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 with an or codon by QuickChange Mutagenesis to create and mutant using mutagenic primer pairs and and respectively. These primer pairs created a or site to facilitate screening. The pair of mutagenic was also used to the of plasmid was used in PCNA FEN-1 proteins amino acids or with C-terminal polyhistidine were created by QuickChange Mutagenesis of pET-FCH to codon or with six histidine codons immediately by a stop codon. The mutagenic primer pairs used to create the FEN-1 were and and These primer pairs each created a site to facilitate screening. The amino acid fragment of human XPG was amplified by polymerase chain reaction with primer and primer using plasmid Park M.S. S. Cell. Biol. PubMed Scopus Google Scholar, Shen B. Park M.S. Res. 1995; PubMed Scopus Google Scholar) as The was ligated with pGEX-4T-1 to create plasmid that expresses a fusion protein of GST and The XPG codon arginine in this plasmid was with an or codon by QuickChange Mutagenesis to create R992A and R992E mutant using mutagenic primer pairs and and respectively. These primer pairs created an or site to facilitate screening. PCNA was amplified by polymerase chain reaction with primer and primer using plasmid by as was ligated to a The three nucleotides of a C-terminal site to facilitate of of PCNA for were to a stop codon by QuickChange mutagenesis with and This primer pair a site to facilitate screening. The plasmid a stop codon in the and expression of human PCNA or and site-directed mutagenesis were by DNA was used as the to PCNA, FEN-1 and and all GST fusion Expression was with were in for FEN-1 and and for and with and at a of 1 The cell were by at GST fusion protein binding of was with of GST fusion protein cell and of PCNA cell The same procedure was used for protein binding that of acid metal chelation was from and and was to the were for at and then six with of were by to with of Nature. PubMed Scopus Google Scholar) and by gel and using CA) molecular In the of synthetic to residues of was to one of the In this of was of each was and of was to the to In the was used for and as was used for binding and In the or synthetic to residues of human p21 by was to at the and GST-FEN328–363 proteins used in experiments were by PCNA was from by gel and Stillman B. Cell. Biol. 1992; PubMed Scopus Google Scholar). were by a and by of human PCNA or bovine serum at in was to and for at to the proteins to the was or for each protein was and then were with were XPG was from as Shen B. Park M.S. Res. 1995; PubMed Scopus Google Scholar), that CA) were used as the were with XPG and at The cell from of was in of 1 A, an of the same was the of to The was at and the was binding of PCNA or was with of cell In of BSA, PCNA, or was to the The were for at and then with of In some a was between the and proteins were with polyacrylamide and to for The was with an XPG amino acid Park M.S. Res. 1996; PubMed Scopus Google Scholar, M.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar), and and then XPG were by PCNA affinity were with cell and three and or of were to the The were for at and were to the in proteins were and the activity of the DNA Inc.) and were at to and as These were by of and and the DNA was by gel and The repair using plasmid has been Park M.S. 1996; PubMed Scopus Google Scholar). expression plasmid was with or of UV light from a XPG function was by a expression plasmid containing human XPG cDNA the of a A of this plasmid was created by to amino acids of the XPG region and a and then the vector R992A and R992E mutations were generated in using mutagenic as for expression plasmids. These mutations were introduced by fragments between plasmids. XPG plasmid were by of at and by gel and The cell Park M.S. S. Cell. Biol. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar) was with a of of of plasmid Park M.S. 1996; PubMed Scopus Google Scholar), and of or mutant XPG plasmid by Park M.S. S. Cell. Biol. PubMed Scopus Google Scholar). each XPG three to six were were after for of and activity Park M.S. 1996; PubMed Scopus Google Scholar). were in and the were each the activity was by the activity to a of DNA repair that is for of the were using We produced a of GST fusion proteins that regions of human FEN-1 and PCNA binding activity using a affinity interaction The binding activity was amino acids 328–355 of FEN-1. fusion proteins that this region to PCNA, whereas of the proteins that the complete amino acid sequence binding activity. the fusion protein containing residues 328–348 of FEN-1 bind PCNA. has been that the PCNA-binding domain of human FEN-1 is residues and that residues 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 is with the the latter is we for in domain mapping we generated FEN-1 proteins to the of this region in of the PCNA-binding region to a form of FEN-1 amino acids PCNA binding activity. a FEN-1 amino acids PCNA as as full-length FEN-1 (amino acids we that the C-terminal region from residues to 380 of FEN-1 is essential for PCNA binding activity and in to this activity. This with the report that FEN-1 (amino acids is to bind to PCNA in gel and affinity (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 for the between and is We to identify residues of FEN-1 that are for interaction with PCNA. 339 of FEN-1, the PCNA-binding region, was found to be crucial for PCNA binding activity. mutagenesis of FEN-1 to convert to or the of the protein to bind PCNA, the of this region of FEN-1 and of this in PCNA binding activity of the and of FEN-1 was each mutant endonuclease activity as by (15Nolan J.P. Shen B. Park M.S. Sklar L.A. Biochemistry. 1996; 35: 11668-11676Crossref PubMed Scopus (61) Google Scholar, B. J.P. Sklar L.A. Park M.S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) using a DNA The amino acid sequence of the PCNA-binding region of FEN-1 is conserved in XPG We to the function of this region is conserved in XPG as We made a GST fusion protein containing the 29-amino acid sequence of human XPG (residues 981–1009) that is homologous to the PCNA-binding region of FEN-1. human PCNA containing these proteins were GST XPG sequence affinity for PCNA. The of and PCNA was by the of as that the XPG of the fusion protein was responsible for the XPG and FEN-1 share a conserved arginine in that was shown by mutagenesis to be for PCNA binding activity in FEN-1 this arginine in with or of PCNA binding activity. The in PCNA from a amino acid in to the of this amino acids bind PCNA, and conserved arginine is essential for this gel of protein to in binding assay. were with from GST GST fusion protein containing residues of human XPG fusion with R992A or fusion with R992E from human PCNA was to each and synthetic to the was to the shown in protein were and by as much fusion protein and PCNA was used in the shown in as in shown in The PCNA-binding domain of FEN-1 binding activity all the binding activity of the region of XPG of and PCNA was in a containing to this produced and or binding to about and of respectively. was in In to the of (residues 328–355) binding in of these from to the of and PCNA, and this complex was to with or affinity was used to the interaction of PCNA and full-length XPG. human PCNA was to XPG was in that been with a that contains XPG cDNA Shen B. Park M.S. Res. 1995; PubMed Scopus Google Scholar). XPG in cell to PCNA to made with The of the PCNA-XPG interaction was in The binding of XPG to PCNA was by the of PCNA to the was by of was also by the of the fusion protein containing the PCNA-binding fragment of XPG. the R992E of this fusion which PCNA binding activity was to compete with XPG for binding to the the mutant of competed with XPG for binding to the PCNA that the FEN-1 domain binds to PCNA with affinity the XPG by GST-FEN328–363 was whereas that by at was PCNA and XPG are DNA-binding we DNA of the PCNA-XPG complex failed to XPG that the proteins were by DNA. to the complex was to DNA and of cell that the of the reaction the used in the was to DNA to and to convert of DNA to a molecular The of of with the that the interaction of XPG and PCNA is PCNA and XPG in excision the that the interaction of these proteins in that we to the role of this interaction in NER in a XPG are to cell and are in the repair of DNA. defects can be by with an XPG expression plasmid Park M.S. S. Cell. Biol. PubMed Scopus Google Scholar, Park M.S. 1996; PubMed Scopus Google Scholar, 1990; PubMed Scopus Google Scholar). This a of NER function in vivo that is XPG. arginine is for PCNA binding activity of we with R992A or R992E mutant of XPG to in which the interaction was to XPG repair activity of and mutant was to the of the interaction in NER In this in vivo repair of a plasmid results in expression of with the of repair. repair activity is by with XPG. of repair activity is shown by with XPG residues The of sequence in this mutant the and the region the nuclear to a mutant protein to the Park M.S. Res. 1996; PubMed Scopus Google Scholar, M.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar). between these of and repair activity were R992A and R992E XPG for NER A small in repair was for each of these This that of the PCNA-XPG interaction NER that the of the is small with complete of The PCNA-binding region of the cyclin-dependent kinase inhibitor p21 has been to amino acids Biol. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). A synthetic to amino acids of p21 with full-length FEN-1 for binding to PCNA (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 domain mapping of FEN-1 and XPG for a sequence between the PCNA-binding regions of these and p21 of the amino acid of the PCNA-binding domains of FEN-1 and p21 a that direct for a binding site PCNA. the mapping between p21 and XPG for binding to PCNA. between the PCNA-binding domains of these proteins was using the affinity interaction the p21 was the XPG in with the fusion protein for binding to PCNA, that the interaction Z. J. Burgers P.M.J. Hurwitz J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) is affinity the the p21 competed the PCNA-binding fusion protein the between p21 and full-length FEN-1 (12Chen J. Chen S. Saha P. Dutta A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11597-11602Crossref PubMed Scopus (115) Google amino acid of p21 with the PCNA-binding domain of XPG for interaction with PCNA. gel of protein to in binding is were with containing PCNA and the fusion protein to amino acids of human p21 was at or and to amino acids of human XPG was at or PCNA a role in DNA replication and DNA damage repair. In to function of DNA δ and to the DNA PCNA also with the cell cycle the replication endonuclease FEN-1, as shown the repair endonuclease XPG. in the of the PCNA-binding domains of FEN-1, and XPG that these proteins all an of key hydrophobic residues to bind to a hydrophobic primarily by the of PCNA, as is to be the for p21 Z. Hurwitz J. J. Cell. 1996; 87: Full Text Full Text PDF PubMed Scopus Google Scholar). This that binding between these proteins be the result of direct for a binding site PCNA, from sites. using the PCNA-binding domains from these proteins are with this In the between FEN-1 and p21 was as to in PCNA, of the same site PCNA (12Chen J. Chen S. Saha P. Dutta A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11597-11602Crossref PubMed Scopus (115) Google Scholar). p21 is to the the in the PCNA-binding regions of p21 and to have by convergent XPG and PCNA are involved in and of NER, excision and repair respectively. The of PCNA binding activity in XPG that the excision and resynthesis with one another in some The and of PCNA binding in FEN-1 and XPG a to this activity. The NER activity for the XPG in vivo that the PCNA-XPG interaction to repair The function of the PCNA-XPG interaction is that PCNA XPG to the NER PCNA is for the of a containing XPG A.F. Sancar A. Nucleic Acids Res. 1992; 20: 2441-2446Crossref PubMed Scopus (181) Google Scholar, 3Shivji M.K.K. Kenny M.K. Wood R.D. 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Oncogene. 1996; Google Scholar). as a cyclin-dependent kinase p21 cell cycle that is to DNA damage by for repair to The of PCNA activity by p21 is a of the p21 the of p21 and PCNA to replicative synthesis and to cell cycle PCNA in an is essential for excision repair. p21 be to DNA repair by PCNA. has been found by some Z.-Q. Li Sancar A. Hurwitz J. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar), all S. Stillman B. Nature. PubMed Scopus Google Scholar, M.K.K. Wood R.D. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), that p21 NER, and this can be by the of PCNA. the a UV repair a that can be by p21 p21 PCNA binding activity T. Res. 1996; Google Scholar). This that the interaction of p21 and PCNA a to the damage The in vivo of the repair or in the expression or of PCNA, and XPG that these proteins to We Hurwitz for synthetic p21 used in the experiments and for PCNA cDNA used in the We also for the endonuclease activity of the FEN-1