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

Abstract

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 β 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 δ and ε 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. 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 β 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 δ and ε 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. 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) 1The 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-β-d-galactopyranoside.1The 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-β-d-galactopyranoside. (reviewed in Refs. 1Lindahl T. Karran P. Wood R.D. Curr. Opin. Genet. Dev. 1997; 7: 158-169Crossref PubMed Scopus (231) Google Scholarand 2Wilson III, D.M. Thompson L.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12754-12757Crossref PubMed Scopus (215) Google Scholar). 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 (3Wilson III, D.M. Takeshita M. Grollman A.P. Demple B. J. Biol. Chem. 1995; 270: 16002-16007Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). 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 β-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 β-elimination reaction catalyzed by DNA polymerase β, leaving a 1-nucleotide gap (4Matsumoto Y. Kim K. Science. 1995; 269: 699-702Crossref PubMed Scopus (646) Google Scholar). Single nucleotide repair synthesis by DNA polymerase β 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 β. Instead, the flap endonuclease/5′-3′ exonuclease FEN-1 (reviewed in Ref. 5Lieber M.R. BioEssays. 1997; 19: 233-240Crossref PubMed Scopus (395) Google Scholar) removes the 5′-terminal dRP moiety along with at least one adjacent nucleotide to leave a gap of two or more nucleotides (6Klungland A. Lindahl T. EMBO J. 1997; 16: 3341-3348Crossref PubMed Scopus (660) Google Scholar, 7Kim K. Biade S. Matsumoto Y. J. Biol. Chem. 1998; 273: 8842-8848Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). 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 β-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 γ-irradiation-induced DNA lesions that can be repaired by BER are resistant to dRP elimination by DNA polymerase β and require FEN-1 for processing (6Klungland A. Lindahl T. EMBO J. 1997; 16: 3341-3348Crossref PubMed Scopus (660) Google Scholar). 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. 8Matsumoto Y. Kim K. Bogenhagen D.F. Mol. Cell. Biol. 1994; 14: 6187-6197Crossref PubMed Scopus (260) Google Scholar and 9Biade S. Sobol R.W. Wilson S.H. Matsumoto Y. J. Biol. Chem. 1998; 273: 898-902Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar), a toroidal homotrimeric DNA-binding protein that encircles template DNA. PCNA forms a holoenzyme complex with DNA polymerases δ or ε in conjunction with replication factor C (RF-C), the PCNA loading factor (reviewed in Ref.10Kelman Z. Oncogene. 1997; 14: 629-640Crossref PubMed Scopus (712) Google Scholar). 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 (11Frosina G. Fortini P. Rossi O. Carrozzini F. Raspaglio G. Cox L.S. Lane D.P. Abbondandolo A. Dogliotti E. J. Biol. Chem. 1996; 271: 9573-9578Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar) or position-specific deoxyribonucleotide incorporation (12Fortini P. Pascucci B. Parlanti E. Sobol R.W. Wilson S.H. Dogliotti E. Biochemistry. 1998; 37: 3575-3580Crossref PubMed Scopus (200) Google Scholar). These results suggest a model in which AP endonuclease, FEN-1, PCNA, RF-C, a PCNA-dependent DNA polymerase, probably DNA polymerase δ, and DNA ligase are used for long-patch BER. Complete repair can be achieved with these proteins (6Klungland A. Lindahl T. EMBO J. 1997; 16: 3341-3348Crossref PubMed Scopus (660) Google Scholar, 7Kim K. Biade S. Matsumoto Y. J. Biol. Chem. 1998; 273: 8842-8848Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 8Matsumoto Y. Kim K. Bogenhagen D.F. Mol. Cell. Biol. 1994; 14: 6187-6197Crossref PubMed Scopus (260) Google Scholar), and cell extracts lacking DNA polymerase β are fully capable of repair (9Biade S. Sobol R.W. Wilson S.H. Matsumoto Y. J. Biol. Chem. 1998; 273: 898-902Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 12Fortini P. Pascucci B. Parlanti E. Sobol R.W. Wilson S.H. Dogliotti E. Biochemistry. 1998; 37: 3575-3580Crossref PubMed Scopus (200) Google Scholar). However, in some assay systems, DNA polymerase β can substitute for the PCNA-DNA polymerase δ holoenzyme complex in FEN-1-dependent BER (6Klungland A. Lindahl T. EMBO J. 1997; 16: 3341-3348Crossref PubMed Scopus (660) Google Scholar). PCNA and FEN-1 are capable of direct physical interaction, as first identified by yeast two-hybrid screening (13Li X. Li J. Harrington J. Lieber M.R. Burgers P.M.J. J. Biol. Chem. 1995; 270: 22109-22112Crossref PubMed Scopus (254) Google Scholar) and confirmed by in vitro binding assays (13Li X. Li J. Harrington J. Lieber M.R. Burgers P.M.J. J. Biol. Chem. 1995; 270: 22109-22112Crossref PubMed Scopus (254) Google Scholar, 14Wu X. Li J. Li X. Hsieh C.-L. Burgers P.M.J. Lieber M.R. Nucleic Acids Res. 1996; 24: 2036-2043Crossref PubMed Scopus (198) Google Scholar, 15Chen J. Chen S. Saha P. Dutta A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11597-11602Crossref PubMed Scopus (115) Google Scholar, 16Warbrick E. Lane D.P. Glover D.M. Cox L.S. Oncogene. 1997; 14: 2313-2321Crossref PubMed Scopus (135) Google Scholar, 17Gary R. Ludwig D.L. Cornelius H.L. MacInnes M.A. Park M.S. J. Biol. Chem. 1997; 272: 24522-24529Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 18Eissenberg J.C. Ayyagari R. Gomes X.V. Burgers P.M.J. Mol. Cell. Biol. 1997; 17: 6367-6378Crossref PubMed Google Scholar). Furthermore, PCNA can stimulate FEN-1 nuclease activity (13Li X. Li J. Harrington J. Lieber M.R. Burgers P.M.J. J. Biol. Chem. 1995; 270: 22109-22112Crossref PubMed Scopus (254) Google Scholar, 14Wu X. Li J. Li X. Hsieh C.-L. Burgers P.M.J. Lieber M.R. Nucleic Acids Res. 1996; 24: 2036-2043Crossref PubMed Scopus (198) Google Scholar, 18Eissenberg J.C. Ayyagari R. Gomes X.V. Burgers P.M.J. Mol. Cell. Biol. 1997; 17: 6367-6378Crossref PubMed Google Scholar). 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. The wild type human PCNA expression plasmid has been described previously (17Gary R. Ludwig D.L. Cornelius H.L. MacInnes M.A. Park M.S. J. Biol. Chem. 1997; 272: 24522-24529Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). 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 (19Shen B. Nolan J.P. Sklar L.A. Park M.S. J. Biol. Chem. 1996; 271: 9173-9176Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 20Nolan J.P. Shen B. Park M.S. Sklar L.A. Biochemistry. 1996; 35: 11668-11676Crossref PubMed Scopus (61) Google Scholar, 21Shen B. Nolan J.P. Sklar L.A. Park M.S. Nucleic Acids Res. 1997; 25: 3332-3338Crossref PubMed Scopus (95) Google Scholar). 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 (22Hiraoka L.R. Harrington J.J. Gerhard D.S. Lieber M.R. Hsieh C.L. Genomics. 1995; 25: 220-225Crossref PubMed Scopus (63) Google Scholar) 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 theNcoI-BamHI restriction fragment from amplified FEN-1 cDNA (20Nolan J.P. Shen B. Park M.S. Sklar L.A. Biochemistry. 1996; 35: 11668-11676Crossref PubMed Scopus (61) Google Scholar), 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 ofaccepted point mutations), only phenylalanine and tryptophan substitutions are tolerated more frequently (23Schulz G.E. Schirmer R.H. Principles of Protein Structure, Table 9-2. Springer-Verlag, New York1979: 172Google Scholar). 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. 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 mmisopropyl-β-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 mmNaCl, 0.2 mg/ml lysozyme, 1 mm benzamidine, 0.5 mm phenylmethylsulfonyl fluoride, 10 μmpepstatin 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. 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 (17Gary R. Ludwig D.L. Cornelius H.L. MacInnes M.A. Park M.S. J. Biol. Chem. 1997; 272: 24522-24529Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). AP endonuclease and the BE-1 fraction from Xenopus laevis ovaries were prepared as described previously (7Kim K. Biade S. Matsumoto Y. J. Biol. Chem. 1998; 273: 8842-8848Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 8Matsumoto Y. Kim K. Bogenhagen D.F. Mol. Cell. Biol. 1994; 14: 6187-6197Crossref PubMed Scopus (260) Google Scholar). The BE-1 fraction contains pol δ, RF-C, and DNA ligase activities. Rat DNA polymerase β was overexpressed in bacteria and purified as described previously (4Matsumoto Y. Kim K. Science. 1995; 269: 699-702Crossref PubMed Scopus (646) Google Scholar). Stimulation of the activity of calf thymus DNA polymerase δ (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 (8Matsumoto Y. Kim K. Bogenhagen D.F. Mol. Cell. Biol. 1994; 14: 6187-6197Crossref PubMed Scopus (260) Google Scholar). 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 [α-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. 32P-Prelabeled covalently closed circular DNA substrates containing a synthetic AP site analog, the 3-hydroxy-2-hydroxymethyltetrahydrofuran residue, were prepared as described previously (8Matsumoto Y. Kim K. Bogenhagen D.F. Mol. Cell. Biol. 1994; 14: 6187-6197Crossref PubMed Scopus (260) Google Scholar). 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 (8Matsumoto Y. Kim K. Bogenhagen D.F. Mol. Cell. Biol. 1994; 14: 6187-6197Crossref PubMed Scopus (260) Google Scholar). 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 μmdATP, 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. laevisBE-1 fraction were used. For the pol β-dependent repair reactions, 7 ng of AP endonuclease, 10 ng of FEN-1, and 50 ng of rat pol β 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. The enzymatic activity of FEN-1 was assayed as described previously (20Nolan J.P. Shen B. Park M.S. Sklar L.A. Biochemistry. 1996; 35: 11668-11676Crossref PubMed Scopus (61) Google Scholar). 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 nucleotides and 20 nucleotides and a primer The 5′-fluoresceinated flap DNA was to and substrate was with enzyme in μl of 50 mm Tris-HCl, pH 10 mm MgCl2, 100 μg/ml bovine serum at was measured in from to by synthetic oligonucleotides were to a DNA substrate for of exonuclease template strand was to a primer The resulting DNA a 1-nucleotide gap at the of the that was used as a site for incorporation of reaction containing μm and 10 units of DNA polymerase S. in 25 pH 7.4, 10 mm MgCl2, 2 50 μg/ml bovine serum albumin, 50 μmdATP, 20 of was incubated for 10 min at 37 °C to a The DNA polymerase was by for 10 min at 70 then was from the using a molar of the primer was to the to an substrate with activity was assayed in a reaction containing DNA substrate and or 4 μm wild type or F343A/F344A mutant FEN-1 in a of mm imidazole, mm 0.5 mm Tris-HCl, mm NaCl, 5 mm MgCl2, 1 mm dithiothreitol, 25 μg/ml bovine serum albumin, pH 7.4. were incubated for 60 min at then were by the addition of 20 μl of 10 mm and to 90 °C for 10 μl of this was on a polyacrylamide 7 10 mm Tris-HCl, 1 mm pH denaturing gel at for 2 The gel was and subjected to autoradiography to or using a was by the of DNA no protein The of with human PCNA has been Z. J. M. J. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). FEN-1 with PCNA by a to that of E. Lane D.P. Glover D.M. Cox L.S. Oncogene. 1997; 14: 2313-2321Crossref PubMed Scopus (135) Google Scholar, 17Gary R. Ludwig D.L. Cornelius H.L. MacInnes M.A. Park M.S. J. Biol. Chem. 1997; 272: 24522-24529Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar), the was used to PCNA to of was into PCNA, and the mutant proteins were for ability to bind to FEN-1. and M. The double point mutant of PCNA was identified as a defect in FEN-1 binding This mutant of PCNA was purified and for as a to investigate the role of the PCNA-FEN-1 interaction in long-patch BER. wild type and mutant human PCNA were purified to 1 The PCNA with an of on a denaturing its is affinity interaction assay was used to the FEN-1 binding of wild type and mutant PCNA. from bacteria expressing FEN-1 and either wild type or mutant PCNA were and metal resin was added to the His6-tagged FEN-1 along with any PCNA. wild type PCNA to FEN-1 1 In contrast, the mutant of PCNA only a of binding to that in negative control assays in which FEN-1 was omitted 1 3 the FEN-1 binding both wild type and mutant PCNA the homotrimeric species when analyzed by gel that interaction was by the site-directed a × 600 mm the which is and which is to was for wild type PCNA and for mutant PCNA. serum albumin, which at is than the PCNA but than the PCNA was used as a standard and a of The mutant of PCNA defective in the ability to stimulate DNA synthesis by DNA polymerase δ with wild type PCNA, the activity of the mutant in this assay was This defect is the result of an of the mutant PCNA to bind to DNA polymerase very have been in yeast and human PCNA J.C. Ayyagari R. Gomes X.V. Burgers P.M.J. Mol. Cell. Biol. 1997; 17: 6367-6378Crossref PubMed Google Scholar, P. Y. Y. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). In human PCNA, the and are and in to interact with DNA polymerase δ P. Y. Y. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). the double mutant of yeast PCNA is defective in physical interaction with DNA polymerase δ, and must be used at greater than wild type PCNA to stimulate DNA polymerase DNA synthesis J.C. Ayyagari R. Gomes X.V. Burgers P.M.J. Mol. Cell. Biol. 1997; 17: 6367-6378Crossref PubMed Google Scholar). However, this mutant normal with RF-C, a complex that PCNA the DNA in an the yeast PCNA mutant binding to yeast FEN-1 J.C. Ayyagari R. Gomes X.V. Burgers P.M.J. Mol. Cell. Biol. 1997; 17: 6367-6378Crossref PubMed Google Scholar), in to the defect for the human PCNA and a of PCNA that with of Z. J. M. J. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) by FEN-1 E. Lane D.P. Glover D.M. Cox L.S. Oncogene. 1997; 14: 2313-2321Crossref PubMed Scopus (135) Google Scholar, 17Gary R. Ludwig D.L. Cornelius H.L. MacInnes M.A. Park M.S. J. Biol. Chem. 1997; 272: 24522-24529Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). the of