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• W2057747091 abstract "Proliferating cell nuclear antigen (PCNA) plays an essential role in nucleic acid metabolism as a component of the DNA replication and DNA repair machinery. As such, PCNA interacts with many proteins that have a sequence motif termed the PCNAinteracting motif (PIM) and also with proteins lacking a PIM. Three regions in human and rat DNA polymerases β (β-pol) that resemble the consensus PIM were identified, and we show here that β-polymerase and PCNA can form a complex both in vitro and in vivo. Immunoprecipitation experiments, yeast two-hybrid analysis, and overlay binding assays were used to examine the interaction between the two proteins. Competition experiments with synthetic PIM-containing peptides suggested the importance of a PIM in the interaction, and studies of a β-polymerase PIM mutant, H222A/F223A, demonstrated that this alteration blocked the interaction with PCNA. The results indicate that at least one of the PIM-like sequences in β-polymerase appears to be a functional PIM and was required in the interaction between β-polymerase and PCNA. Proliferating cell nuclear antigen (PCNA) plays an essential role in nucleic acid metabolism as a component of the DNA replication and DNA repair machinery. As such, PCNA interacts with many proteins that have a sequence motif termed the PCNAinteracting motif (PIM) and also with proteins lacking a PIM. Three regions in human and rat DNA polymerases β (β-pol) that resemble the consensus PIM were identified, and we show here that β-polymerase and PCNA can form a complex both in vitro and in vivo. Immunoprecipitation experiments, yeast two-hybrid analysis, and overlay binding assays were used to examine the interaction between the two proteins. Competition experiments with synthetic PIM-containing peptides suggested the importance of a PIM in the interaction, and studies of a β-polymerase PIM mutant, H222A/F223A, demonstrated that this alteration blocked the interaction with PCNA. The results indicate that at least one of the PIM-like sequences in β-polymerase appears to be a functional PIM and was required in the interaction between β-polymerase and PCNA. DNA polymerase β base excision repair DNA polymerase α DNA polymerase δ DNA polymerase ε apurinic/apyrimidinic AP endonuclease proliferating cell nuclear antigen flap endonuclease-1 PCNA interacting motif Tris-buffered saline Tris-buffered saline-Tween 20 enhanced chemiluminescence dropout medium lacking Trp, Leu, and His bovine serum albumin wild-type DNA repair is vital for cell survival and maintenance of genomic stability. DNA polymerase β (β-pol)1 is known to be involved in short-gap filling DNA synthesis in mammalian cells. The enzyme plays roles in base excision repair (BER) (1Singhal R.K. Prasad R. Wilson S.H. J. Biol. Chem. 1995; 270: 949-957Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar) and in some cases, can function in DNA replication as well as other pathways of DNA repair (2Jenkins T.M. Saxena J.K. Kumar A. Wilson S.H. Ackerman E.J. Science. 1992; 258: 475-478Crossref PubMed Scopus (47) Google Scholar). Base lesions in DNA arise from a variety of physical and chemical agents. These lesions are repaired in part by BER. There are at least two subpathways of BER, differentiated by the repair patch sizes and the enzymes involved, and these subpathways are classified as “single-nucleotide” and “long patch” BER, respectively (3Frosina G. Fortini P. Rossi O. Carrozzino 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, 4Wilson S.H. Mutat. Res. 1998; 407: 203-215Crossref PubMed Scopus (264) Google Scholar). Four purified human enzymes can reconstitute single-nucleotide BER of uracil-DNA (5Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (689) Google Scholar, 6Nicholl I.D. Nealon K. Kenny M.K. Biochemistry. 1997; 36: 7557-7566Crossref PubMed Scopus (77) Google Scholar, 7Srivastava D.K. Vande Berg B.J. Prasad R. Molina J.T. Beard W.A. Tomkinson A.E. Wilson S.H. J. Biol. Chem. 1998; 273: 21203-21209Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). This repair pathway is a sequential process initiated by uracil-DNA glycosylase base removal and formation of the apurinic/apyrimidinic (AP) site, this was followed by AP endonuclease (APE) incision of the AP site (8Doetsch P.W. Cunningham R.P. Mutat. Res. 1990; 236: 173-201Crossref PubMed Scopus (324) Google Scholar, 9Mosbaugh D.W. Bennett S.E. Prog. Nucleic Acids Res. Mol. Biol. 1994; 48: 315-370Crossref PubMed Scopus (97) Google Scholar). The resulting single nucleotide gap is filled by β-pol, and the enzyme also conducts another required enzymatic step, removal of the sugar phosphate from the incised AP site (10Matsumoto Y. Kim K. Science. 1995; 269: 699-702Crossref PubMed Scopus (646) Google Scholar, 11Piersen C.E. Prasad R. Wilson S.H. Lloyd R.S. J. Biol. Chem. 1996; 271: 17811-17815Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Finally, DNA ligase I or the x-ray cross-complementing factor 1-DNA ligase III complex completes this BER subpathway (5Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (689) Google Scholar, 12Caldecott K.W. Aoufouchi S. Johnson P. Shall S. Nucleic Acids Res. 1996; 24: 4387-4394Crossref PubMed Scopus (546) Google Scholar, 13Dimitriadis E.K. Prasad R. Vaske M.K. Chen L. Tomkinson A.E. Lewis M.S. Wilson S.H. J. Biol. Chem. 1998; 273: 20540-20550Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 14Prasad R. Singhal R.K. Srivastava D.K. Molina J.T. Tomkinson A.E. Wilson S.H. J. Biol. Chem. 1996; 271: 16000-16007Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). It has been proposed that the various sequential steps in the single-nucleotide BER subpathway are coordinated through protein-protein interactions. A direct interaction between β-pol/DNA ligase I has been described, as has interaction between β-pol and x-ray cross-complementing factor 1-ligase III (5Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (689) Google Scholar, 12Caldecott K.W. Aoufouchi S. Johnson P. Shall S. Nucleic Acids Res. 1996; 24: 4387-4394Crossref PubMed Scopus (546) Google Scholar, 13Dimitriadis E.K. Prasad R. Vaske M.K. Chen L. Tomkinson A.E. Lewis M.S. Wilson S.H. J. Biol. Chem. 1998; 273: 20540-20550Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 14Prasad R. Singhal R.K. Srivastava D.K. Molina J.T. Tomkinson A.E. Wilson S.H. J. Biol. Chem. 1996; 271: 16000-16007Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). These interactions could have biological consequences, as cells deficient in the proteins, β-pol, DNA ligase I, DNA ligase III, or x-ray cross-complementing factor 1, are hypersensitive to DNA alkylating agents (15Ochs K. Sobol R.W. Wilson S.H. Kaina B. Cancer Res. 1999; 59: 1544-1551PubMed Google Scholar, 16Sobol R.W. Horton J.K. Kuhn R., Gu, H. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Nature. 1996; 379: 183-186Crossref PubMed Scopus (783) Google Scholar, 17Sobol R.W. Prasad R. Evenski A. Baker A. Yang X.P. Horton J.K. Wilson S.H. Nature. 2000; 405: 807-810Crossref PubMed Scopus (299) Google Scholar, 18Teo I.A. Arlett C.F. Harcourt S.A. Priestley A. Broughton B.C. Mutat. Res. 1983; 107: 371-386Crossref PubMed Scopus (51) Google Scholar, 19Thompson L.H. Brookman K.W. Dillehay L.E. Carrano A.V. Mazrimas J.A. Mooney C.L. Minkler J.L. Mutat. Res. 1982; 95: 427-440Crossref PubMed Scopus (285) Google Scholar, 20Tomkinson A.E. Chen L. Dong Z. Leppard J.B. Levin D.S. Mackey Z.B. Motycka T.A. Prog. Nucleic Acid Res. Mol. Biol. 2001; 68: 151-164Crossref PubMed Google Scholar), and extracts from the cells are defective in BERin vitro (21Cappelli E. Taylor R. Cevasco M. Abbondandolo A. Caldecott K. Frosina G. J. Biol. Chem. 1997; 272: 23970-23975Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 22Prigent C. Satoh M.S. Daly G. Barnes D.E. Lindahl T. Mol. Cell. Biol. 1994; 14: 310-317Crossref PubMed Scopus (129) Google Scholar). Also, an interaction between β-pol and DNA-bound APE has been reported (23Bennett R.A. Wilson III, D.M. Wong D. Demple B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7166-7169Crossref PubMed Scopus (325) Google Scholar), yet these two proteins do not directly interact in solution. Finally, uracil-DNA glycosylase has been proposed to recruit APE to the AP site after release of uracil from uracil-DNA (24Parikh S.S. Putnam C.D. Tainer J.A. Mutat. Res. 2000; 460: 183-199Crossref PubMed Scopus (113) Google Scholar). The long patch BER subpathway involves multiple proteins, in addition to those described above for single-nucleotide BER. These include replication factor C, PCNA, DNA polymerases δ/ε (pol δ/ε), flap endonuclease-1 (FEN-1), and poly(ADP-ribose) polymerase-1 (25Dantzer F. de La Rubia G. Menissier-De Murcia J. Hostomsky Z. de Murcia G. Schreiber V. Biochemistry. 2000; 39: 7559-7569Crossref PubMed Scopus (405) Google Scholar, 26Lavrik O.I. Prasad R. Sobol R.W. Horton J.K. Ackerman E.J. Wilson S.H. J. Biol. Chem. 2001; 276: 25541-25548Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 27Matsumoto Y. Kim K. Hurwitz J. Gary R. Levin D.S. Tomkinson A.E. Park M.S. J. Biol. Chem. 1999; 274: 33703-33708Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 28Pascucci B. Stucki M. Jonsson Z.O. Dogliotti E. Hubscher U. J. Biol. Chem. 1999; 274: 33696-33702Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 29Prasad R. Dianov G.L. Bohr V.A. Wilson S.H. J. Biol. Chem. 2000; 275: 4460-4466Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 30Prasad R. Lavrik O.I. Kim S.J. Kedar P. Yang X.P. Vande Berg B.J. Wilson S.H. J. Biol. Chem. 2001; 276: 32411-32414Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). PCNA is known to interact with some of the BER enzymes, including FEN-1 and DNA ligase I (20Tomkinson A.E. Chen L. Dong Z. Leppard J.B. Levin D.S. Mackey Z.B. Motycka T.A. Prog. Nucleic Acid Res. Mol. Biol. 2001; 68: 151-164Crossref PubMed Google Scholar). Klungland and Lindahl (31Klungland A. Lindahl T. EMBO J. 1997; 16: 3341-3348Crossref PubMed Scopus (660) Google Scholar) found that PCNA enhances β-pol-dependent long patch BER of AP sites by stimulating FEN-1 activity. No role, however, has been proposed for PCNA in single-nucleotide BER. PCNA is also well known as a component of the DNA replication system in mammalian cells, and it plays roles in multiple cellular pathways in addition to DNA replication and BER, including the following: nucleotide excision repair (32Nichols A.F. Sancar A. Nucleic Acids Res. 1992; 20: 2441-2446Crossref PubMed Scopus (181) Google Scholar, 33Shivji K.K. Kenny M.K. Wood R.D. Cell. 1992; 69: 367-374Abstract Full Text PDF PubMed Scopus (732) Google Scholar), mismatch repair (34Umar A. Buermeyer A.B. Simon J.A. Thomas D.C. Clark A.B. Liskay R.M. Kunkel T.A. Cell. 1996; 87: 65-73Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar), cell cycle control (35Chen I.T. Smith M.L. O'Connor P.M. Fornace Jr., A.J. Oncogene. 1995; 11: 1931-1937PubMed Google Scholar, 36Hall P.A. Kearsey J.M. Coates P.J. Norman D.G. Warbrick E. Cox L.S. Oncogene. 1995; 10: 2427-2433PubMed Google Scholar, 37Watanabe H. Pan Z.Q. Schreiber-Agus N. DePinho R.A. Hurwitz J. Xiong Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1392-1397Crossref PubMed Scopus (144) Google Scholar), apoptosis (38Scott M. Bonnefin P. Vieyra D. Boisvert F.M. Young D. Bazett-Jones D.P. Riabowol K. J. Cell Sci. 2001; 114: 3455-3462Crossref PubMed Google Scholar), and transcription (39Hasan S. Hassa P.O. Imhof R. Hottiger M.O. Nature. 2001; 410: 387-391Crossref PubMed Scopus (148) Google Scholar). Thus, PCNA has been termed a “cellular communicator” by virtue of its ability to connect various cellular processes (40Jonsson Z.O. Hindges R. Hubscher U. EMBO J. 1998; 17: 2412-2425Crossref PubMed Scopus (235) Google Scholar). Finally, PCNA is known to function as a processivity factor for DNA polymerases such as pol δ and pol ε in vitro (41Stucki M. Pascucci B. Parlanti E. Fortini P. Wilson S.H. Hubscher U. Dogliotti E. Oncogene. 1998; 17: 835-843Crossref PubMed Scopus (160) Google Scholar). Whereas evaluating the question of potential interacting partners for mammalian β-pol in BER, we identified three short sequences (7–9 residues) that were similar to the PCNAinteracting motif or PIM in some of the known PCNA-binding proteins, and we subsequently found, by cell extract immunoprecipitation and yeast two-hybrid experiments, that PCNA appeared to interact with β-pol in vivo. A possible direct interaction between β-pol and PCNA was examined using the purified samples of the two proteins and a combination of co-immunoprecipitation and overlay assay binding techniques. We found that the proteins interact directly and mapped a region of β-pol responsible for its interaction with PCNA to a sequence resembling a consensus PIM. These results and possible implications of the β-pol and PCNA interaction are discussed. Dulbecco's modified Eagle's medium and GlutaMAX-1 were from Invitrogen. Fetal bovine serum was from Summit Biotechnology (Ft. Collins, CO) and hygromycin was from Roche Molecular Biochemicals (Indianapolis, IN). Anti-β-pol affinity purified polyclonal antibody has been described previously (14Prasad R. Singhal R.K. Srivastava D.K. Molina J.T. Tomkinson A.E. Wilson S.H. J. Biol. Chem. 1996; 271: 16000-16007Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar); anti-PCNA polyclonal antibody (Ab-5) and anti-PCNA monoclonal antibody (Ab-2) were from Oncogene Research Products (Boston, MA); anti-PCNA monoclonal antibody (SC-56) was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-α-pol mouse monoclonal antibody (SJK132-20) and rabbit monoclonal antibody (DPN) were gifts from Dr. W. C. Copeland, NIEHS, National Institutes of Health. Anti-FEN-1 monoclonal antibody (FEN-1–4EP) was from Genetex (San Antonio, TX). Matchmaker two-hybrid systems were from CLONTECH (Palo Alto, CA). The mouse IgG secondary antibody used was goat anti-mouse IgG (H+L) binding grade affinity purified horseradish peroxidase conjugate, and the rabbit IgG secondary antibody used was goat anti-rabbit IgG (H+L)-horseradish peroxidase conjugate, both from Bio-Rad. Protein A-Sepharose CL-4B and SP-Sepharose (fast flow) were from AmershamBiosciences. Protein G-agarose and the protease inhibitor complete (EDTA-free) were from Roche Molecular Diagnostics. Leupeptin, aprotinin, and phenylmethylsulfonyl fluoride were from Calbiochem (La Jolla, CA). Normal goat serum was from Vector Laboratories (Burlingame, CA). Human β-pol, rat β-pol, and human PCNA were purified as described previously (42Beard W.A. Wilson S.H. Methods Enzymol. 1995; 262: 98-107Crossref PubMed Scopus (159) Google Scholar, 43Fien K. Stillman B. Mol. Cell. Biol. 1992; 12: 155-163Crossref PubMed Scopus (190) Google Scholar, 44Kumar A. Widen S.G. Williams K.R. Kedar P. Karpel R.L. Wilson S.H. J. Biol. Chem. 1990; 265: 2124-2131Abstract Full Text PDF PubMed Google Scholar). Special care was taken to remove DNA from the PCNA preparation, because some DNA persisted in co-elution with purified PCNA. DNA was removed by chromatography on phenyl-Sepharose, Resource S, and Superdex S200 columns (Amersham Biosciences) in buffer containing 50 mmTris-HCl, pH 7.5, 1 mm EDTA, 1 mmdithiothreitol, and 100 mm KCl. UV spectra were measured before and after each column step. The final preparation was free of DNA as measured by spectral analysis and by ethidium bromide staining after native gel electrophoresis. The peptide derivative of p21, KRRQTSMTDFYHSKRRLIFS (amino acids 141–160 of p21) contains the site for p21WAF1 and PCNA interaction (34Umar A. Buermeyer A.B. Simon J.A. Thomas D.C. Clark A.B. Liskay R.M. Kunkel T.A. Cell. 1996; 87: 65-73Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar, 45Warbrick E. Lane D.P. Glover D.M. Cox L.S. Curr. Biol. 1995; 5: 275-282Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). The negative control or “jumbled” peptide, QDKTRYFHRTMSRSKSIRLF, had the same amino acid composition as the p21 peptide. The peptide derivative of human MSH6, MSRQSTLYSFFPKSPALSDA, contains the site for MSH6 and PCNA interaction (46Clark A.B. Valle F. Drotschmann K. Gary R.K. Kunkel T.A. J. Biol. Chem. 2000; 275: 36498-36501Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). A peptide containing the PIM-like sequence found in human β-pol (Fig. 2, region II), HQVVEQLQKVHFITDTLSKGE, was obtained. All peptides were from Research Genetics Inc. (Huntsville, AL); purity was found to be greater than 75% by high performance liquid chromatography. Peptides were weighed and dissolved in water at 5–10 mg/ml and stored at −80 °C. The cell lines used were a clone of the wild-type (WT) mouse embryonic fibroblast cell line Mβ16tsA, a clone of the isogenic β-pol null line Mβ19tsA described previously (16Sobol R.W. Horton J.K. Kuhn R., Gu, H. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Nature. 1996; 379: 183-186Crossref PubMed Scopus (783) Google Scholar), and a β-pol null cell line (termed 19HB3) stably transfected with a FLAG-β-pol vector and expressing a high level of the protein (26Lavrik O.I. Prasad R. Sobol R.W. Horton J.K. Ackerman E.J. Wilson S.H. J. Biol. Chem. 2001; 276: 25541-25548Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Cells were routinely grown at 34 °C in a 10% CO2incubator in Dulbecco's modified Eagle's medium supplemented with GlutaMAX-1, 10% fetal bovine serum, and hygromycin (80 μg/ml). All cells were routinely tested and found to be free of mycoplasma contamination. The WT, β-pol null, and 19HB3 cells were harvested and washed two times in phosphate-buffered saline. Cell lysates were prepared in a lysis buffer (47Watters D. Khanna K.K. Beamish H. Birrell G. Spring K. Kedar P. Gatei M. Stenzel D. Hobson K. Kozlov S. Zhang N. Farrell A. Ramsay J. Gatti R. Lavin M. Oncogene. 1997; 14: 1911-1921Crossref PubMed Scopus (169) Google Scholar) (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 25 mm NaF, 0.1 mmsodium orthovanadate, 0.2% Triton X-100, 0.3% Nonidet P-40) containing protease inhibitors, 0.1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, and 5 μg/ml leupeptin. Cells in the lysis buffer were incubated on ice for 30 min. The lysates were centrifuged at 14,000 rpm for 30 min at 4 °C and the supernatant fraction was transferred to another tube. The protein concentration in the extract was determined using the Bio-Rad protein assay, with bovine serum albumin (BSA) as standard. For co-immunoprecipitations, equal amounts (1 mg of protein) of cell lysate were mixed with 0.7 μg of affinity purified anti-β-pol polyclonal antibody or rabbit nonimmune IgG. The mixture was incubated with rotation for 4 h at 4 °C. The immunocomplex was adsorbed onto protein A-Sepharose and protein G-agarose beads by incubating the mixture overnight at 4 °C. The beads were washed four times with lysis buffer containing protease inhibitors. Finally, the beads were resuspended in SDS sample buffer, heated for 5 min, and the soluble proteins were separated by 4–12% SDS-PAGE. The proteins were then transferred onto a nitrocellulose membrane in a transblot apparatus for 3 h at 25 V. The membrane was incubated with 5% nonfat dry milk in Tris-buffered saline (TBS) containing 0.1% (v/v) Tween 20 (TBS-T) and eventually probed with the anti-PCNA monoclonal antibody (1:1,000 dilution). Goat anti-rabbit IgG conjugated to horseradish peroxidase (1:10,000 dilution) was used as secondary antibody and immobilized horseradish peroxidase activity was detected by enhanced chemiluminescence (ECL). The same blot was stripped by incubating with buffer containing 62.5 mmTris-HCl, pH 6.8, 100 mm β-mercaptoethanol, and 1% SDS for 30 min at 50 °C, followed by two washes with TBS-T at room temperature. The presence of β-pol was confirmed by incubating the membrane with mouse anti-β-pol monoclonal antibody 18S (48Srivastava D.K. Evans R.K. Kumar A. Beard W.A. Wilson S.H. Biochemistry. 1996; 35: 3728-3734Crossref PubMed Scopus (17) Google Scholar). Similarly, the cell lysate was immunoprecipitated with anti-PCNA polyclonal antibody, Ab-5, as described above. The blot was developed with anti-β-pol monoclonal antibody 18S to detect β-pol. After stripping the blot, the presence of PCNA was confirmed using the anti-PCNA monoclonal antibody SC-56. The same method was used for immunoprecipitation and probing with anti-α-pol and anti-FEN-1 antibodies. Co-immunoprecipitation of purified PCNA and β-pol was performed in the presence of binding buffer (25 mm Tris, pH 8, 10% glycerol, 100 mm NaCl, 0.01% Nonidet P-40) containing protease inhibitors (0.1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, and 5 μg/ml leupeptin). To the mixture of 1.5 μm β-pol and 1.5 μm PCNA in a final volume of 50 μl either anti-β-pol or anti-PCNA antibody were added, and the mixture was incubated with rotation for 4 h at 4 °C. The protein complex was adsorbed onto protein A-Sepharose and protein G-agarose beads by incubating the mixture overnight at 4 °C. The beads were washed four times with binding buffer containing protease inhibitors. The beads were suspended in SDS sample buffer, heated for 5 min, and the soluble proteins were separated by 4–12% SDS-PAGE. After transferring the proteins to nitrocellulose membrane, the membrane was blocked in 5% milk in TBS-T. Immunoblotting was performed with the appropriate antibody as described above. The β-pol two-hybrid constructs used in this study were prepared from a full-length human β-pol cDNA as a restriction fragment. Adapters were used as needed for in-frame insertion relative to the GAL4 activation domain encoded in the pACT2 yeast two-hybrid vector plasmid (CLONTECH). The β-pol 31-kDa domain construct codes for Arg102 to Glu335 and was prepared by insertion of a Xhol restriction fragment of β-pol. The β-pol-(1–251) construct codes for Met1 to Asp251 and was prepared by insertion of aNcoI-EcoRV restriction fragment of β-pol and contains a vector encoded stop codon. The β-pol-(251–335) construct codes for Asp251 to Glu335 and was prepared by insertion of an EcoRV-XhoI restriction fragment of β-pol. Full-length β-pol and the N-terminal 8-kDa domain were also prepared as in-frame inserts into pACT2. The 8-kDa domain construct codes for Met1 to Arg102. The full-length PCNA two-hybrid construct used in this study was prepared from a full-length human cDNA as a restriction fragment inserted in-frame relative to the GAL4-binding domain in the pAS2-1 yeast two-hybrid vector plasmid (CLONTECH). All of the constructs were confirmed by sequencing. The yeast media used to determine nutritional requirements in the directed two-hybrid selections were prepared following established recipes. Chemical reagents for transformation were obtained from Sigma, and the yeast plasmid vectors and host cells were obtained from CLONTECH. Before testing for protein interactions, each construct was first checked for background Hisexpression on defined medium without histidine. No histidine expression or colony formation were observed for any of the constructs tested, where transformation was always confirmed by reversion to the Trp+ or Leu+ phenotype for the PCNA-binding domain or β-pol activation domain constructs, respectively. Protein interactions were tested by selection for his+revertants following co-transformation of yeast strain CG1945, carrying the his3 and lacZ reporter genes under control of the GAL4 responsive element, with the PCNA-binding domain and β-pol activation domain constructs. Co-transformed cells were plated on dropout medium containing 2.5 mm His3 inhibitor, 3-amino-1,2,4-triazole, and lacking Trp, Leu, and His (DO3). This was compared with an equal volume of transformants plated on dropout medium lacking Trp and Leu (DO2). The preparation of competent cells and transformations were performed by the LiCl method as described in the Matchmaker GAL4 two-hybrid user manual (CLONTECH PT3061-1). The transformation reactions were split and added in equal amounts to the DO2 and DO3 selection plates, which were grown at 30 °C and photographed after 5 days. All protein interactions detected by nutritional selection were confirmed by β-galactosidase assays performed using the Gal-ScreenTM protocol with detection on a TR717TM Microplate Luminometer (Applera Corp.). The overlay protein binding assay was performed as described previously (49Shi J. Sugrue S.P. J. Biol. Chem. 2000; 275: 14910-14915Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Briefly, purified β-pol (24 μg) was digested with trypsin (substrate to trypsin ratio, 10:1, w/w) in a final volume of 105 μl in 25 mm Tris-HCl, pH 7.5, 25 mm NaCl, 4 mm MgCl2, and 1 mm EDTA (44Kumar A. Widen S.G. Williams K.R. Kedar P. Karpel R.L. Wilson S.H. J. Biol. Chem. 1990; 265: 2124-2131Abstract Full Text PDF PubMed Google Scholar, 48Srivastava D.K. Evans R.K. Kumar A. Beard W.A. Wilson S.H. Biochemistry. 1996; 35: 3728-3734Crossref PubMed Scopus (17) Google Scholar). The reaction was carried out at room temperature. Aliquots were withdrawn at 0, 1, 5, 15, 30, 60, and 120 min, mixed with SDS sample buffer, boiled for 5 min, and proteins were separated by 12% SDS-PAGE. The proteins were transferred to a nitrocellulose membrane. The membrane was incubated in a buffer containing 10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm MgCl2, 0.1% (v/v) Tween 20, and 5% nonfat dry milk at 4 °C for 16 h. Membranes were then incubated at room temperature for 4 h in the same buffer with 0.1 μm PCNA, either with or without competing peptide, with buffer alone, or with IgG as a control (in a buffer containing protease inhibitors and 0.1% Tween 20, 1% BSA, and 0.5% Triton X-100). After incubation, the membrane was washed five times with the same buffer and subjected to immunoblot analysis using anti-PCNA monoclonal antibody diluted 1:1000 in TBS-T. Blots were incubated in 5% normal goat serum in TBS prior to secondary antibody (goat anti-mouse IgG, 1:10,000) incubation, followed by ECL. Equimolar amounts (1.5 μm) of β-pol and PCNA were incubated in binding buffer (100 mm NaCl, 50 mm Tris-HCl, pH 7.5, 0.1% Nonidet P-40, and the protease inhibitors) in a final volume of 50 μl. A suspension of SP-Sepharose (30 μl) pre-equilibrated with binding buffer was added and the mixture was incubated overnight at 4 °C on a rotating shaker. Increasing amounts of peptide II or jumbled peptide were added to the incubation mixture as indicated in the figure legends. Protein-bound Sepharose beads were washed three times with binding buffer; SDS sample buffer was added, the mixture was heated for 5 min at 95 °C, and soluble proteins were resolved by 4–12% SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane and the blots were developed as described above. The mutant β-pol (H222A/F223A) expression construct was prepared with the assistance of Dr. T. G. Wood, University of Texas Medical Branch, as described (50Beard W.A. Osheroff W.P. Prasad R. Sawaya M.R. Jaju M. Wood T.G. Kraut J. Kunkel T.A. Wilson S.H. J. Biol. Chem. 1996; 271: 12141-12144Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). The β-pol mutant protein was overexpressed and purified, as described (42Beard W.A. Wilson S.H. Methods Enzymol. 1995; 262: 98-107Crossref PubMed Scopus (159) Google Scholar). A partial BER reaction was reconstituted with purified proteins under the following conditions: the reaction mixture (10 μl) contained 50 mm Hepes, pH 7.5, 10 mm MgCl2, 2 mm dithiothreitol, 20 mm KCl, 100 μg/ml BSA, 4 mm ATP, 250 nm 34-base pair DNA substrate with a uracil residue at position 16, 20 μm each of dATP, dGTP, and dTTP, and 2.3 μm [α-32P]dCTP (specific activity: 1 × 106 dpm/pmol). Uracil-containing DNA substrate was pretreated with uracil-DNA glycosylase as described previously (30Prasad R. Lavrik O.I. Kim S.J. Kedar P. Yang X.P. Vande Berg B.J. Wilson S.H. J. Biol. Chem. 2001; 276: 32411-32414Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). The reaction mixture was assembled on ice by mixing 10 nmAPE and 2.5 nm wild-type β-pol or H222A/F223A mutant β-pol. Incubation was for 20 min at 37 °C and was within a linear range for product formation as a function of time and enzyme concentration. The reaction products were separated by electrophoresis in a 15% denaturing polyacrylamide gel as described previously (30Prasad R. Lavrik O.I. Kim S.J. Kedar P. Yang X.P. Vande Berg B.J. Wilson S.H. J. Biol. Chem. 2001; 276: 32411-32414Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Because PCNA is involved in many DNA repair functions, we considered it to be a candidate for interaction with β-pol. To explore this possibility, we conducted co-immunoprecipitation experiments with anti-β-pol antibody and extracts from WT and β-pol null mouse cells and also with extract from null cells (19HB3) stably transfected with a β-pol expression vector. With WT cell extract the anti-β-pol antibody immunoprecipitated PCNA (Fig 1A, panel 1, lanes 2 and 3). A protein of similar size was not detected in the immunoprecipitate prepared with preimmune IgG and WT extract (Fig.1A, panel 1, lane 1) or with immunoprecipitates of β-pol null cell extract prepared with anti-β-pol antibody (Fig. 1A, panel 1, lanes 4 and 5). Extract of null cells supplemented with purified β-pol or extract from null cells stably transfected with a β-pol expression vector also showed immunoprecipitation of PCNA (Fig.1A, lanes 7 and 8). To verify that β-pol" @default.
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• W2057747091 date "2002-08-01" @default.
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• W2057747091 title "Direct Interaction between Mammalian DNA Polymerase β and Proliferating Cell Nuclear Antigen" @default.
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• W2057747091 doi "https://doi.org/10.1074/jbc.m201497200" @default.
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