is excised by the structure-specific endonuclease FEN-1 and approximately 2-8 nucleotides are incorporated by proliferating cell nuclear antigen (PCNA)-dependent synthesis. PCNA is an accessory factor of DNA polymerases delta  and epsilon  that is required for DNA replication and repair. PCNA binds to FEN-1 and stimulates its nuclease activity, but the physiological significance of this interaction is unknown. The importance of the PCNA-FEN-1 interaction in BER was investigated. In a reconstituted BER assay system containing FEN-1, omission of PCNA caused the accumulation of pre-excision reaction intermediates which could be converted to completely repaired product by addition of PCNA. When dNTPs were omitted from the reaction to suppress repair synthesis, PCNA was required for the formation of excised reaction intermediates. In contrast, a PCNA mutant that could not bind to FEN-1 was unable to stimulate excision. To further study this effect, a mutant of FEN-1 was identified that retained full nuclease activity but was specifically defective in binding to PCNA. The mutant FEN-1 exhibited one-tenth the specific activity of wild type FEN-1 in the reconstituted BER assay, and this repair defect was due to a kinetic block at the excision step as evidenced by the accumulation of pre-excision intermediates when dNTPs were omitted. These results indicate that PCNA facilitates excision during long-patch BER through its interaction with FEN-1.

    INTRODUCTION
Top
Abstract
Introduction
References

Damage to DNA bases can occur spontaneously under physiological conditions. Bases may be modified by deamination, or lost entirely due to hydrolysis of the N-glycosidic bond that links them to the sugar-phosphate backbone. Reactive oxygen species that are the by-products of normal cellular respiration also damage DNA. Exogenous agents such as chemical mutagens and ionizing radiation are additional sources of base modification. Aberrant DNA bases generated by such processes can be restored by base excision repair (BER)1 (reviewed in Refs. 1 and 2). This repair system includes several types of DNA glycosylases that recognize and remove many types of modified bases to leave an apurinic or apyrimidinic site (AP site). Alternatively, an AP site may be the direct result of damage. The AP site must be removed during the course of repair. AP endonuclease initiates the process by hydrolyzing the phosphodiester bond immediately to the 5' side of the abasic site. This enzyme is capable of recognizing various classes of abasic lesions (3). After this incision has been made, there are two mechanistically distinct methods for AP site removal that distinguish short-patch BER from long-patch BER in higher eukaryotes. Short-patch BER is a DNA polymerase beta -dependent pathway that requires an unaltered deoxyribose phosphate (dRP) sugar moiety as the AP site. The 5'-terminal dRP resulting from AP endonuclease incision is removed in a beta -elimination reaction catalyzed by DNA polymerase beta , leaving a 1-nucleotide gap (4). Single nucleotide repair synthesis by DNA polymerase beta  fills the gap, and the nick is sealed by the DNA ligase III/XRCC1 heterodimer or other ligase to complete repair.

In long-patch BER, the repair patch size is typically 2-8 nucleotides in length. This pathway also utilizes AP endonuclease for 5'-incision, but the AP site is not removed by DNA polymerase beta . Instead, the flap endonuclease/5'-3' exonuclease FEN-1 (reviewed in Ref. 5) removes the 5'-terminal dRP moiety along with at least one adjacent nucleotide to leave a gap of two or more nucleotides (6, 7). Because it relies on phosphodiester bond hydrolysis for excision, long-patch BER can repair regular AP sites and also altered AP sites that are not susceptible to beta -elimination. Sites with oxidized or reduced sugar groups and those with fragmented bases or sugars can be repaired only by long-patch BER. This versatility may be especially important in the repair of DNA damage caused by ionizing radiation. For example, about 80% of the gamma -irradiation-induced DNA lesions that can be repaired by BER are resistant to dRP elimination by DNA polymerase beta  and require FEN-1 for processing (6). The investigation of long-patch BER in cell extracts and reconstituted assay systems has been aided by the availability of synthetic AP site analogs such as 3-hydroxy-2-hydroxymethyltetrahydrofuran that cannot be repaired by short-patch BER. Repair of such sites requires proliferating cell nuclear antigen (PCNA) (Refs. 8 and 9), a toroidal homotrimeric DNA-binding protein that encircles template DNA. PCNA forms a holoenzyme complex with DNA polymerases delta  or epsilon  in conjunction with replication factor C (RF-C), the PCNA loading factor (reviewed in Ref. 10). PCNA dependence in long-patch BER has also been demonstrated using a regular AP site as substrate and selectively observing repair patches of greater than 1 nucleotide in length by exploiting strategically placed restriction sites (11) or position-specific deoxyribonucleotide incorporation (12). These results suggest a model in which AP endonuclease, FEN-1, PCNA, RF-C, a PCNA-dependent DNA polymerase, probably DNA polymerase delta , and DNA ligase are used for long-patch BER. Complete repair can be achieved with these proteins (6-8), and cell extracts lacking DNA polymerase beta  are fully capable of repair (9, 12). However, in some assay systems, DNA polymerase beta  can substitute for the PCNA-DNA polymerase delta  holoenzyme complex in FEN-1-dependent BER (6).

PCNA and FEN-1 are capable of direct physical interaction, as first identified by yeast two-hybrid screening (13) and confirmed by in vitro binding assays (13-18). Furthermore, PCNA can stimulate FEN-1 nuclease activity (13, 14, 18). Both proteins participate in DNA replication and long-patch BER, and thus it is likely that these proteins may co-exist within a macromolecular assembly during either of these processes. Because PCNA and FEN-1 have a proven capacity for interaction, it is tempting to suppose that direct physical interaction of these proteins occurs during one or more steps of replication or repair. The most direct test of this hypothesis is to selectively abolish the ability of PCNA and FEN-1 to interact with one another, and then to determine whether loss of this function affects replication or repair efficiency. We have used this strategy to investigate the potential role of this interaction in long-patch BER. In this report, we show that the PCNA binding activity of FEN-1 is important for excision in BER, thus establishing a specific repair function for the interaction of these two proteins.

    EXPERIMENTAL PROCEDURES

Bacterial Expression Plasmids-- The wild type human PCNA expression plasmid has been described previously (17). The L126D/I128E double point mutant derivative of this plasmid was generated by site-directed mutagenesis using the QuickChange Mutagenesis protocol (Stratagene, La Jolla, CA) with the high-fidelity Pfu DNA polymerase and mutagenic oligonucleotides 5'-GGATTTAGATGTTGAACAGGATGGAGAACCAGAACAG-3' and 5'-CTGTTCTGGTTCTCCATCCTGTTCAACATCTAAATCC-3'. This primer pair created an FokI restriction site to facilitate screening. The plasmid pET-FCH to express wild type human FEN-1 with a 6-histidine C-terminal purification tag has been described (19-21). This plasmid was modified by site-directed mutagenesis to create the F343A/F344A mutant FEN-1 expression plasmid by QuickChange mutagenesis using oligonucleotides 5'-GGCCGCCTGGATGATGCCGCCAAAGTGACCGGCTCACTC-3' and 5'-GAGTGAGCCGGTCACTTTGGCGGCATCATCCAGGCGGCC-3'. This primer pair was designed to destroy a naturally occurring BstEII restriction site in order to facilitate screening. The expression constructs were verified by DNA sequencing. In both the pET-FCH wild type FEN-1 parental vector and the F343A/F344A mutant derivative, a single nucleotide disagreement with the published human FEN-1 sequence (22) was observed. Nucleotide 247 of the FEN-1 open reading frame was "C" rather than "T" as found in the originally published DNA sequence. The resultant CAT codon specifies a histidine 83 residue in both of the proteins (wild type and mutant FEN-1) used in the present study, in place of tyrosine-83 obtained by conceptual translation of the published DNA sequence. The C-247 nucleotide was present in the original plasmid prepared by direct subcloning of the NcoI-BamHI restriction fragment from amplified FEN-1 cDNA (20), raising the possibility that there may be polymorphism at this position. Supporting this possibility, histidine in place of tyrosine is a very conservative amino acid substitution; at an evolutionary distance of 2 PAM (percentage of accepted point mutations), only phenylalanine and tryptophan substitutions are tolerated more frequently (23). In any case, the histidine substitution had no effect on the catalytic activity of FEN-1, based on comparison of enzymatic specific activity with tyrosine 83 wild type FEN-1 created with mutagenic oligonucleotides 5'-AACGGCATCAAGCCCGTGTACGTATTTGATGGCAAGCCGCCA-3' and 5'-TGGCGGCTTGCCATCAAATACGTACACGGGCTTGATGCCGTT-3' (data not shown). This primer pair created a SnaBI restriction site to facilitate screening.

Affinity Interaction Binding Assay-- Escherichia coli strain BL21(DE3) cells harboring either wild type or mutant PCNA expression plasmid, wild type or mutant FEN-1 plasmid, or pET28b vector (Novagen, Madison, WI) without cDNA insert were grown to a density of approximately 0.6 absorbance units at 600 nm, then 0.8 mM isopropyl-beta -D-thiogalactopyranoside was added to induce protein expression and growth was continued for an additional 3 h at 37 °C. Cells were harvested by centrifugation and cell pellets were resuspended in 50 mM Tris-HCl, 150 mM NaCl, 0.2 mg/ml lysozyme, 1 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride, 10 µM pepstatin A, 10 µg/ml chymostatin, 10 µg/ml aprotinin, pH 7.4, at a ratio of 1 ml/35-ml culture and briefly sonicated. The cell lysates were clarified by two cycles of centrifugation at 16,000 × g. Binding assay mixtures consisted of 70 µl of 50% NiSO4-charged iminodiacetic acid metal chelating Sepharose resin (Pharmacia Biotech, Piscataway, NJ), 150 µl of lysate from cells expressing His6-tagged wild type or mutant FEN-1, or negative control lysate from pET28b vector-containing cells, 150 µl of lysate from cells expressing untagged wild type or mutant PCNA, and 150 µl of 50 mM Tris-HCl, 150 mM NaCl, pH 7.4, added to increase mixing efficiency. Mixtures were incubated for 90 min at 4 °C with continuous gentle rocking, then washed six times with 0.8 ml of 50 mM Tris-HCl, 150 mM NaCl, 60 mM imidazole, pH 7.4. Protein complexes were eluted by heating to 100 °C with 80 µl of 2 × Laemmli SDS gel sample buffer, and analyzed on 12% gels by SDS-polyacrylamide gel electrophoresis (PAGE) and Coomassie Blue staining.

Protein Purification-- His6-tagged wild type and mutant FEN-1 were purified with HisBind affinity resin (Novagen) according to the manufacturer's instructions. Wild type and mutant PCNA were purified by a method previously described for wild type PCNA (17). AP endonuclease and the BE-1 fraction from Xenopus laevis ovaries were prepared as described previously (7, 8). The BE-1 fraction contains pol delta , RF-C, and DNA ligase activities. Rat DNA polymerase beta  was overexpressed in bacteria and purified as described previously (4).

DNA Polymerase delta  Stimulation Assay-- Stimulation of the activity of calf thymus DNA polymerase delta  (a generous gift from Drs. Chen-Keat Tan and Antero G. So of the University of Miami) by PCNA proteins was measured as described previously (8). Briefly, each assay contained 0.5 µg of substrate consisting of poly(dA) template and oligo(dT) primer at 5:1 molar ratio in 50 mM bis-Tris-HCl, 10 mM KCl, 6 mM MgCl2, 0.4 mg/ml bovine serum albumin, 1 mM dithiothreitol, 50 µM [alpha -32P]TTP, pH 6.5. One unit of polymerase activity corresponds to the incorporation of 1 pmol of TMP into acid-precipitable material in 30 min at 37 °C.

AP Site Repair Assay-- 32P-Prelabeled covalently closed circular DNA substrates containing a synthetic AP site analog, the 3-hydroxy-2-hydroxymethyltetrahydrofuran residue, were prepared as described previously (8). Among these substrates, the 5'-labeled DNA has a 32P at the position 5 nucleotides away from the lesion toward 5' while the 3'-labeled DNA has a 32P at the position 10 nucleotides away from the lesion toward 3'. Repair assays with these DNA substrates were conducted as described previously (8). Briefly, indicated proteins were incubated with either the 5'- or 3'-labeled DNA containing a 3-hydroxy-2-hydroxymethyltetrahydrofuran site for 30 min at 25 °C in the presence of 20 µM dATP, 20 µM dCTP, 20 µM dGTP, 20 µM TTP, and 2 mM ATP. In a standard reaction, 7 ng of X. laevis AP endonuclease, 10 ng of human PCNA, 10 ng of human FEN-1, and 0.4 µg of protein of the X. laevis BE-1 fraction were used. For the pol beta -dependent repair reactions, 7 ng of AP endonuclease, 10 ng of FEN-1, and 50 ng of rat pol beta  were used. After the reaction was stopped by the addition of SDS, DNA was recovered by phenol/chloroform extraction and ethanol precipitation, digested with HinfI and subjected to electrophoresis in a denaturing 20% polyacrylamide gel. Subsequently, the gel was subjected to autoradiography with an x-ray film and was quantitatively analyzed with a Fuji BAS1000 phosphorimage analyzer.

Flap Endonuclease Assay-- The enzymatic activity of FEN-1 was assayed as described previously (20). Briefly, a three-oligonucleotide 5'-flap substrate was assembled by annealing a 3'-biotinylated 31-mer template strand, a 5'-fluoresceinated 34-mer flap strand (14 nucleotides annealed and 20 nucleotides displaced), and a 16-mer upstream annealed primer strand. The 5'-fluoresceinated flap DNA was attached to streptavidin-coated microspheres, and 50-100 pM substrate was mixed with enzyme in 500 µl of 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 100 µg/ml bovine serum albumin at room temperature. Bead-associated fluorescence was measured in real time from 0 to 500 s by flow cytometry.

Exonuclease Assay-- Three synthetic oligonucleotides were annealed to build a nicked DNA substrate for measurement of exonuclease activity. A 57-mer template strand (5'-TCGAGTTATTTAAACCATGCATCTAATGTTTTTTGCTTAGTTTTGTTTGCAAGCTTG-3') was annealed to a perfectly complementary 36-mer primer (5'-AGCAAAAAACATTAGATGCATGGTTTAAATAACTCG-3'). The resulting duplex DNA possessed a 1-nucleotide gap at the 3'-end of the 36-mer that was used as a labeling site for incorporation of [alpha -32P]dATP. A 100-µl reaction containing 12 µM duplex and 10 units of Sequenase version 2.0 DNA polymerase (U. S. Biochemical, Cleveland, OH) in 25 mM HEPES, pH 7.4, 10 mM MgCl2, 2 mM dithiothreitol, 50 µg/ml bovine serum albumin, 50 µM dATP, 20 µCi of [alpha -32P]dATP was incubated for 10 min at 37 °C to generate a 3'-end 32P-labeled 37-mer strand. The DNA polymerase was inactivated by incubation for 10 min at 70 °C, then unincorporated dATP was separated from the labeled duplex using a 0.8-ml Sephadex G-50 spin column. A 2-fold molar excess of the perfectly complementary upstream primer 20-mer (5'-CAAGCTTGCAAACAAAACTA-3') was annealed to the labeled 37-mer/57-mer duplex to generate an internally nicked substrate with blunt ends. Exonuclease activity was assayed in a 20-µl reaction volume containing 300 nM 32P-labeled DNA substrate and 0, 1, 2, or 4 µM wild type or F343A/F344A mutant FEN-1 in a buffer of 28 mM imidazole, 12.5 mM HEPES, 0.5 mM Tris-HCl, 14 mM NaCl, 5% glycerol, 5 mM MgCl2, 1 mM dithiothreitol, 25 µg/ml bovine serum albumin, pH 7.4. Samples were incubated for 60 min at 32 °C, then reactions were terminated by the addition of 20 µl of 94% formamide, 10 mM EDTA, 0.1% xylene cyanol, 0.1% bromphenol blue and heated to 90 °C for 10 min. 15 µl of this mixture (approximately 10,000 cpm per lane) was run on a 15% polyacrylamide (19:1 acrylamide:bis-acrylamide), 7 M urea, 10 mM Tris-HCl, 1 mM EDTA, pH 8.0, denaturing gel at 65 watts constant power for 2 h. The gel was dried and subjected to autoradiography (3 days exposure to Kodax BioMax film) or quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Substrate remaining was calculated as: 37-mer area intensity/total integrated area intensity divided by the mean (37-mer area intensity/total integrated area intensity) of input DNA (the no protein lanes).

    RESULTS

Characterization of the I126D/L128E Mutant of PCNA-- The crystal structure of p21 peptide complexed with human PCNA has been reported (24). FEN-1 interacts with PCNA by a mechanism analogous to that of p21 (16, 17), so the PCNA-p21 structural information was used to design PCNA mutants predicted to exhibit FEN-1-binding defects. A series of mutations was introduced into PCNA, and the mutant proteins were tested for their ability to bind to FEN-1.2 The I126D/L128E double point mutant of PCNA was identified as having a particularly severe defect in FEN-1 binding activity. This mutant of PCNA was purified and characterized (Fig. 1) for use as a tool to investigate the role of the PCNA-FEN-1 interaction in long-patch BER.


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Fig. 1.   Purification and characterization of the I126D/L128E mutant of PCNA. A, purification to homogeneity. Coomassie Blue-stained SDS-PAGE gel shows 4 µg of purified human wild type (lane 1) or I126D/L128E mutant (lane 2) PCNA. The migration positions of molecular mass markers (in kDa) are indicated. B, the I126D/L128E mutant of PCNA does not bind to FEN-1. Nickel-charged metal chelate resin was incubated with cleared bacterial lysate from IPTG-induced cells containing a His6-tagged human FEN-1 expression plasmid (lanes 1 and 2) or vector alone as a control (lanes 3 and 4). An equal aliquot of lysate from bacteria expressing wild type (lanes 1 and 3) or I126D/L128E mutant (lanes 2 and 4) PCNA was added to each assay. After washing, protein complexes were denatured, separated by SDS-PAGE, and detected by Coomassie Blue staining. The expression levels and solubilities of wild type and I126D/L128E mutant PCNA were similar, as shown by SDS-PAGE analysis of 7 µl of each induced cell lysate (lanes 5 and 6, respectively). C, the I126D/L128E mutant of PCNA does not stimulate DNA polymerase delta  activity. DNA polymerase delta  activity is shown as a function of wild type (filled circles) or mutant (open circles) PCNA added per reaction. Polymerase activity was quantitated as the incorporation of 32P-labeled TTP nucleotide into a poly(dA)/oligo(dT) template/primer.

Bacterially expressed wild type and I126D/L128E mutant human PCNA were purified to apparent homogeneity (Fig. 1A). The PCNA subunit migrates with an apparent molecular mass of 34 kDa on a denaturing SDS-PAGE gel, although its calculated molecular mass is 29 kDa. An affinity interaction assay was used to compare the FEN-1 binding activities of wild type and mutant PCNA. Lysates from bacteria expressing polyhistidine (His6)-tagged FEN-1 and either wild type or mutant PCNA were mixed, and nickel-charged metal chelate resin was added to capture the His6-tagged FEN-1 along with any associated PCNA. As expected, wild type PCNA bound avidly to FEN-1 (Fig. 1B, lane 1). In contrast, the I126D/L128E mutant of PCNA displayed only a background level of nonspecific binding (Fig. 1B, lane 2), similar to that observed in negative control assays in which FEN-1 was omitted (Fig. 1B, lanes 3 and 4). Thus, the I126D/L128E mutation virtually abolished FEN-1 binding activity. Nonetheless, both wild type and mutant PCNA formed the characteristic 87-kDa homotrimeric species when analyzed by gel filtration chromatography, confirming that intersubunit interaction was unaffected by the site-directed mutagenesis. On a Sephacryl S-300 HR 16 × 600 mm column, the parameter K, which is given by: (elution volume) - (void volume)/(solvent accessible volume) and which is inversely proportional to log MW, was 0.389 for wild type PCNA and 0.384 for mutant PCNA. Bovine serum albumin, which at 66 kDa is smaller than the 87-kDa PCNA trimer but larger than the 29-kDa PCNA monomer, was used as a standard and gave a K value of 0.405.

The I126D/L128E mutant of PCNA was, however, severely defective in the ability to stimulate DNA synthesis by DNA polymerase delta  (Fig. 1C). Compared with wild type PCNA, the activity of the mutant in this assay was negligible. This defect is the result of an inability of the mutant PCNA to bind to DNA polymerase delta . Recently, very similar mutations have been characterized independently in yeast and human PCNA (18, 25). In human PCNA, the L126S and I128A mutations are moderately and severely impaired, respectively, in their abilities to interact with DNA polymerase delta  (25). Likewise, the I126A/L128A double mutant of yeast PCNA is severely defective in physical interaction with DNA polymerase delta , and must be used at concentrations 1000-fold greater than wild type PCNA to stimulate DNA polymerase delta -mediated DNA synthesis (18). However, this mutant retains normal functional interactions with RF-C, a 5-subunit complex that loads PCNA onto the DNA primer-template junction in an ATP-dependent manner. Interestingly, the I126A/L128A yeast PCNA mutant retains binding to yeast FEN-1 (18), in contrast to the FEN-1-binding defect reported here for the I126D/L128E human PCNA mutant. Residues 126 and 128 line a hydrophobic pocket of PCNA that interacts with protruding hydrophobic residues of PCNA-binding ligands p21 (24) and, by analogy, FEN-1 (16, 17). Thus, the introduction of charged acidic residues at these positions more strongly interferes with the binding of complementary hydrophobic FEN-1 residues at this site.

Base Excision Repair Activities of Wild Type and Mutant PCNA-- The 3-hydroxy-2-hydroxymethyltetrahydrofuran abasic site analog can be completely repaired by long-patch BER in the presence of dNTPs, AP endonuclease, FEN-1, PCNA, and a protein fraction partially purified through four chromatographic steps that has been designated BE-1 (7, 8). BE-1 provides RF-C, DNA polymerase delta , and DNA ligase activities. BER reaction intermediates were observed by selectively omitting various components of the reconstituted system (Fig. 2). In the first step of BER, AP endonuclease makes an incision immediately 5' of the abasic site, producing a nick in the damaged strand. This nick leaves a 3'-hydroxyl that serves as the primer for repair synthesis, and a 5'-terminal AP site that must be excised before repair can be completed. In order to permit observation of modifications to either side of the nick, 32P label was incorporated either 5 nucleotides to the 5' side of the abasic site (5'-labeled; Fig. 2A) or 10 nucleotides to the 3' side of the abasic site (3'-labeled; Fig. 2B) within a covalently closed circular DNA substrate. After termination of the repair reaction, HinfI restriction digestion was used to release the 32P-labeled DNA for analysis by gel electrophoresis. Thus, incision by AP endonuclease cleaves the HinfI-HinfI DNA segment into two fragments that can be observed selectively in accordance with the placement of the 32P label. AP endonuclease recognized and incised the 3-hydroxy-2-hydroxymethyltetrahydrofuran lesion very efficiently, as virtually all of the 32P-labeled substrate was nicked to produce 5'-labeled (Fig. 2A, lane 1) or 3'-labeled (Fig. 2B, lane 1) incised fragment. The 3'-terminal hydroxyl of the 5'-32P-labeled incised fragment was extended via repair synthesis in the presence of dNTPs, BE-1 (providing RF-C and DNA polymerase delta ), and wild type PCNA (Fig. 2A, lane 3). As expected, mutant PCNA was unable to support DNA synthesis (Fig. 2A, lane 4). Complete repair to yield the ligated full-length HinfI-HinfI DNA fragment was very efficient with all of the constituents of the repair reaction present (Fig. 2A, lane 6).


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Fig. 2.   Analysis of repair intermediates of wild type or mutant PCNA. A, repair of the circular DNA substrate labeled with 32P on the 5' side of the synthetic AP site. B, repair of the circular DNA substrate labeled with 32P on the 3' side of the synthetic AP site. Repair reactions were conducted as described under "Experimental Procedures," with various combinations of proteins in the absence or presence of dNTPs as indicated. Proteins used in these experiments were AP endonuclease (AP endo.), FEN-1, protein fraction BE-1 (containing RF-C, DNA polymerase delta , and DNA ligase), and wild type (wt) or I126D/L128E mutant (mut.) PCNA. After termination of the repair reaction, DNA was digested with HinfI, electrophoresed in an 8 M urea-containing 20% polyacrylamide gel, and subjected to autoradiography. Positions of repaired products and intermediates are indicated for each autoradiogram. The ligation intermediate is a product resulting from addition of AMP to the 5'-terminal AP site of the incised fragment by DNA ligase. Diagrams indicate the relative positions of 32P label, the AP site, and the HinfI restriction sites for each panel.

The 5'-32P-labeled substrate is most useful for the observation of repair synthesis. Because removal of the synthetic AP site requires a second cleavage event to the 3' side of the initial incision, 3'-32P-labeled substrate must be used to analyze excision. AP endonuclease treatment produced a 3'-labeled incised fragment with a 5'-terminal AP site (Fig. 2B, lane 1). Although this AP end cannot be ligated, DNA ligase can covalently attach the AMP moiety from ATP to the 5'-terminal AP site via a pyrophosphoryl linkage to form a species referred to as a ligation intermediate (7), by analogy with the intermediate generated during normal ligation of base-containing ends. Addition of BE-1, which contains DNA ligase, converted most of the incised fragment to ligation intermediate (Fig. 2B, lane 2). Despite the presence of FEN-1 nuclease in this reaction, little if any excision was observed. However, the inclusion of wild type PCNA greatly stimulated excision by FEN-1 (Fig. 2B, lane 3). Excision was evident as the conversion of ligation intermediate to smaller product and as the appearance of an excised band that corresponds in size to the removal of the 5'-terminal AP site plus one additional nucleotide (characterized in Ref. 7). It is likely that some of the excised product became modified by DNA ligase to generate a novel post-excision ligation intermediate that co-migrates with the incised product; this explains the apparent increase in the intensity of the incised band upon activation of excision (compare lanes 2 and 3 of Fig. 2B). Minor excision products corresponding to removal of successive additional nucleotides are sometimes seen in this assay system (7). The inclusion of the I126D/L128E mutant of PCNA stimulated the production of only a small amount of excised product (Fig. 2B, lane 4). The slight stimulation of excision observed with the mutant PCNA may indicate that interaction with FEN-1, while dramatically reduced, is not completely abolished by this mutation. Consistent with the results shown in Fig. 2A, wild type but not mutant PCNA supported complete repair in the fully reconstituted reaction containing all components (Fig. 2B, lanes 6 and 7).

The I126D/L128E mutant of PCNA, unable to interact with DNA polymerase delta  and FEN-1, produced reaction intermediates that indicated both a repair synthesis defect (Fig. 2A) and an excision defect (Fig. 2B). This second observation suggested that the excision step of BER may be facilitated by PCNA and, furthermore, that this activity of PCNA may be mediated through direct physical interaction with FEN-1. To further investigate this possibility, a series of mutations was introduced into FEN-1 and the ability of the mutant proteins to bind to PCNA was evaluated.2 From these studies, the F343A/F344A double point mutant derivative of FEN-1 was identified. By analogy with p21, the substituted phenylalanines were predicted to be critical for the interaction of FEN-1 with the hydrophobic ligand-binding pocket of PCNA.

Characterization of the F343A/F344A Mutant of FEN-1-- The His6-tagged wild type human FEN-1 and its F343A/F344A derivative were purified to near homogeneity by metal chelate affinity chromatography (Fig. 3A). The F343A/F344A mutant of FEN-1 was severely defective in binding to PCNA. In the affinity interaction assay, wild type FEN-1 bound well to PCNA, whereas mutant FEN-1 showed little if any specific binding to PCNA (Fig. 3B). However, the catalytic behavior of the F343A/F344A mutant of FEN-1 was identical to that of the wild type enzyme as judged by several criteria. The enzyme concentration dependence of 5'-flap DNA substrate cleavage was measured in real time using a flow cytometry-based endonuclease assay (Fig. 4) as described previously (20). The apparent first-order rate constant (kobs) was estimated from the half-time of the reaction using the formula kobs = ln2/(t1/2) and plotted as a function of enzyme concentration. Although the reaction mechanism is likely to be more complex, the data can be interpreted in terms of a two-step mechanism consisting of a binding step and a cleavage step (20, 21). At low enzyme concentrations, the reaction kinetics are dominated by the binding step and are thus sensitive to enzyme concentration. At high enzyme concentrations, the binding step becomes very fast and the reaction kinetics become limited by the cleavage rate. Thus, the plot of kobs versus [enzyme] is similar to a binding curve, and the data can be described well by fitting to a single site binding curve which has the shape of a hyperbola (y = (a)(x)/(b + x), where y = kobs, chi  = [FEN-1], a = kobs(max), and b = [FEN-1] at 1/2 kobs(max)) (20, 21). The best fit curve indicates a saturating kobs of 0.07 s-1 for both the wild type and mutant enzymes, in good agreement with previous estimates of the cleavage rate constant for wild type FEN-1 (20). Half-maximal binding was 173 nM (±45, S.E.) for wild type and 136 nM (± 51) for mutant FEN-1, showing that substrate affinities were identical within the limits of experimental error. Wild type and mutant FEN-1 were also compared using 32P-labeled 5'-flap DNA as substrate so that reaction products could be analyzed by gel electrophoresis and autoradiography. These products were of the same size and intensity, indicating no difference in cleavage site preference between the two proteins (data not shown). In an exonuclease assay using nicked substrate, the activities of wild type and F343A/F344A mutant FEN-1 were again indistinguishable (Fig. 5). 5'-3' exonucleolytic degradation from the nick site yielded an identical pattern of reaction products regardless of protein used. Enzymatic specific activities were the same as determined by 2-fold serial dilution of proteins over the concentration range of interest. The amount of undegraded substrate remaining after reaction with wild type FEN-1 at 1, 2, or 4 µM was 90.2, 71.3, and 11.8%, respectively, whereas substrate remaining after reaction with mutant FEN-1 at 1, 2, or 4 µM was 95.5, 80.6, and 12.4%. Thus, the introduction of the F343A/F344A mutation selectively modified FEN-1 such that binding to PCNA was abolished while all aspects of catalytic activity were unaffected. Because the catalytic and PCNA-binding regions of FEN-1 are well defined and distinct (16, 17), it is not surprising that such specificity can be achieved.


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Fig. 3.   Purification and characterization of the F343A/F344A mutant of FEN-1. A, purification to near homogeneity. Coomassie Blue-stained SDS-PAGE gel shows 7 µg of purified His6-tagged human wild type (lane 1) or F343A/F344A mutant (lane 2) FEN-1. The migration positions of molecular mass markers (in kDa) are indicated. B, the F343A/F344A mutant of FEN-1 does not bind to PCNA. Nickel-charged metal chelate resin was incubated with cleared bacterial lysate from IPTG-induced cells containing vector alone (lane 1), wild type (lane 2), or F343A/F344A mutant (lane 3) His6-tagged FEN-1 expression plasmid. An equal aliquot of lysate from bacteria expressing wild type PCNA was added to each assay (lanes 1-3). After washing, protein complexes were denatured, separated by SDS-PAGE, and detected by Coomassie Blue staining.


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Fig. 4.   The F343A/F344A mutant of FEN-1 has wild type endonuclease activity. Beads bearing immobilized 5'-fluoresceinated flap DNA substrate were incubated with purified enzyme at room temperature, and bead-associated fluorescence was monitored as a function of time by flow cytometry. Assays were conducted using wild type (A) or F343A/F344A mutant (B) FEN-1 at 360 nM (filled circles), 180 nM (open circles), 36 nM (filled triangles), 18 nM (open triangles), 7.2 nM (filled squares), or 3.6 nM (open squares). Substrate fluorescence was stable over time in the absence of enzyme (filled diamonds). C, the observed first-order rate constant based on the time to achieve 50% substrate consumption is shown for wild type (filled circles) and F343A/F344A mutant (open circles) FEN-1 at each enzyme concentration. A hyperbolic substrate binding model (described under "Results") was used to generate a best fit curve with equal weighting to each enzyme concentration for wild type (solid line) and F343A/F344A mutant (dashed line) FEN-1 rate constant data.


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Fig. 5.   The F343A/F344A mutant of FEN-1 has wild type exonuclease activity. The exonuclease activity of wild type and mutant FEN-1 was assayed using a linear double-stranded oligonucleotide substrate bearing a single nick on one strand. As illustrated in the diagram, a 32P label was incorporated at the 3'-end of the strand that was susceptible to degradation by the 5'-3' exonuclease activity of FEN-1. The DNA substrate was incubated with no protein (lanes 1 and 5), wild type FEN-1 (lanes 2-4), or F343A/F344A mutant FEN-1 (lanes 6-8) at enzyme concentrations of 1 µM (lanes 2 and 6), 2 µM (lanes 3 and 7), or 4 µM (lanes 4 and 8). Reaction products were electrophoresed in a 7 M urea-containing 15% polyacrylamide gel, and subjected to autoradiography. The specific activities and exonuclease cleavage site preferences of wild type and mutant FEN-1 were essentially identical.

Base Excision Repair Activities of Wild Type and Mutant FEN-1-- Wild type and F343A/F344A mutant FEN-1 were combined with other BER reaction components to repair the 3'-32P-labeled substrate (Fig. 6). Each reaction contained the standard amount of AP endonuclease, PCNA, and protein fraction BE-1 (RF-C, DNA polymerase delta , and DNA ligase) plus varying amounts of FEN-1. Repair synthesis was suppressed by omission of dNTPs (Fig. 6, left panel) or permitted by dNTP inclusion (Fig. 6, right panel). All reaction intermediates are the same as those described for Fig. 2B. In the absence of FEN-1, two types of pre-excision intermediates accumulate: the incised product and the ligation intermediate (Fig. 6, lanes 1 and 12). As noted earlier (Fig. 2B), AP endonuclease cleaves the substrate at the AP site to generate the incised fragment, some of which undergoes 5'-phosphoryl-AMP modification by ligase to form the ligation intermediate. These pre-excision reaction intermediates could be further processed by FEN-1 to give an excised fragment (Fig. 6, left panel) or repaired product (Fig. 6, right panel). The excision step was PCNA-dependent, as demonstrated earlier (compare lanes 2 and 3 of Fig. 2B). Mutant FEN-1 was markedly deficient at PCNA-dependent excision (Fig. 6, left panel) and complete repair (Fig. 6, right panel) over a range of concentrations up to 1 ng/reaction. The BER concentration dependence curve for mutant FEN-1 was shifted to the right by about 1 log unit compared with that of wild type FEN-1. This indicates that the PCNA binding-defective mutant had only one-tenth the specific activity of wild type FEN-1 in supporting complete repair, even though basal catalytic activity of the two proteins was identical (Figs. 4 and 5). These results show that the physical interaction of PCNA and FEN-1 was required for efficient excision and repair.


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Fig. 6.   Analysis of repair intermediates of wild type or mutant FEN-1 in the PCNA-dependent pathway. All reactions used the circular DNA substrate labeled with 32P on the 3' side of the synthetic AP site, as illustrated in Fig. 2B. Repair reactions were conducted as described under "Experimental Procedures," in the absence (lanes 1-11) or presence (lanes 12-22) of dNTPs, with AP endonuclease, wild type PCNA, and protein fraction BE-1 (containing RF-C, DNA polymerase delta , and DNA ligase) present in each. Reactions included either wild type (wt; lanes 2-6 and 13-17) or F343A/F344A mutant (mut; lanes 7-11 and 18-22) FEN-1 as follows: no FEN-1 (lanes 1 and 12), 0.01 ng (lanes 2, 7, 13, and 18), 0.03 ng (lanes 3, 8, 14, and 19), 0.1 ng (lanes 4, 9, 15, and 20), 0.3 ng (lanes 5, 10, 16, and 21), and 1 ng of FEN-1 (lanes 6, 11, 17, and 22). After termination of the repair reaction, DNA was digested with HinfI, electrophoresed in an 8 M urea-containing 20% polyacrylamide gel, and subjected to autoradiography. Positions of repaired products and intermediates are indicated on the side of the autoradiogram. The excised product (left panel) and repaired product (right panel) were quantitated with a phosphorimage analyzer and plotted as percent of total radioactivity for wild type FEN-1 (filled circles) and mutant FEN-1 (open circles).

With 1 ng/reaction wild type FEN-1, more than 80% of the DNA was repaired in the complete reaction (Fig. 6, right panel), yet, in the absence of dNTPs, only about 50% excision was observed at the same FEN-1 concentration (Fig. 6, left panel). However, the intensity of the signal in the excised band underestimates the total amount of excision that has taken place, because the excised fragment can undergo 5'-phosphoryl-AMP modification by DNA ligase to generate a type of post-excision ligation intermediate that co-migrates with the incised band (evidence for this was discussed for Fig. 2B). Thus, there was a very good quantitative correlation between excision and repair for wild type FEN-1. This was not true for the mutant FEN-1, however. Mutant FEN-1 failed to produce significant excision in the absence of dNTPs, even at 10 ng/reaction (data not shown). These results suggest that wild type and mutant FEN-1 employ different excision mechanisms during PCNA-dependent BER (see "Discussion" for details).

Wild Type and Mutant FEN-1 Behave Similarly in a DNA Polymerase beta -Dependent Repair System-- Recently, it has been shown that AP endonuclease, DNA polymerase beta , and DNA ligase can process reduced AP site analogs if the reaction is supplemented with FEN-1 (6, 7). Using X. laevis proteins, we have demonstrated that FEN-1 can excise a 5'-terminal AP site from the flap structure formed by DNA synthesis with DNA polymerase beta , and that this excision reaction does not require PCNA (7). We tested whether F343A/F344A mutant FEN-1 still retained this PCNA-independent excision activity. At 10 ng/reaction, neither wild type nor mutant FEN-1 exhibited excision activity in the absence of dNTPs (Fig. 7A, lanes 1-3). This contrasts with the excision activity observed at much lower concentration (0.1-1 ng/reaction) for wild type but not mutant FEN-1 under similar conditions in the PCNA-dependent system (Fig. 6, left panel). When dNTPs were included to permit strand-displacing synthesis by DNA polymerase beta  (Fig. 7B), both wild type and mutant FEN-1 proteins efficiently removed approximately 5 nucleotides that included the AP site. The number of nucleotides excised by FEN-1 was in a good agreement with the length of DNA synthesis catalyzed by DNA polymerase beta  in a processive manner (26, 27).


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Fig. 7.   Analysis of repair intermediates of wild type or mutant FEN-1 in the DNA polymerase beta -dependent pathway. All reactions used the circular DNA substrate labeled with 32P on the 3' side of the synthetic AP site, as illustrated in Fig. 2B. Positions of repaired products and intermediates are indicated for each autoradiogram. A, effect of DNA synthesis by DNA polymerase beta  on FEN-1 activity. Repair reactions were conducted as described under "Experimental Procedures," with various combinations of proteins in the absence or presence of dNTPs as indicated. Proteins used in these experiments were AP endonuclease (AP endo.), DNA polymerase beta  (Pol beta ), and wild type (wt) or F343A/F344A mutant (mut) FEN-1. After termination of the repair reaction, DNA was digested with HinfI, electrophoresed in an 8 M urea-containing 20% polyacrylamide gel, and subjected to autoradiography. B, titration of FEN-1 proteins. Reactions were conducted in the presence of dNTPs with AP endonuclease, DNA polymerase beta , and either wild type (wt; lanes 3-7 and 14-18) or F343A/F344A mutant (mut; lanes 8-12 and 19-23) FEN-1 as follows: no FEN-1 (lanes 2 and 13), 0.01 ng (lanes 3, 8, 14, and 19), 0.03 ng (lanes 4, 9, 15, and 20), 0.1 ng (lanes 5, 10, 16, and 21), 0.3 ng (lanes 6, 11, 17, and 22), and 1 ng of FEN-1 (lanes 7, 12, 18, and 23). In lanes 13-23, 10 ng of wild type PCNA was added to the reactions. In lane 1, the DNA substrate was treated with AP endonuclease only to show the incised product. Reaction products were analyzed as described for panel A. Excised product was quantitated with a phosphorimage analyzer and plotted as percent of total radioactivity for wild type FEN-1 (filled circles) and mutant FEN-1 (open circles).

The addition of PCNA did not stimulate nor inhibit FEN-1-mediated excision in this system. Two distinct modes of PCNA-FEN-1 interaction can be envisioned. PCNA and FEN-1 can form a complex that is free of DNA, or the DNA-bound form of PCNA can bind to FEN-1. In studies that use short oligonucleotide substrates (6), it is not clear which mode of interaction may be applicable, because PCNA can bind to DNA with free ends to establish an equilibrium between DNA-bound and DNA-free PCNA. The PCNA loading factor RF-C is not required for this type of DNA binding. In Fig. 7B, a closed circular DNA substrate was used in the absence of RF-C, so DNA-bound PCNA was precluded. PCNA not bound to DNA was unable to stimulate FEN-1 nuclease activity. Given this result, one might expect that PCNA could be inhibitory in this system, if nonproductive DNA-free PCNA-FEN-1 complexes are formed. However, at 0.1 ng/reaction of FEN-1 (the half-maximal concentration), a nearly 50-fold molar excess of PCNA trimer (i.e. 10 ng/reaction) had no effect on excision.

    DISCUSSION

FEN-1 is a structure-specific endonuclease and 5'-3' exonuclease that is involved in Okazaki fragment processing during lagging strand synthesis of DNA replication. Its role in BER was first suggested by genetic experiments in Saccharomyces cerevisiae. Strains lacking RAD27, the yeast homolog of FEN-1, showed increased sensitivity to methyl methanesulfonate, a DNA alkylating agent that generates BER-repairable lesions (28, 29). Recent work has confirmed the requirement for FEN-1 in long-patch BER in vertebrates (6, 7). The DNA-binding protein PCNA has long been recognized as an essential co-factor of the replication and repair DNA polymerases delta  and epsilon . The involvement of PCNA in DNA damage repair has been documented for nucleotide excision repair (30, 31), mismatch repair (32-34), double-strand break repair (35), and long-patch BER (7-9, 11, 12). Repair synthesis mediated by PCNA, RF-C, and DNA polymerase delta  or epsilon  has been shown for many of these processes. Recently, additional functions for PCNA in repair have begun to emerge. PCNA acts in repair synthesis and also at an early presynthesis step in mismatch repair (32-34), and its interaction with the repair endonuclease XPG has functional significance during nucleotide excision repair (17). Here we show that the FEN-1 binding activity of PCNA is important in BER and, furthermore, that this interaction serves to facilitate excision.

The removal of the AP site lesion by excision appears to be a rate-limiting step in BER when the concentration of FEN-1 is not in excess. PCNA and FEN-1 interaction domains were disrupted by mutagenesis, and the non-interacting PCNA and FEN-1 mutant proteins each exhibited excision defects (Figs. 2 and 6). At 0.1, 0.3, and 1.0 ng/reaction, mutant FEN-1 was defective in both excision and overall repair as compared with wild type FEN-1 (Fig. 6). Wild type FEN-1 was about 10-fold more potent in reconstituting repair activity than the mutant FEN-1 that was unable to bind to PCNA. These results demonstrate that direct physical interaction of PCNA and FEN-1 is required for maximum repair efficiency. Thus, physical contact between these proteins appears to be a feature of the BER excision mechanism.

FEN-1 can act as a 5'-3' exonuclease at nicks, and as a structure-specific endonuclease to cleave the unannealed strand of branched structures such as the 5'-flap or pseudo Y. FEN-1 is unable to remove a 5'-terminal AP site as a free sugar phosphate, but it can cleave farther down the strand to excise the AP site as part of a short oligonucleotide, predominantly a dinucleotide (7, 36-38). This property of FEN-1 activity helps to justify in mechanistic terms the clear distinction between short- and long-patch BER; the 1 nucleotide repair patch size in short-patch BER arises because the DNA polymerase beta -catalyzed elimination reaction removes the 5'-terminal AP site only, whereas FEN-1-catalyzed phosphodiester bond hydrolysis releases two or more nucleotides to yield the patch size that defines long-patch BER. The best substrate for FEN-1 nuclease activity in vitro is a 5'-flap structure (39, 40), which has a displaced single strand bearing a free 5'-end and an annealed upstream primer abutting the single-strand/double-strand transition. Strand-displacing synthesis during BER could create this favored 5'-flap substrate, thereby allowing excision by FEN-1 endonuclease action. Such an endo mechanism is clearly involved in the DNA polymerase beta -dependent BER system, because excision was strictly dependent upon strand-displacing synthesis (Fig. 7A).

In the PCNA-dependent BER system, is FEN-1-mediated excision the result of exo- or endonuclease-type activity? In the absence of dNTPs, strand-displacing DNA synthesis cannot proceed, so only an exonuclease-type substrate can be available for excision in such assays. Under these conditions, excision by wild type FEN-1 was well correlated with overall repair observed in parallel reactions containing dNTPs (Fig. 6; as discussed under "Results," the quantitation of the excised band intensity that is plotted underestimates the total extent of excision, because there is another post-excision species that co-migrates with the incised band). Thus, the exonuclease-type excision activity of wild type FEN-1 (Fig. 6, left panel) was adequate to account for most if not all of the excision that was required during complete repair (Fig. 6, right panel). This was not the case for mutant FEN-1, however. Under exo-specific conditions, no appreciable excision activity was observed for mutant FEN-1 at 1 ng/reaction (Fig. 6, lane 11) or 10 ng/reaction (data not shown). However, 1 ng/reaction mutant FEN-1 supported 60% repair (Fig. 6, lane 22) under conditions in which strand-displacing DNA synthesis was possible. Because excision is a prerequisite for complete repair, these data strongly suggest that mutant FEN-1 employed endonuclease-type excision activity during BER. Consistent with this, under the endo-specific conditions of the DNA polymerase beta -dependent assay, wild type and mutant FEN-1 at 0.1-1 ng/reaction were equally efficient at excising the displaced strand (Fig. 7B). In the PCNA-dependent BER pathway, it appears that wild type FEN-1 makes use of a more efficient exo mechanism predominantly, whereas mutant FEN-1 must use a less efficient endo mechanism. If this explanation is correct, BER exonuclease activity requires interaction with PCNA.

In reconstituted systems using purified proteins, either DNA polymerase beta  alone or the PCNA-DNA polymerase delta  complex are capable of providing repair synthesis activities for long-patch BER (6, 7). With respect to excision, these two systems appear to be mechanistically distinct, as the former requires strand-displacing DNA synthesis whereas the latter does not. In long-patch BER assay systems that use cell extracts, PCNA dependence and DNA polymerase beta -independence are both evident (9, 11, 12). Such studies confirm the physiological relevance of the PCNA-dependent pathway for long-patch BER. The new result that PCNA functionally interacts with the essential long-patch repair protein FEN-1 further supports this view. In addition, we have found that yeast strains possessing a mutant rad27 allele that is specifically defective in binding to PCNA grow slowly in the presence of methyl methanesulfonate, suggesting a role for the PCNA-FEN-1 interaction during BER in vivo.3

The short-patch BER pathway is mechanistically well established. An AP site attracts AP endonuclease, which makes the 5'-incision. AP endonuclease physically interacts with DNA polymerase beta  (41), providing a recruitment mechanism for the next enzyme in the sequence of events. After provision of dRP elimination and single nucleotide repair synthesis by DNA polymerase beta , ligation by either DNA ligase I or the DNA ligase III/XRCC1 heterodimer completes repair. The DNA ligase III/XRCC1 heterodimer appears to be the relevant short-patch BER ligase in cell extracts (42), and it binds directly to DNA polymerase beta  (43). Thus, a complete chain of physical interactions is known among short-patch BER proteins that are necessary and sufficient to process the AP site completely. These interactions no doubt increase the efficiency of repair by placing repair enzymes at the appropriate sites in the appropriate sequence, thereby coordinating repair events. An important consequence of such coordination may be that repair intermediates exist only transiently in vivo. Nucleotide gaps and unligated nicks, if allowed to persist while awaiting further processing, might be subject to nonspecific exonuclease action, and would likely be recombinogenic DNA structures as well. The high-efficiency BER processing aided by protein-protein interaction may be important in suppressing such unwanted secondary events. A similar situation may exist in long-patch BER as well. A chain of protein-protein interactions that facilitates long-patch BER is beginning to unfold, with PCNA as the central element. PCNA binds to and functionally interacts with RF-C and DNA polymerase delta , and these proteins together can perform long-patch BER repair synthesis (6-8). PCNA also binds to the essential long-patch BER nuclease FEN-1, an interaction that is functionally important, as shown here. Ligation during long-patch BER probably utilizes DNA ligase I, because XRCC1 mutant cell extracts exhibit a ligation defect affecting DNA polymerase beta -dependent but not PCNA-dependent BER (42), indicating that the DNA ligase III/XRCC1 heterodimer does not act in the latter pathway. PCNA binds to DNA ligase I (44, 45), so this ligase possesses a potential recruitment mechanism that would continue the protein-protein interaction chain that centers about PCNA during long-patch BER.

    ACKNOWLEDGEMENTS

We thank Dr. John Nolan for FEN-1 enzymatic assay data analysis, and Dr. Suman Lee for characterization of the FEN-1 cleavage site in the 5'-flap endonuclease assay. We also thank Drs. Chen-Keat Tan and Antero G. So for providing calf thymus DNA polymerase delta .

    FOOTNOTES

* This work was supported by the Office of Biological and Environmental Research of the U. S. Department of Energy and National Institutes of Health Grants CA71630 (to M. S. P.) and CA63154 (to Y. M.).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: Life Sciences Div., M888, Los Alamos National Laboratory, Los Alamos, NM 87545. Tel.: 505-665-7015; Fax: 505-665-3024; E-mail: gary{at}telomere.lanl.gov.

The abbreviations used are: BER, base excision repair; AP, apurinic/apyrimidinic; dRP, deoxyribose phosphate; FEN-1, flap endonuclease-1; PCNA, proliferating cell nuclear antigen; RF-C, replication factor C; PAGE, polyacrylamide gel electrophoresis; PAM, percentage of accepted point mutations; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol; IPTG, isopropyl-1-thio-beta -D-galactopyranoside.

2 R. Gary and M. Park, unpublished data.

3 R. Gary and D. Gordenin, unpublished data.

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Abstract
Introduction
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