Title: The DNA Repair Endonuclease XPG Binds to Proliferating Cell Nuclear Antigen (PCNA) and Shares Sequence Elements with the PCNA-binding Regions of FEN-1 and Cyclin-dependent Kinase Inhibitor p21
Abstract: Proliferating cell nuclear antigen (PCNA) is a DNA polymerase accessory factor that is required for DNA replication during S phase of the cell cycle and for resynthesis during nucleotide excision repair of damaged DNA. PCNA binds to flap endonuclease 1 (FEN-1), a structure-specific endonuclease involved in DNA replication. Here we report the direct physical interaction of PCNA with xeroderma pigmentosum (XP) G, a structure-specific repair endonuclease that is homologous to FEN-1. We have identified a 28-amino acid region of human FEN-1 (residues 328–355) and a 29-amino acid region of human XPG (residues 981–1009) that contains the PCNA binding activity. These regions share key hydrophobic residues with the PCNA-binding domain of the cyclin-dependent kinase inhibitor p21Waf1/Cip1, and all three competed with one another for binding to PCNA. A conserved arginine in FEN-1 (Arg339) and XPG (Arg992) was found to be crucial for PCNA binding activity. R992A and R992E mutant forms of XPG failed to fully reconstitute nucleotide excision repair in an in vivo complementation assay. These results raise the possibility of a mechanistic linkage between excision and repair synthesis that is mediated by PCNA. Proliferating cell nuclear antigen (PCNA) is a DNA polymerase accessory factor that is required for DNA replication during S phase of the cell cycle and for resynthesis during nucleotide excision repair of damaged DNA. PCNA binds to flap endonuclease 1 (FEN-1), a structure-specific endonuclease involved in DNA replication. Here we report the direct physical interaction of PCNA with xeroderma pigmentosum (XP) G, a structure-specific repair endonuclease that is homologous to FEN-1. We have identified a 28-amino acid region of human FEN-1 (residues 328–355) and a 29-amino acid region of human XPG (residues 981–1009) that contains the PCNA binding activity. These regions share key hydrophobic residues with the PCNA-binding domain of the cyclin-dependent kinase inhibitor p21Waf1/Cip1, and all three competed with one another for binding to PCNA. A conserved arginine in FEN-1 (Arg339) and XPG (Arg992) was found to be crucial for PCNA binding activity. R992A and R992E mutant forms of XPG failed to fully reconstitute nucleotide excision repair in an in vivo complementation assay. These results raise the possibility of a mechanistic linkage between excision and repair synthesis that is mediated by PCNA. Exposure to UV light causes damage to DNA primarily in the form of cyclobutane pyrimidine dimers and (6-4) photoproducts. These types of DNA lesions, as well as bulky adducts produced by some chemical mutagens, are processed by nucleotide excision repair (NER). 1The abbreviations used are: NER, nucleotide excision repair; XP, xeroderma pigmentosum; PCNA, proliferating cell nuclear antigen; FEN-1, flap endonuclease 1; GST, glutathioneS-transferase; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin.1The abbreviations used are: NER, nucleotide excision repair; XP, xeroderma pigmentosum; PCNA, proliferating cell nuclear antigen; FEN-1, flap endonuclease 1; GST, glutathioneS-transferase; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin. The human genetic disorder xeroderma pigmentosum (XP) is the result of defects in this DNA damage repair pathway. Symptoms of XP include extreme sensitivity to sunlight exposure and a greatly elevated risk of skin cancer. In the past few years, much progress has been made in understanding the molecular events associated with NER (1Sancar A. J. Biol. Chem. 1995; 270: 15915-15918Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). The DNA-binding protein XPA is involved in damage recognition. In concert with replication protein A, which binds single-stranded DNA, and helicases XPB and XPD, a ∼27–29-base oligonucleotide segment containing the lesion is excised as the result of dual incision by structure-specific endonucleases XPF-ERCC1 and XPG. The XPF-ERCC1 complex cleaves the damaged strand at a 5′ site about 23 nucleotides from the lesion, whereas XPG cleaves the strand approximately 5 nucleotides to the 3′ side of the damage. The resultant gap is filled in by the action of DNA polymerase δ or ε, and then DNA ligase seals the nick to complete repair. The resynthesis step requires proliferating cell nuclear antigen (PCNA; Refs. 2Nichols A.F. Sancar A. Nucleic Acids Res. 1992; 20: 2441-2446Crossref PubMed Scopus (181) Google Scholar and 3Shivji M.K.K. Kenny M.K. Wood R.D. Cell. 1992; 69: 367-374Abstract Full Text PDF PubMed Scopus (732) Google Scholar), a ring-shaped homotrimeric protein that encircles DNA and acts as a "sliding clamp" that links the polymerase to the DNA template (4Kelman Z. Oncogene. 1997; 14: 629-640Crossref PubMed Scopus (717) Google Scholar). PCNA performs the same essential function in replicative DNA synthesis during S phase of the cell cycle. PCNA requires replication factor C, a primer recognition protein that loads the PCNA trimer onto DNA in an ATP-dependent manner (5Tsurimoto T. Stillman B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1023-1027Crossref PubMed Scopus (199) Google Scholar, 6Lee S.-H. Hurwitz J. Proc. Natl. Acad. Sci U. S. A. 1990; 87: 5672-5676Crossref PubMed Scopus (175) Google Scholar, 7Lee S.-H. Kwong A.D. Pan Z.-Q. Hurwitz J. J. Biol. Chem. 1991; 266: 594-602Abstract Full Text PDF PubMed Google Scholar). XPG is homologous to another structure-specific endonuclease, FEN-1. FEN-1 is involved in Okazaki fragment processing during DNA replication (8Bambara R.A. Murante R.S. Henricksen L.A. J. Biol. Chem. 1997; 272: 4647-4650Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar), and it is required for avoidance of duplication-type insertion mutations in yeast (9Tishkoff D.X. Filosi N. Gaida G.M. Kolodner R.D. Cell. 1997; 88: 253-263Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). FEN-1 binds to PCNA (10Li X. Li J. Harrington J. Lieber M.R. Burgers P.M.J. J. Biol. Chem. 1995; 270: 22109-22112Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 11Wu 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, 12Chen J. Chen S. Saha P. Dutta A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11597-11602Crossref PubMed Scopus (115) Google Scholar), and this complex can be disrupted by p21Waf1/Cip1 (12Chen J. Chen S. Saha P. Dutta A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11597-11602Crossref PubMed Scopus (115) Google Scholar), a bifunctional protein that has a C-terminal PCNA-binding domain and an N-terminal domain that inhibits cyclin-dependent protein kinases (13Chen J. Jackson P.K. Kirschner M.W. Dutta A. Nature. 1995; 374: 386-388Crossref PubMed Scopus (511) Google Scholar, 14Luo Y. Hurwitz J. Massague J. Nature. 1995; 375: 159-161Crossref PubMed Scopus (515) Google Scholar). Here we report domain mapping experiments to pinpoint the PCNA-binding region of FEN-1 and show that the small region responsible for activity is conserved in XPG. This domain in XPG as well as the full-length XPG protein are shown to bind to PCNA. We also provide evidence fromin vivo studies indicating that the PCNA-XPG interaction has a role in repair of UV damage. Finally, we identify a convergent evolutionary relationship between the PCNA-binding domains of the DNA damage-inducible inhibitor p21 and the repair endonuclease XPG and show that these domains compete for binding to PCNA. TheEcoRI-XhoI fragment of human FEN-1 expression plasmid pET-FCH (15Nolan J.P. Shen B. Park M.S. Sklar L.A. Biochemistry. 1996; 35: 11668-11676Crossref PubMed Scopus (61) Google Scholar) was ligated withEcoRI/XhoI-linearized pGEX-4T-1 vector (Pharmacia Biotech Inc.) to create a plasmid that expresses a fusion protein of glutathione S-transferase (GST) and residues 206–380 of FEN-1. The amino acid fragments 254–363, 254–380, 290–363, 290–380, 254–328, 290–328, 328–363, and 328–380 of FEN-1 were amplified by polymerase chain reaction using pET-FCH as template and primer pairs F2 (5′-CCAGAATTCAAGAGCATCGAGGAGATCGTG-3′) and R2 (5′-GTGCTCGAGTTAAGATCCCTTGGGTTCTGGCTC-3′), F2 and R3 (5′-GTGCTCGAGTTATTTTCCCCTTTTAAACTTCCC-3′), F3 (5′-CCAGAATTCGACCCAGAGTCTGTGGAGCTG-3′) and R2, F3 and R3, F2 and R5 (5′-GTGCTCGAGTCACAGCCTCTTGACCCCACTG-3′), F3 and R5, F5 (5′-TGGAATTCCTGAGTAAGAGCCGCCAA-3′) and R2, and F5 and R3, respectively. The products were digested with EcoRI andXhoI and subcloned intoEcoRI/XhoI-linearized pGEX-4T-1 to make GST fusion protein expression plasmids. Expression plasmids for the production of GST fusion proteins containing FEN-1 residues 328–348 and 328–355 were generated by site-directed mutagenesis of the GST-FEN328–363 plasmid to convert codon 349 or 356 to a stop codon. The stop codons were introduced using the QuickChange Mutagenesis procedure (Stratagene, La Jolla, CA) and mutagenic primer pairs 5′-CTTCAAGGTGACCGGCTGACTCTCTTCAGCTAAGC-3′ and 5′-GCTTAGCTGAAGAGAGTCAGCCGGTCACCTTGAAG-3′, 5′-TCACTCTCTTCAGCTAAGCGCTAGCAGCCAGAACCCAAGGGATCC-3′ and 5′-GGATCCCTTGGGTTCTGGCTGCTAGCGCTTAGCTGAAGAGAGTGA-3′, respectively. The latter primer pair created an NheI site to facilitate screening. Expression plasmids for the production of GST fusion proteins containing FEN-1 residues 354–380 and 363–380 were generated by ligating annealed synthetic oligonucleotides withEcoRI/XhoI-linearized pGEX-4T-1. The oligonucleotides corresponded to each strand of the FEN-1 cDNA sequence for the specified region, included the stop codon immediately after codon 380 and were flanked by EcoRI andXhoI sites. Plasmid pET-FCH produces full-length FEN-1 (amino acids 1–380) with six histidine residues appended to the C terminus (15Nolan J.P. Shen B. Park M.S. Sklar L.A. Biochemistry. 1996; 35: 11668-11676Crossref PubMed Scopus (61) Google Scholar). The polyhistidine tag binds tightly to metal chelation affinity resin. The FEN-1 codon arginine 339 in this plasmid was replaced with either an alanine or glutamate codon by QuickChange Mutagenesis to create R339A and R339E single point mutant derivatives using mutagenic primer pairs 5′-CCAAGGCAGCACCCAGGGCGCGCTGGATGATTTCTTCAA-3′ and 5′-CCTTGAAGAAATCATCCAGCGCGCCCTGGGTGCTGCCTT-3′, 5′-CCAAGGCAGCACCCAGGGCGAGCTCGATGATTTCTTCAA-3′ and 5′-CCTTGAAGAAATCATCGAGCTCGCCCTGGGTGCTGCCTT-3′, respectively. These primer pairs created a BssHII or SacI site to facilitate screening. The second pair of mutagenic primers was also used to generate the R339E derivative of plasmid GST-FEN328–363, whose product was used in PCNA bead competition experiments. Truncated FEN-1 proteins comprising amino acids 1–328 or 1–363 with C-terminal polyhistidine tags were created by QuickChange Mutagenesis of pET-FCH to replace codon 329 or 364, respectively, with six histidine codons followed immediately by a stop codon. The mutagenic primer pairs used to create the truncated FEN-1 constructs were 5′-GCAGTGGGGTCAAGAGGCTGCACCATCACCACCATCACTAGTGCAGCACCCAGGGCCGCC-3′ and 5′-GGCGGCCCTGGGTGCTGCACTAGTGATGGTGGTGATGGTGCAGCCTCTTGACCCCACTGC-3′, 5′-AGCCAGAACCCAAGGGATCCCACCATCACCACCATCACTAGTGGGGCAGCAGGGAAGT-3′ and 5′-ACTTCCCTGCTGCCCCACTAGTGATGGTGGTGATGGTGGGATCCCTTGGGTTCTGGCT-3′. These primer pairs each created a SpeI site to facilitate screening. The amino acid 981–1009 fragment of human XPG was amplified by polymerase chain reaction with forward primer 5′-CCAGAATTCTTAAAGCAACTCGATG-3′ and reverse primer 5′-GTGCTCGAGTTAACGTTTAGCATCTTCTTTCTC-3 using plasmid pBSK-XPGA (16MacInnes M.A. Dickson J.A. Hernandez R.R. Learmonth D. Lin G.Y. Mudgett J.S. Park M.S. Schauer S. Reynolds R.J. Strniste G.F. Yu J.Y. Mol. Cell. Biol. 1993; 13: 6393-6402Crossref PubMed Scopus (49) Google Scholar, 17Cloud K.G. Shen B. Strniste G.F. Park M.S. Mutat. Res. 1995; 347: 55-60Crossref PubMed Scopus (36) Google Scholar) as template. The EcoRI/XhoI-digested product was ligated with EcoRI/XhoI-linearized pGEX-4T-1 to create plasmid GST-XPG that expresses a fusion protein of GST and XPG981–1009. The XPG codon arginine 992 in this plasmid was replaced with either an alanine or glutamate codon by QuickChange Mutagenesis to create R992A and R992E single point mutant derivatives using mutagenic primer pairs 5′-GCCCAGCAGACACAGCTCGCGATTGATTCCTTCTTTAGATTAG-3′ and 5′-CTAATCTAAAGAAGGAATCAATCGCGAGCTGTGTCTGCTGGGC-3′, 5′-GCCCAGCAGACACAGCTCGAGATTGATTCCTTCTTTAGATTAG-3′ and 5′-CTAATCTAAAGAAGGAATCAATCTCGAGCTGTGTCTGCTGGGC-3′, respectively. These primer pairs created an NruI or XhoI site to facilitate screening. Human PCNA was amplified by polymerase chain reaction with forward primer 5′-GGAATTCATGAGTCACCACCACCACCACCACATGTTCGAGGCGCGCCTGG-3′ and reverse primer 5′-TTGCGAAGCTTACTCGAGAGATCCTTCTTCATCCTCG-3′ using plasmid kindly provided by Dr. Suk-Hee Lee (St. Jude Children's Research Hospital, Memphis, TN) as template. TheAflIII/HindIII-digested product was ligated toNcoI/HindIII-linearized pET28b (Novagen, Madison, WI) to produce a subcloning intermediate. The first three nucleotides of a C-terminal XhoI site (introduced to facilitate generation of tagged variants of PCNA for other experiments) were converted to a stop codon by QuickChange mutagenesis with primers 5′-ATCGAGGATGAAGAAGGATCTTAGGAGTCAGCTTGCGGCCGCACTCGA-3′ and 5′-TCGAGTGCGGCCGCAAGCTGACTCCTAAGATCCTTCTTCATCCTCGAT-3′. This primer pair destroyed a HindIII site to facilitate screening. The resulting plasmid contained a stop codon in the naturally occurring position and directed high level inducible expression of wild-type human PCNA without tags or extensions. All subcloning and site-directed mutagenesis procedures were confirmed by DNA sequencing. BL21(DE3) Escherichia coli was used as the host strain to express PCNA, polyhistidine-tagged FEN-1 and derivatives, and all GST fusion proteins. Expression was induced with 0.8 mmisopropyl-β-d-thiogalactopyranoside. Cells were lysed in wash buffer (50 mm Tris-HCl, 150 mm NaCl, pH 7.4, for FEN-1 and its derivatives and 50 mm Tris-HCl, 100 mmKH2PO4/K2HPO4, 150 mm NaCl, pH 7.4, for GST-XPG and its derivatives) supplemented with 2 mm EDTA and 0.2 mg/ml lysozyme at a ratio of 1 ml/25-ml culture. The cell lysates were clarified by centrifugation at 16,000 × g. For GST fusion protein binding assays, 80 μl of 40% glutathione-agarose beads (Sigma) was mixed with 450 μl of GST fusion protein cell lysate and 450 μl of PCNA cell lysate. The same procedure was used for polyhistidine-tagged protein binding assays, except that 80 μl of 40% NiSO4-charged iminodiacetic acid metal chelation resin (HisBind; Novagen) replaced glutathione-agarose beads, EDTA was omitted from lysis and wash buffers, and 60 mm imidazole was added to the wash buffer. Mixtures were incubated for 2 h at 4 °C and then washed six times with 0.8 ml of wash buffer. Protein complexes were eluted by heating to 100 °C with 80 μl of 2 × Laemmli sample buffer (18Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206999) Google Scholar) and analyzed on 12% gels by SDS-polyacrylamide gel electrophoresis (PAGE) and Coomassie Blue staining using Mark12 (Novex, San Diego, CA) molecular weight standards. In the first competition experiment, 0.24 mg of synthetic peptide LKQLDAQQTQLRIDSFFRLAQQEKEDAKR (Research Genetics, Huntsville, AL), corresponding to residues 981–1009 of XPG, was added to one of the lysate mixtures. In this set of assays, NaCl concentration was 300 mm, 150 μl of each lysate was used, and 150 μl of wash buffer was added to increase the volume to improve mixing. In the salt dependence experiment, low salt wash buffer (20 mm Tris-HCl, 60 mm NaCl, pH 7.4) plus 0.2 mg/ml lysozyme was used for lysis, and low salt wash buffer supplemented as indicated was used for subsequent binding and washing steps. In the second competition experiment, XPG981–1009 peptide or biotinylated synthetic peptide GRKRRQTSMTDFYHSKRRLIFS corresponding to residues 139–160 of human p21 (kindly provided by Dr. Jerard Hurwitz, Memorial Sloan-Kettering Cancer Center) was added to lysate mixtures at the concentrations indicated. GST-XPG981–1009, GST-XPG981–1009(R992E), GST-FEN328–363, and GST-FEN328–363 (R339E) proteins used in competition experiments were purified by glutathione-agarose chromatography. Human PCNA was purified from bacterial lysate by QSepharose, S-300 Sephacryl gel filtration, and Phenyl-Superose chromatography (19Fein K. Stillman B. Mol. Cell. Biol. 1992; 12: 155-163Crossref PubMed Scopus (190) Google Scholar). Protein concentrations were determined by a Bradford-based assay (Bio-Rad) and verified by Coomassie Blue staining of SDS-PAGE gels. Purified human PCNA or protease-free bovine serum albumin (BSA; Boehringer Mannheim) at 4 mg/ml in 25 mm NaHCO3, 200 mm NaCl, pH 8.3, was added to cyanogen bromide-activated agarose beads (Sigma) and incubated for 16 h at 4 °C to covalently attach the proteins to the beads. Coupling efficiency was 80% or higher for each protein. Unbound protein was removed, and then remaining reactive sites were blocked with 0.1 m glycine, 65 mmTris-HCl, 150 mm NaCl, pH 8.0. Washed beads were stored on ice. Human XPG was expressed from recombinant baculovirus as described previously (17Cloud K.G. Shen B. Strniste G.F. Park M.S. Mutat. Res. 1995; 347: 55-60Crossref PubMed Scopus (36) Google Scholar), except that Trichoplusia ni (BTI-TN-5B1-4) insect cells ("High Five" cells, Invitrogen, San Diego, CA) were used as the host. Cells were infected with XPG recombinant baculovirus and collected at 35.5 h post-infection. The cell pellet from 50 ml of culture was lysed in 4 ml of 0.5% Nonidet P-40, 100 mmpotassium phosphate, 300 mm KCl, 2 mm EDTA, 1 mm benzamidine, 0.5 mm phenylmethylsulfonyl fluoride, 10 μm pepstatin A, 10 μg/ml chymostatin, 10 μg/ml aprotinin, pH 7.4. After lysis, an equal volume of the same buffer without detergent was added, reducing the final concentration of Nonidet P-40 to 0.25%. The lysate was centrifuged at 90,000 ×g, and the supernatant was 0.45 μm filtered. For binding assays, 80 μl of 20% PCNA or BSA beads was mixed with 430 μl of XPG-containing insect cell supernatant. In competition experiments, 0.4 mg of purified BSA, PCNA, GST-XPG981–1009, GST-XPG981–1009(R992E), GST-FEN328–363, or GST-FEN328–363(R339E) was added to the lysate mixture. The mixtures were incubated for 2.5 h at 4 °C and then washed five times with 0.8 ml of 100 mmKH2PO4/K2HPO4, 300 mm KCl, pH 7.4. In some experiments, a DNase I treatment was performed between the third and fourth washes. Noncovalently bound proteins were eluted with Laemmli sample buffer, resolved on 4–12% polyacrylamide gradient gels, and transferred to Immobilon-P (Millipore, Bedford, MA) polyvinylidene fluoride membrane for Western blot analysis. The membrane was sequentially incubated with 10% nonfat dry milk, 1:2000 rabbit polyclonal XPG1322 (IgG fraction) raised against an XPG amino acid 1147–1163 peptide (20Knauf J.A. Pendergrass S.H. Marrone B.L. Strniste G.F. MacInnes M.A. Park M.S. Mutat. Res. 1996; 363: 67-75Crossref PubMed Scopus (24) Google Scholar, 21Park M.S. Knauf J.A. Pendergrass S.H. Coulon C.H. Strniste G.F. Marrone B.L. MacInnes M.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8368-8373Crossref PubMed Scopus (48) Google Scholar), and 1:2000 peroxidase-conjugated goat anti-rabbit IgG (Life Technologies, Inc.), and then XPG bands were detected by enhanced chemiluminescence (Amersham Corp.). PCNA affinity beads were incubated with insect cell lysate and washed three times. 10 mmMgCl2 and 0 or 250 units of DNase I (Boehringer Mannheim) were added to the washed beads. The beads were incubated for 20 min at 10 °C and were mixed periodically to keep the beads in suspension. After two more washes, proteins were eluted and analyzed. To confirm the activity of DNase I under the conditions used,HindIII-digested Lambda DNA (Life Technologies, Inc.) and DNase I were added at various ratios to washed beads and incubated as described. These confirmatory reactions were terminated by addition of 25 mm EDTA and 0.5% SDS, and the DNA was evaluated by agarose gel electrophoresis and ethidium bromide staining. The repair assay using UV-damaged luciferase reporter plasmid has been described previously (22Ludwig D.L. Mudgett J.S. Park M.S. Perez-Castro A.V. MacInnes M.A. Mamm. Genome. 1996; 7: 644-649Crossref PubMed Scopus (9) Google Scholar). Briefly, luciferase expression plasmid pGL-2 (Promega, Madison, WI) was irradiated with 0, 400, or 800 J/m2 of 254-nm UV light from a calibrated source. XPG function was provided by pBactin-XPG, a mammalian expression plasmid containing human XPG cDNA under the control of a β-actin promoter. A truncated derivative of this plasmid was created by BamHI digestion to remove amino acids 948–1186 of the XPG coding region and a downstream polyadenylation signal and then recircularizing the vector fragment. R992A and R992E mutations were generated in pBSK-XPGA using mutagenic primers as described for bacterial expression plasmids. These mutations were introduced into pBactin-XPG by swappingEcoRV-KpnI fragments between plasmids. XPG plasmid concentrations were determined by measurement of absorbance at 260 nm and verified by agarose gel electrophoresis and ethidium bromide staining. The XPG-deficient CHO cell line UV135 (16MacInnes M.A. Dickson J.A. Hernandez R.R. Learmonth D. Lin G.Y. Mudgett J.S. Park M.S. Schauer S. Reynolds R.J. Strniste G.F. Yu J.Y. Mol. Cell. Biol. 1993; 13: 6393-6402Crossref PubMed Scopus (49) Google Scholar, 23Busch D. Greiner C. Lewis K. Ford R. Adair G. Thompson L. Mutagenesis. 1989; 4: 349-354Crossref PubMed Scopus (79) Google Scholar) was transfected with a mixture of 150 ng of luciferase plasmid, 150 ng of β-galactosidase plasmid (22Ludwig D.L. Mudgett J.S. Park M.S. Perez-Castro A.V. MacInnes M.A. Mamm. Genome. 1996; 7: 644-649Crossref PubMed Scopus (9) Google Scholar), and 30 ng of wild-type or mutant XPG plasmid by calcium phosphate precipitation (16MacInnes M.A. Dickson J.A. Hernandez R.R. Learmonth D. Lin G.Y. Mudgett J.S. Park M.S. Schauer S. Reynolds R.J. Strniste G.F. Yu J.Y. Mol. Cell. Biol. 1993; 13: 6393-6402Crossref PubMed Scopus (49) Google Scholar). For each XPG transfection set, three to six 60-mm dishes were used. Cells were lysed 48 h after transfection for analysis of luciferase and β-galactosidase activity (22Ludwig D.L. Mudgett J.S. Park M.S. Perez-Castro A.V. MacInnes M.A. Mamm. Genome. 1996; 7: 644-649Crossref PubMed Scopus (9) Google Scholar). Activity assays were performed in duplicate, and the values were averaged. For each transfection, the luciferase activity was divided by the corresponding β-galactosidase activity to give relative luciferase activity, a measure of DNA repair that is normalized for transfection efficiency. Statistical analyses of the data were performed using Microsoft Excel version 4. We produced a series of GST fusion proteins that contain various regions of human FEN-1 and assayed their PCNA binding activity using a "pull-down" affinity bead interaction assay (Fig. 1). The binding activity was contained entirely within amino acids 328–355 of FEN-1. All fusion proteins that contained this region bound to PCNA, whereas none of the proteins tested that lacked the complete 28 amino acid sequence displayed binding activity. For example, the fusion protein containing only residues 328–348 of FEN-1 did not bind PCNA. It has been reported previously that the PCNA-binding domain of human FEN-1 is contained within residues 307–380 and that residues 364–380 are essential for PCNA binding (12Chen J. Chen S. Saha P. Dutta A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11597-11602Crossref PubMed Scopus (115) Google Scholar). The former conclusion is consistent with the observations reported here, but the latter conclusion is not. Because we observed no requirement for 364–380 in our domain mapping studies, we generated truncated FEN-1 proteins to address the importance of this region in more detail (Fig.2). Deletion of the PCNA-binding region to give a truncated form of FEN-1 comprising only amino acids 1–328 abolished PCNA binding activity. However, a truncated FEN-1 comprising amino acids 1–363 bound PCNA as effectively as full-length FEN-1 (amino acids 1–380). Thus, we conclude that the C-terminal region from residues 364 to 380 of FEN-1 is not essential for PCNA binding activity and in fact makes little if any contribution to this activity. This contrasts with the previous report that truncated FEN-1 (amino acids 1–363) is unable to bind to PCNA in gel filtration and affinity bead pull-down assays (12Chen J. Chen S. Saha P. Dutta A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11597-11602Crossref PubMed Scopus (115) Google Scholar). The reason for the difference between those observations and our own is not apparent. We next sought to identify specific residues of FEN-1 that are most important for interaction with PCNA. Arginine 339 of FEN-1, lying within the PCNA-binding region, was found to be crucial for PCNA binding activity. Single point mutagenesis of FEN-1 to convert Arg339 to either alanine or glutamate dramatically decreased the ability of the protein to bind PCNA, showing the importance of this region of FEN-1 and of this residue in particular. Although PCNA binding activity of the R339A and R339E mutants of FEN-1 was severely impaired, each mutant retained endonuclease activity as determined by rapid kinetic flow cytometry (15Nolan J.P. Shen B. Park M.S. Sklar L.A. Biochemistry. 1996; 35: 11668-11676Crossref PubMed Scopus (61) Google Scholar, 24Shen 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) using a fluoresceinated 5′-flap DNA substrate (data not shown). The amino acid sequence of the PCNA-binding region of FEN-1 is significantly conserved in XPG (Fig. 3). We sought to determine whether the function of this region is conserved in XPG as well. We made a GST fusion protein (GST-XPG) containing the 29-amino acid sequence of human XPG (residues 981–1009) that is homologous to the PCNA-binding region of FEN-1. GST-XPG bound human PCNA very efficiently when bacterial lysates containing these proteins were mixed (Fig.4). GST alone lacking XPG sequence had no observable affinity for PCNA. The association of GST-XPG and PCNA was blocked by the addition of XPG981–1009 peptide as competitor, confirming that the XPG moiety of the fusion protein was responsible for the binding. XPG and FEN-1 share a conserved arginine (Arg992 in XPG) that was shown by mutagenesis to be important for PCNA binding activity in FEN-1 (Fig. 2). Replacing this arginine in GST-XPG with alanine or glutamate caused almost total loss of PCNA binding activity. The dramatic decrease in bound PCNA resulting from a single amino acid substitution in GST-XPG attests to the specificity of this assay.Figure 4XPG amino acids 981–1009 bind PCNA, and conserved arginine 992 is essential for this activity.Coomassie-stained gel of protein complexes bound to glutathione-agarose beads in pull-down binding assay. Beads were mixed with lysate from bacteria expressing GST (lane 1), GST fusion protein containing residues 981–1009 of human XPG (lanes 2–4), GST-XPG981–1009 fusion with single point mutation R992A (lane 5), or GST-XPG981–1009 fusion with single point mutation R992E (lane 6). Lysate from bacteria expressing human PCNA was added to each assay, and synthetic peptide corresponding to the XPG981–1009 29-mer was added to the assay shown in lane 3. After washing, protein complexes were eluted and analyzed by SDS-PAGE. Three times as much fusion protein and PCNA lysate was used in the assays shown in lanes 4–6 as in those shown in lanes 1–3.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The PCNA-binding domain of FEN-1 displayed robust binding activity under all conditions tested; however, the binding activity of the corresponding region of XPG exhibited profound salt dependence (Fig.5). Binding of GST-XPG and PCNA was almost undetectable in a buffer containing 60 mm NaCl. Adding 100 mm potassium phosphate to this buffer produced maximal binding, and adding 25 mm potassium phosphate, 100 mm KCl, or 300 mm KCl increased binding to about 25, 25, and 100% of maximum, respectively. Thus, divalent anion was especially effective in aiding binding. In contrast to the behavior of GST-XPG, GST-FEN (residues 328–355) displayed nearly maximal binding in any of these buffers. Varying pH from 6.0 to 8.0 had little effect on the association of GST-XPG and PCNA, and this complex was stable to repeated washing with either 1% Nonidet P-40 detergent or 1.0 m NaCl (data not shown). An affinity bead assay was used to evaluate the interaction of PCNA and full-length XPG. Purified human PCNA was covalently attached to beads. Human XPG was expressed in insect cells that had been infected with a recombinant baculovirus strain that contains XPG cDNA (17Cloud K.G. Shen B. Strniste G.F. Park M.S. Mutat. Res. 1995; 347: 55-60Crossref PubMed Scopus (36) Google Scholar). XPG present in baculovirus-infected cell lysates bound to PCNA beads but not to control beads made with BSA (Fig.6 A). The specificity of the PCNA-XPG interaction was further demonstrated in competition experiments. The binding of XPG to PCNA beads was blocked by the addition of free PCNA to the lysate but was unaffected by addition of BSA (Fig. 6 B). Binding was also inhibited by the addition of GST-XPG981–1009, the fusion protein containing the PCNA-binding fragment of XPG. However, the R992E derivative of this fusion protein, which lacks PCNA binding activity (Fig. 4), was unable to compete with XPG for binding to the beads. Similarly, GST-FEN328–363, but not the R339E mutant of GST-FEN328–363, competed with XPG for binding to the PCNA beads. It appe