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• W2077338748 abstract "The interaction between proliferating cell nuclear antigen (PCNA) and DNA polymerase δ is essential for processive DNA synthesis during DNA replication/repair; however, the identity of the subunit of DNA polymerase δ that directly interacts with PCNA has not been resolved until now. In the present study we have used reciprocal co-immunoprecipitation experiments to determine which of the two subunits of core DNA polymerase δ, the 125-kDa catalytic subunit or the 50-kDa small subunit, directly interacts with PCNA. We found that PCNA co-immunoprecipitated with human p50, as well as calf thymus DNA polymerase δ heterodimer, but not with p125 alone, suggesting that PCNA directly interacts with p50 but not with p125. A PCNA-binding motif, similar to the sliding clamp-binding motif of bacteriophage RB69 DNA polymerase, was identified in the N terminus of p50. A 22-amino acid oligopeptide containing this sequence (MRPFL) was shown to bind PCNA by far Western analysis and to compete with p50 for binding to PCNA in co-immunoprecipitation experiments. The binding of p50 to PCNA was inhibited by p21, suggesting that the two proteins compete for the same binding site on PCNA. These results establish that the interaction of PCNA with DNA polymerase δ is mediated through the small subunit of the enzyme. The interaction between proliferating cell nuclear antigen (PCNA) and DNA polymerase δ is essential for processive DNA synthesis during DNA replication/repair; however, the identity of the subunit of DNA polymerase δ that directly interacts with PCNA has not been resolved until now. In the present study we have used reciprocal co-immunoprecipitation experiments to determine which of the two subunits of core DNA polymerase δ, the 125-kDa catalytic subunit or the 50-kDa small subunit, directly interacts with PCNA. We found that PCNA co-immunoprecipitated with human p50, as well as calf thymus DNA polymerase δ heterodimer, but not with p125 alone, suggesting that PCNA directly interacts with p50 but not with p125. A PCNA-binding motif, similar to the sliding clamp-binding motif of bacteriophage RB69 DNA polymerase, was identified in the N terminus of p50. A 22-amino acid oligopeptide containing this sequence (MRPFL) was shown to bind PCNA by far Western analysis and to compete with p50 for binding to PCNA in co-immunoprecipitation experiments. The binding of p50 to PCNA was inhibited by p21, suggesting that the two proteins compete for the same binding site on PCNA. These results establish that the interaction of PCNA with DNA polymerase δ is mediated through the small subunit of the enzyme. DNA polymerase δ proliferating cell nuclear antigen chromatin assembly factor 1 DNA polymerase δ (pol δ),1 the principal DNA replicase in eukaryotes, also participates in several DNA repair pathways, including nucleotide excision repair, mismatch repair, and long patch base excision repair (1Waga S. Stillman B. Annu. Rev. Biochem. 1998; 67: 721-751Crossref PubMed Scopus (667) Google Scholar, 2Burgers P.M. Chromosoma. 1998; 107: 218-227Crossref PubMed Scopus (163) Google Scholar). For both replication and repair functions, pol δ requires an accessory protein, the proliferating cell nuclear antigen (PCNA), to carry out highly processive DNA synthesis (3Kelman Z. Oncogene. 1997; 14: 629-640Crossref PubMed Scopus (727) Google Scholar, 4Tsurimoto T. Front. Biosci. 1999; 4: D849-D858Crossref PubMed Google Scholar). Crystallographic studies have demonstrated that PCNA forms a homotrimeric ring that encircles double-stranded DNA and functions to tether the DNA polymerase to its template/primer, thus dramatically increasing the processivity of the enzyme (5Krishna T.S. Kong X.P. Gary S. Burgers P.M.J. Kuriyan J. Cell. 1994; 79: 1233-1243Abstract Full Text PDF PubMed Scopus (755) Google Scholar, 6Gulbis J.M. Kelman Z. Hurwitz J. O'Donnell M. Kuriyan J. Cell. 1996; 87: 297-306Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar). In addition to functioning as a processivity factor for pol δ (7Tan C.-K. Castillo C., So, A.G. Downey K.M. J. Biol. Chem. 1986; 261: 12310-12316Abstract Full Text PDF PubMed Google Scholar, 8Prelich G. Tan C.-K. Kostura M. Mathews M.B., So, A.G. Downey K.M. Stillman B. Nature. 1987; 326: 517-520Crossref PubMed Scopus (893) Google Scholar), PCNA also interacts with other replication/repair proteins such as DNA ligase 1 (9Levin D.S. Bai W. Yao N. O'Donnell M. Tomkinson A.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12863-12868Crossref PubMed Scopus (200) Google Scholar), the structural flap endonuclease 1 (10Li X., Li, J. Harrington J. Lieber M.R. Burgers P.M. J. Biol. Chem. 1995; 270: 22109-22112Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar), and replication factor C (11Fotedar R. Mossi R. Fitzgerald P. Pousselle T. Maga G. Brickner H. Messier H. Kasibhatla S. Hubscher U. Fotedar A. EMBO J. 1996; 15: 4423-4433Crossref PubMed Scopus (90) Google Scholar), as well as with several repair proteins,e.g. the mismatch repair proteins MSH3 and MSH6 (12Clark A.B. Valle E. Drotschmann K. Garg R.K. Kunkel T.A. J. Biol. Chem. 2000; 275: 36498-37501Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 13Kleczkowska H.E. Marra G. Lettieri T. Jiricny J. Genes Dev. 2001; 15: 724-736Crossref PubMed Scopus (198) Google Scholar) and the nucleotide excision repair endonuclease XPG (14Gary 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). PCNA also interacts with proteins that are involved in postreplication processes such as DNA (cytosine-5) methyl transferase (15Chuang L.S.H. Ian H.I. Koh T.W., Ng, H.H., Xu, G.L. Li B.F.L. Science. 1997; 277: 1996-2000Crossref PubMed Scopus (787) Google Scholar) and chromatin assembly factor 1 (CAF1) (16Shibahara K. Stillman B. Cell. 1999; 96: 575-585Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar, 17Moggs J. Grandi P. Quivy J.-P. Jonsson Z.O. Hubscher U. Becker P.B. Almouzni G. Mol. Cell. Biol. 2000; 20: 1206-1218Crossref PubMed Scopus (257) Google Scholar), as well as with several proteins that are involved in cell cycle control, e.g. the cyclin-dependent kinase inhibitors p21WAF1,CIP1,SDI (18Xiong Y. Zhang H. Beach D. Cell. 1993; 71: 505-514Abstract Full Text PDF Scopus (902) Google Scholar, 19Waga S. Hannon G.J. Beach D. Stillman B. Nature. 1994; 364: 574-578Crossref Scopus (1591) Google Scholar, 20Flores-Rozas H. Kelman Z. Dean F.B. Pan Z.-Q. Harper J.W. Elledge S.J. O'Donnell M. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6655-6659Crossref Scopus (417) Google Scholar) and p57 (21Watanabe H. Pan Z.Q. Schreiber-Aqus N. DePinho R.A. Hurwitz J. Xiong Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1392-1397Crossref PubMed Scopus (144) Google Scholar) and the DNA damage response protein GADD45 (22Smith M.L. Chen I.T. Zhan Q. Bae I. Chen C.Y. Gilmer T.M Kastan M.D. O'Connor P.M. Fornace A.G., Jr. Science. 1994; 266: 1376-1380Crossref PubMed Scopus (896) Google Scholar). Many of these proteins have been shown to contain a consensus PCNA-binding motif initially identified in p21 (23Warbrick E. Lane D.P. Glover D.M. Cox L.S. Curr. Biol. 1995; 5: 275-282Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar) that specifically binds at the interdomain connector loop of PCNA (6Gulbis J.M. Kelman Z. Hurwitz J. O'Donnell M. Kuriyan J. Cell. 1996; 87: 297-306Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar), suggesting that PCNA may coordinate DNA replication with DNA repair as well as with cell cycle progression by functioning as a regulatory target (1Waga S. Stillman B. Annu. Rev. Biochem. 1998; 67: 721-751Crossref PubMed Scopus (667) Google Scholar, 24Warbrick E. BioEssays. 2000; 22: 997-1006Crossref PubMed Scopus (353) Google Scholar). Despite the fact that PCNA was identified as a processivity factor for pol δ and a replication protein essential for in vitroSV40 DNA replication nearly 15 years ago (8Prelich G. Tan C.-K. Kostura M. Mathews M.B., So, A.G. Downey K.M. Stillman B. Nature. 1987; 326: 517-520Crossref PubMed Scopus (893) Google Scholar, 25Prelich G. Kostura M. Marshak D.R. Mathews M. Stillman B. Nature. 1987; 326: 471-475Crossref PubMed Scopus (356) Google Scholar), the site on pol δ that interacts with PCNA is still unresolved, as is the quaternary structure of the pol δ species that interacts with PCNA. Purified pol δ from calf thymus tissue has been shown to be a heterodimer comprised of a catalytic subunit of 125 kDa containing the DNA polymerase and 3′ to 5′ exonuclease active sites, and a 50-kDa subunit of unknown function (26Lee M.Y. Tan C.-K. Downey K.M. So A.G. Biochemistry. 1984; 23: 1906-1913Crossref PubMed Scopus (174) Google Scholar, 27Ng L. Tan C.-K. Downey K.M. Fisher P.A. J. Biol. Chem. 1991; 266: 11699-11704Abstract Full Text PDF PubMed Google Scholar). The heterodimeric core enzyme (p125/p50), which is essentially distributive, can be transformed into a highly processive DNA polymerase by PCNA (8Prelich G. Tan C.-K. Kostura M. Mathews M.B., So, A.G. Downey K.M. Stillman B. Nature. 1987; 326: 517-520Crossref PubMed Scopus (893) Google Scholar, 28Zhou J.-Q., He, H. Tan C.-K. Downey K.M. So A.G. Nucleic Acids Res. 1997; 25: 1090-1099Crossref Scopus (57) Google Scholar), suggesting that functional interaction of pol δ with PCNA is mediated through interaction with either the 125- or the 50-kDa subunit of the enzyme or with both. In contrast, highly purified pol δ from Schizosaccharomyces pombe is composed of four subunits: the homologues of p125 (Pol3/Cdc6) and p50 (Cdc1) and two additional subunits, Cdc27 and Cdm 1 (29Zuo S. Bermudez V. Zhang G. Kelman Z. Hurwitz J. J. Biol. Chem. 2000; 275: 5153-5162Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). In Saccharomyces cerevisiae, active pol δ can be isolated either as a heterodimer composed of the homologues of mammalian p125 and p50, i.e. Pol3 and Pol31, or as a heterotrimer with, in addition to Pol3 and Pol31, Pol32, the homologue of S. pombe Cdc27 (30Gerik K.J., Li, X.Y. Pautz A. Burgers P.M.J. J. Biol. Chem. 1998; 273: 19747-19755Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Interestingly, althoughcdc27+ is an essential gene in S. pombe (31MacNeill S.A. Moreno S. Reynolds N. Nurse P. Fantes P.A. EMBO J. 1996; 15: 4613-4628Crossref PubMed Scopus (87) Google Scholar), POl32 is not an essential gene in S. cerevisiae (30Gerik K.J., Li, X.Y. Pautz A. Burgers P.M.J. J. Biol. Chem. 1998; 273: 19747-19755Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Recently, it was reported that a homologue ofS. pombe Cdc27 or S. cerevisiae Pol32 is present in preparations of mammalian pol δ, i.e. p66 (32Hughes P. Tratner J. Ducous M. Piard K. Baldacci G. Nucleic Acids Res. 1999; 27: 2108-2114Crossref PubMed Scopus (76) Google Scholar). Studies aimed at identifying the subunit of pol δ that interacts with PCNA have produced conflicting answers. Binding studies using recombinant human p125 and PCNA have demonstrated direct interaction between these two polypeptides by protein overlay experiments using biotinylated PCNA and by biochemical cross-linking experiments (33Zhang P., Mo, J.Y. Perez A. Leon A. Liu L. Mazloum N., Hu, H. Lee M.Y.T. J. Biol. Chem. 1999; 274: 26647-26653Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar); however, pull-down assays using PCNA-linked beads have yielded both positive (34Shikata K. Ohta S. Yamada K Obuse C. Yoshikawa H. Tsurimoto T. J. Biochem. (Tokyo). 2001; 129: 699-708Crossref PubMed Scopus (39) Google Scholar) and negative (35Ducoux M Urbach S. Baldacci G Hubscher U Koundrioukoff S. Christensen J. Hughes P. J. Biol. Chem. 2001; 276: 49258-49266Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) results with respect to the binding of PCNA and p125. Similar studies with S. cerevisiae (30Gerik K.J., Li, X.Y. Pautz A. Burgers P.M.J. J. Biol. Chem. 1998; 273: 19747-19755Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 36Eissenberg J.C. Ayyagari R. Gomes X.V. Burgers P.M.J. Mol. Cell. Biol. 1997; 17: 6367-6378Crossref PubMed Google Scholar) and S. pombe (37Tratner I. Piard K. Grenon M. Perderiset M. Baldacci G. Biochem. Biophys. Res. Commun. 1997; 231: 321-328Crossref PubMed Scopus (20) Google Scholar, 38Reynolds N. Warbrick E. Fantes P.A. MacNeill S.A. EMBO J. 2000; 19: 1108-1118Crossref PubMed Scopus (53) Google Scholar) pol δ failed to detect any interaction between PCNA and the catalytic subunits of the yeast enzymes. The small subunits of pol δ from both budding yeast (Pol31) and mammalian (p50) sources were found not to directly bind PCNA (30Gerik K.J., Li, X.Y. Pautz A. Burgers P.M.J. J. Biol. Chem. 1998; 273: 19747-19755Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar,33Zhang P., Mo, J.Y. Perez A. Leon A. Liu L. Mazloum N., Hu, H. Lee M.Y.T. J. Biol. Chem. 1999; 274: 26647-26653Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 34Shikata K. Ohta S. Yamada K Obuse C. Yoshikawa H. Tsurimoto T. J. Biochem. (Tokyo). 2001; 129: 699-708Crossref PubMed Scopus (39) Google Scholar, 36Eissenberg J.C. Ayyagari R. Gomes X.V. Burgers P.M.J. Mol. Cell. Biol. 1997; 17: 6367-6378Crossref PubMed Google Scholar). A third subunit of pol δ, initially identified in S. pombe(Cdc27), was found to interact with Cdc1 (homologue of mammalian p50 and S. cerevisiae Pol31) and to bind PCNA through a consensus PCNA-binding motif, suggesting that the third subunit mediates the interaction between pol δ and PCNA (31MacNeill S.A. Moreno S. Reynolds N. Nurse P. Fantes P.A. EMBO J. 1996; 15: 4613-4628Crossref PubMed Scopus (87) Google Scholar, 38Reynolds N. Warbrick E. Fantes P.A. MacNeill S.A. EMBO J. 2000; 19: 1108-1118Crossref PubMed Scopus (53) Google Scholar). Homologues of Cdc27 in S. cerevisiae (Pol32) and mammalian cells (p66) also contain a consensus PCNA-binding motif (30Gerik K.J., Li, X.Y. Pautz A. Burgers P.M.J. J. Biol. Chem. 1998; 273: 19747-19755Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 32Hughes P. Tratner J. Ducous M. Piard K. Baldacci G. Nucleic Acids Res. 1999; 27: 2108-2114Crossref PubMed Scopus (76) Google Scholar), and Pol32 has also been found to interact with both Pol31 and PCNA (30Gerik K.J., Li, X.Y. Pautz A. Burgers P.M.J. J. Biol. Chem. 1998; 273: 19747-19755Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Functional studies with recombinant p125 from human (28Zhou J.-Q., He, H. Tan C.-K. Downey K.M. So A.G. Nucleic Acids Res. 1997; 25: 1090-1099Crossref Scopus (57) Google Scholar, 39Zhou J.-Q. Tan C.-K., So, A.G. Downey K.M. J. Biol. Chem. 1996; 271: 29740-29745Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 40Sun Y.B. Jiang Y.Q. Zhang P. Zhang S.J. Zhou Y., Li, B.Q. Toomey N.L. Lee M. J. Biol. Chem. 1997; 272: 13013-13018Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), mouse (41Hindges R. Hubscher U. Gene (Amst.). 1995; 158: 241-246Crossref PubMed Scopus (22) Google Scholar), and S. pombe (42Arroyo M.P. Downey K.M., So, A.G. Wang T.S.F. J. Biol. Chem. 1996; 271: 1571-1580Abstract Full Text Full Text PDF Scopus (35) Google Scholar) sources have shown that DNA synthesis catalyzed by p125 alone is not significantly stimulated by PCNA. On the other hand, PCNA was shown to stimulate the activity and processivity of the recombinant human heterodimer to the same extent as the native two-subunit enzyme isolated from calf thymus (28Zhou J.-Q., He, H. Tan C.-K. Downey K.M. So A.G. Nucleic Acids Res. 1997; 25: 1090-1099Crossref Scopus (57) Google Scholar), suggesting the possibility that the interaction of PCNA with pol δ is mediated through the small subunit. Similarly, heterodimeric pol δ from S. cerevisiae (Pol3/Pol31) was found to be highly processive in the presence of PCNA (43Burgers P.M.J. Gerik K.J. J. Biol. Chem. 1998; 273: 19756-19762Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). In fact, heterodimeric pol δ was found to be as processive as heterotrimeric pol δ (Pol3/Pol31/Pol32), the only difference being that the latter enzyme required considerably lower concentrations of PCNA for highly processive synthesis (43Burgers P.M.J. Gerik K.J. J. Biol. Chem. 1998; 273: 19756-19762Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). In contrast, recent functional studies in which recombinant human enzymes containing either two subunits (p125/p50) or three subunits (p125/p50/p66) were directly compared demonstrated that only the three-subunit enzyme could be stimulated by PCNA (34Shikata K. Ohta S. Yamada K Obuse C. Yoshikawa H. Tsurimoto T. J. Biochem. (Tokyo). 2001; 129: 699-708Crossref PubMed Scopus (39) Google Scholar, 35Ducoux M Urbach S. Baldacci G Hubscher U Koundrioukoff S. Christensen J. Hughes P. J. Biol. Chem. 2001; 276: 49258-49266Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The reasons for these discrepancies are not clear. In the present study we have used antibodies to the individual subunits of mammalian pol δ and PCNA to examine the interactions among these polypeptides by co-immunoprecipitation to identify the PCNA-binding site on pol δ. The plasmids pT7/hPCNA and pETp21His were the kind gifts of Drs. Bruce Stillman and Shou Waga (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The plasmid pET16b-p50 was constructed by subcloning the human p50 cDNA (44Zhang J. Tan C.-K. McManus B. Downey K.M. So A.G. Genomics. 1995; 29: 179-186Crossref PubMed Scopus (30) Google Scholar) into theNdeI site of pET16b (Novagen). Calf thymus pol δ was purified through step 7 as described by Downey and So (45Downey K.M. So A.G. Methods Enzymol. 1995; 262: 84-92Crossref PubMed Scopus (13) Google Scholar). Recombinant human p125 and p50 were overexpressed in Sf9 cells using the baculoviruses AcN-p125-14 and AcN-p50-1 and purified as described previously (28Zhou J.-Q., He, H. Tan C.-K. Downey K.M. So A.G. Nucleic Acids Res. 1997; 25: 1090-1099Crossref Scopus (57) Google Scholar, 39Zhou J.-Q. Tan C.-K., So, A.G. Downey K.M. J. Biol. Chem. 1996; 271: 29740-29745Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Recombinant human PCNA was produced in Escherichia coliusing the plasmid pT7/hPCNA and purified as described in Brush et al. (46Brush G.S. Kelly T.J. Stillman B. Methods Enzymol. 1995; 262: 522-548Crossref PubMed Scopus (45) Google Scholar). Polyhistidine-tagged p21 (His-p21) was expressed inE. coli using the plasmid pETp21His and purified according to Waga et al. (19Waga S. Hannon G.J. Beach D. Stillman B. Nature. 1994; 364: 574-578Crossref Scopus (1591) Google Scholar). Polyhistidine-tagged p50 (His-p50) was expressed in E. coli using the plasmid pET16b-p50. The fusion protein was purified by binding to a nickel-chelate-nitrilotriacetic acid column (Qiagen) in 50 mm Tris-HCl, pH 7.8, 7.5% glycerol, and 2 mmphenylmethylsulfonyl fluoride. After washing with the same buffer containing 40 mm imidazole, His-p50 was eluted with the same buffer containing 400 mm imidazole and dialyzed against phosphate-buffered saline (137 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, 1.8 mm KH2PO4, pH 7.5) containing 2 mm dithiothreitol. Human autoantiserum to PCNA was a generous gift of Dr. Irving Kushner (Cleveland Metropolitan General Hospital, Cleveland, OH). Rabbit antibodies to the N-terminal one-third of human p125, expressed as a glutathione S-transferase fusion protein, were prepared as described previously (28Zhou J.-Q., He, H. Tan C.-K. Downey K.M. So A.G. Nucleic Acids Res. 1997; 25: 1090-1099Crossref Scopus (57) Google Scholar). Polyclonal antibodies to His-p50 were raised in rabbits. IgGs from human anti-PCNA, rabbit anti-Hisp50, and rabbit anti-glutathione S-transferase p125 were purified on protein A-Sepharose 4B (Sigma) and then coupled to protein A-Sepharose 4B beads using dimethyl pimelidate (Pierce) according to Harlow and Lane (47Harlow E. Lane D. Using Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1999: 321-325Google Scholar). Control beads were prepared by cross-linking preimmune rabbit IgG or normal human IgG to protein A-Sepharose beads. Monoclonal anti-PCNA antibody (PC-10) was from Sigma. Monoclonal anti-p125 antibody (clone 22) was from BD Bioscience. Chicken anti-p50 antibodies were prepared by Alpha Diagnostic International (San Antonio, TX). Peroxidase-conjugated rabbit anti-mouse IgG and peroxidase-conjugated donkey anti-chicken IgY were from Jackson Immunoresearch (West Grove, PA). Purified recombinant proteins were mixed with antibody-linked protein A-Sepharose beads at 4 °C for 3 h in phosphate-buffered saline containing 0.2% Nonidet P-40 (200 μl of total volume). After extensive washing with the same buffer, the beads were mixed with an equal volume of 1× Laemmli SDS sample buffer and denatured at 90 °C for 10 min. The proteins in the supernatants were separated by 10% SDS-PAGE and electroblotted onto a nitrocellulose membrane (Bio-Rad). After incubation for 2 h at room temperature with blocking buffer, Tris-buffered saline (25 mm Tris-HCl, pH 7.5, 150 mm NaCl) containing 5% nonfat dry milk, the membrane was incubated with primary antibody for 2 h at room temperature in the same buffer, washed extensively with Tris-buffered saline containing 0.05% Tween 20 (TBST), and incubated with peroxidase-conjugated secondary antibody for 1 h. After extensive washing with TBST, the proteins were detected by SuperSignal enhanced chemiluminescent substrate (Pierce). Peptides or proteins were slot-blotted onto a polyvinylidene difluoride membrane (Bio-Rad) and processed as described by He et al. (48He H. Tan C.-K. Downey K.M. So A.G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11979-11984Crossref PubMed Scopus (48) Google Scholar). Wild type p50 peptide (amino acids 51–72) (AHIYATRLIQMRPFLENRAQQH), mutated p50 peptide (F64A,L65A) (AHIYATRLIQMRPAAENRAQQH), an unrelated peptide (AGSYIVPEDKREMWMACIKEAA), and p21 peptide (amino acids 139–160) (GRKRRQTSMTDFYHSKRRLIFS) were synthesized by ResGen (Huntsville, AL). The protein concentration was determined using the Bio-Rad DC protein assay kit with bovine serum albumin as a standard. To identify the subunit(s) of pol δ that interact(s) with PCNA, we have used purified recombinant human p125, p50, and PCNA and polyclonal antibodies to each of these polypeptides linked to protein A-Sepharose beads to detect co-immunoprecipitation of PCNA with one or both of the subunits of the pol δ core enzyme. As shown in Fig.1 A, anti-PCNA beads co-immunoprecipitated p50 in the presence but not in the absence of PCNA, and the amount of p50 co-immunoprecipitated increased with increasing amounts of PCNA in the reaction mixture. To confirm the association of PCNA with p50, reciprocal immunoprecipitation experiments were carried out using anti-p50 polyclonal antibodies. As shown in Fig. 1 B, anti-p50 beads co-immunoprecipitated PCNA in the presence but not in the absence of p50. These experiments provide strong evidence for a direct interaction of p50 and PCNA. As shown in Fig. 1 C, anti-p125 antibodies failed to co-immunoprecipitate PCNA in the presence of purified p125; however, anti-p125 antibodies effectively co-immunoprecipitated PCNA in the presence of calf thymus pol δ, i.e. when p125 is complexed with p50 in the heterodimeric core enzyme. Reciprocal experiments were carried out to further substantiate that the interaction of PCNA with p125 is mediated through p50. As shown in Fig. 1 D, anti-PCNA beads failed to co-immunoprecipitate recombinant p125 in the presence of PCNA but efficiently co-immunoprecipitated p125 when it was complexed with p50 in native calf thymus pol δ. These experiments suggest that the interaction of PCNA with p125 is mediated through p50. It has been shown that p21 and human pol δ both bind to the interdomain connector loop of PCNA (6Gulbis J.M. Kelman Z. Hurwitz J. O'Donnell M. Kuriyan J. Cell. 1996; 87: 297-306Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar, 49Zhang P. Sun Y. Hsu H. Zhang L. Zhang Y Lee M.Y.W.T. J. Biol. Chem. 1998; 273: 713-719Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) and that p21 inhibits pol δ-catalyzed DNA synthesis in vitro (19Waga S. Hannon G.J. Beach D. Stillman B. Nature. 1994; 364: 574-578Crossref Scopus (1591) Google Scholar, 20Flores-Rozas H. Kelman Z. Dean F.B. Pan Z.-Q. Harper J.W. Elledge S.J. O'Donnell M. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6655-6659Crossref Scopus (417) Google Scholar). If the interaction of PCNA with pol δ is indeed mediated through p50, one would expect that the binding of p50 to PCNA would be disrupted by the addition of p21. That this is the case is shown in Fig. 2. The addition of molar ratios of p21 to PCNA monomer greater than 1:1 resulted in inhibition of binding of p50 to PCNA, and complete disruption was achieved when the molar ratio of p21 to PCNA exceeded 3:1 (Fig. 2 A). The addition of an oligopeptide containing the p21 PCNA-binding motif also inhibited the binding of PCNA to p50 (Fig. 2 B), and the p21 peptide was nearly as efficient as the p21 protein in inhibiting PCNA binding to p50, i.e.binding was ∼50% inhibited at 22 nm p21 protein and 35 nm p21 peptide (Fig. 2 C). It has been demonstrated that p21 effectively competes with a number of replication/repair proteins such as flap endonuclease 1 (50Warbrick E. Lane D.P. Glover D.M. Cox L.S. Oncogene. 1997; 14: 2313-2321Crossref PubMed Scopus (136) Google Scholar) and DNA ligase 1 (9Levin D.S. Bai W. Yao N. O'Donnell M. Tomkinson A.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12863-12868Crossref PubMed Scopus (200) Google Scholar), repair proteins such as XPG (14Gary 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), as well as the postreplication processing protein DNA (cytosine-5) methyl transferase (15Chuang L.S.H. Ian H.I. Koh T.W., Ng, H.H., Xu, G.L. Li B.F.L. Science. 1997; 277: 1996-2000Crossref PubMed Scopus (787) Google Scholar), all of which share a consensus p21-like PCNA-binding motif, for binding to PCNA at the interdomain connector loop. The observation that p21 can dissociate the p50-PCNA complex suggests that the PCNA-binding motif of p50 targets the same site on PCNA as p21. Examination of the amino acid sequence of human p50 (44Zhang J. Tan C.-K. McManus B. Downey K.M. So A.G. Genomics. 1995; 29: 179-186Crossref PubMed Scopus (30) Google Scholar) did not identify the consensus eight-amino acid PCNA-binding motif (Q1 XX(I/L/M)4 XX(F/H)7(F/Y)8) originally identified in p21 and found in most proteins that interact with PCNA (24Warbrick E. BioEssays. 2000; 22: 997-1006Crossref PubMed Scopus (353) Google Scholar, 51Warbrick E. BioEssays. 1998; 20: 195-197Crossref PubMed Scopus (315) Google Scholar). However, a hydrophobic five-amino acid sequence (MRPFL) that is homologous to a recently identified motif in the C termini of RB69 DNA polymerase (LFDMF) and T4 DNA polymerase (LDFLF) and that is necessary for interaction of these polymerases with their respective sliding clamps (52Shamoo Y. Steitz T.A. Cell. 1999; 99: 155-166Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar) was identified in human p50. However, the motif was found at the N terminus (residues 61–65) rather than the C terminus of p50. Similar to the sliding clamp-binding sites of p21 and RB69 DNA polymerase, the p50 sequence is also in a helical conformation. To determine whether the MRPFL sequence in human p50 indeed represents a PCNA-binding motif, far Western analysis was carried out. A 22-amino acid peptide containing the putative PCNA-binding motif was synthesized, as was a mutated peptide, an unrelated peptide, and the p21 peptide as a positive control. The peptides, along with p50 protein and p21 protein, were slot-blotted to a polyvinylidene difluoride membrane, overlaid with PCNA, and probed with a monoclonal antibody to PCNA. The results (Fig. 3) demonstrated that the peptide containing MRPFL did interact with PCNA, whereas the mutated peptide and the unrelated peptide did not. Although the p50 peptide, the p21 peptide, and p21 protein bound PCNA, p50 protein did not, possibly because of denaturation or improper folding of the protein on the membrane. To further examine whether the interaction of p50 with PCNA is mediated through the sequence motif MRPFL, we investigated the effects of the addition of increasing amounts of the oligopeptide containing the identified PCNA-binding motif on the interaction of p50 with PCNA in co-immunoprecipitation experiments (Fig.4). As shown in Fig. 4 A, the p50 peptide, but not the mutated peptide in which residues Phe and Leu are substituted by alanines, effectively blocked the binding of p50 to PCNA. A 50% inhibition of binding was achieved at 200 nmpeptide (Fig. 4 B), corresponding to a molar ratio of p50 peptide to p50 protein of 10:1. These results establish that the MRPFL motif is responsible for the binding of p50 to PCNA. In this report we have demonstrated that PCNA interacts directly with the small subunit of mammalian pol δ (p50) by co-immunoprecipitation of these two proteins using polyclonal antibodies to each of them. We have further demonstrated, using polyclonal antibodies to either p125 or PCNA, that the catalytic subunit can only be co-immunoprecipitated with PCNA when it is complexed with p50 in heterodimeric pol δ and not when it exists as a single subunit. These results suggest that PCNA does not directly interact with the catalytic subunit and that the interaction between pol δ and its sliding clamp is mediated through the small subunit. The demonstration of a direct intera" @default.
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• W2077338748 date "2002-07-01" @default.
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• W2077338748 title "Direct Interaction of Proliferating Cell Nuclear Antigen with the Small Subunit of DNA Polymerase δ" @default.
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