Proliferating Cell Nuclear Antigen (PCNA) May Function as a Double Homotrimer Complex in the Mammalian Cell

The diverse function of proliferating cell nuclear antigen (PCNA) may be regulated by interactions with different protein partners. Interestingly, the binding sites for all known PCNA-associating proteins are on the outer surface or the C termini (“front”) sides of the PCNA trimer. Using cell extracts and purified human PCNA protein, we show here that two PCNA homotrimers form a back-to-back doublet. Mutation analysis suggests that the Arg-5 and Lys-110 residues on the PCNA back side are the contact points of the two homotrimers in the doublet. Furthermore, short synthetic peptides encompassing either Arg-5 or Lys-110 inhibit double trimer formation. We also found that a PCNA double trimer, but not a homotrimer alone, can simultaneously accommodate chromatin assembly factor-1 and polymerase δ. Together, our data supports a model that chromatin remodeling by chromatin assembly factor-1 (and, possibly, many other cellular activities) are tightly coupled with DNA replication (and repair) through a PCNA double trimer complex. The diverse function of proliferating cell nuclear antigen (PCNA) may be regulated by interactions with different protein partners. Interestingly, the binding sites for all known PCNA-associating proteins are on the outer surface or the C termini (“front”) sides of the PCNA trimer. Using cell extracts and purified human PCNA protein, we show here that two PCNA homotrimers form a back-to-back doublet. Mutation analysis suggests that the Arg-5 and Lys-110 residues on the PCNA back side are the contact points of the two homotrimers in the doublet. Furthermore, short synthetic peptides encompassing either Arg-5 or Lys-110 inhibit double trimer formation. We also found that a PCNA double trimer, but not a homotrimer alone, can simultaneously accommodate chromatin assembly factor-1 and polymerase δ. Together, our data supports a model that chromatin remodeling by chromatin assembly factor-1 (and, possibly, many other cellular activities) are tightly coupled with DNA replication (and repair) through a PCNA double trimer complex. Proliferating cell nuclear antigen (PCNA) 1The abbreviations used are: PCNA, proliferating cell nuclear antigen; CAF-1, chromatin assembly factor; CHO, Chinese hamster ovary cell; pol, polymerase; GFP, green fluorescent protein; PBS, phosphate-buffered saline. is involved in the regulation of a wide spectrum of biological functions, including DNA replication, repair, cell-cycle control, and chromatin remodeling (1.Tsurimoto T. Front Biosci. 1999; 4: 849-858Crossref PubMed Google Scholar, 2.Stucki M. Stagljar I. Jonsson Z.O. Hubscher U. Prog. Nucleic Acids Res. Mol. Biol. 2001; 65: 261-298Crossref PubMed Google Scholar, 3.Ellison V. Stillman B. Cell. 2001; 106: 655-660Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 4.Jonsson Z.O. Hubscher U. BioEssays. 1997; 19: 967-975Crossref PubMed Scopus (218) Google Scholar, 5.Maga G. Hubscher U. J. Cell Sci. 2003; 116: 3051-3060Crossref PubMed Scopus (877) Google Scholar). PCNA may exist in the cell as a homotrimer ring, which functions as a sliding clamp and, possibly, a docking station for many proteins (6.Fukuda K. Morioka H. Imajou S. Ikeda S. Ohtsuka E. Tsurimoto T. J. Biol. Chem. 1995; 270: 22527-22534Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 7.Gulbis 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, 8.Krishna T.S. Kong X.P. Gary S. Burgers P.M. Kuriyan J. Cell. 1994; 79: 1233-1243Abstract Full Text PDF PubMed Scopus (755) Google Scholar, 9.Schurtenberger P. Egelhaaf S.U. Hindges R. Maga G. Jonsson Z.O. May R.P. Glatter O. Hubscher U. J. Mol. Biol. 1998; 275: 123-132Crossref PubMed Scopus (58) Google Scholar). It is thought that the regulation of protein binding to PCNA may be directly relevant to its diverse biological functions. Consistent with this idea, PCNA-associated proteins possess a wide range of different functions such as replication factor-C, DNA polymerases δ and ϵ (pol δ and ϵ), chromatin assembly factor-1 (CAF-1), p21WAF1, DNA ligase, FEN1, XPG, MSH2, cyclins, and cyclin-dependent kinases (1.Tsurimoto T. Front Biosci. 1999; 4: 849-858Crossref PubMed Google Scholar, 4.Jonsson Z.O. Hubscher U. BioEssays. 1997; 19: 967-975Crossref PubMed Scopus (218) Google Scholar, 5.Maga G. Hubscher U. J. Cell Sci. 2003; 116: 3051-3060Crossref PubMed Scopus (877) Google Scholar, 10.Loor G. Zhang S.J. Zhang P. Toomey N.L. Lee M.Y. Nucleic Acids Res. 1997; 25: 5041-5046Crossref PubMed Scopus (77) Google Scholar, 11.Warbrick E. BioEssays. 2000; 22: 997-1006Crossref PubMed Scopus (353) Google Scholar, 12.Paunesku T. Mittal S. Protic M. Oryhon J. Korolev S.V. Joachimiak A. Woloschak G.E. Int. J. Radiat. Biol. 2001; 77: 1007-1021Crossref PubMed Scopus (290) Google Scholar, 13.Ohta S. Shiomi Y. Sugimoto K. Obuse C. Tsurimoto T. J. Biol. Chem. 2002; 277: 40362-40367Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 14.Chapados B.R. Hosfield D.J. Han S. Qiu J. Yelent B. Shen B. Tainer J.A. Cell. 2004; 116: 39-50Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 15.Clark 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, 16.Flores-Rozas H. Clark D. Kolodner R.D. Nat. Genet. 2000; 26: 375-378Crossref PubMed Scopus (196) Google Scholar). Interestingly, all of these proteins bind to the front side or outer surface of the PCNA trimer ring, which faces the replication primer. Thus far, there is no direct evidence that any protein binds to the back side. The connection between CAF-1 and DNA replication was initially suggested to be that CAF-1 was found in the replication foci (17.Krude T. Exp. Cell Res. 1995; 220: 304-311Crossref PubMed Scopus (135) Google Scholar). It is now clear that the process of chromatin assembly is tightly coupled to DNA replication or repair, for which the association of PCNA and CAF-1 is required (18.Shibahara K. Stillman B. Cell. 1999; 96: 575-585Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar, 19.Moggs J.G. 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, 20.Zhang Z. Shibahara K. Stillman B. Nature. 2000; 408: 221-225Crossref PubMed Scopus (394) Google Scholar). The CAF-1 complex is composed of three subunits, p150, p60, and p48 (21.Smith S. Stillman B. Cell. 1989; 58: 15-25Abstract Full Text PDF PubMed Scopus (524) Google Scholar), and the largest subunit is directly associated with PCNA (20.Zhang Z. Shibahara K. Stillman B. Nature. 2000; 408: 221-225Crossref PubMed Scopus (394) Google Scholar). Here we present evidence that PCNA exists as a double trimer complex and that pol δ binds to one homotrimer and CAF-1 binds to the other homotrimer simultaneously, allowing chromatin remodeling to be coupled with DNA replication. Cells and Plasmids—CHO cells were maintained in minimal essential medium as described previously (22.Naryzhny S.N. Lee H. Electrophoresis. 2001; 22: 1764-1775Crossref PubMed Scopus (39) Google Scholar). Escherichia coli strain BL21 (DE3) transformed by pT7hPCNA (23.Fien K. Stillman B. Mol. Cell. Biol. 1992; 12: 155-163Crossref PubMed Scopus (190) Google Scholar) was a kind gift of Dr. B. Stillman (Cold Spring Harbor Laboratory). The plasmid pEGFPCNA (a kind gift of Dr. C. Cardoso, Max Delbruck Center for Molecular Medicine, Germany) was created by joining the GFP and PCNA open reading frames with a flexible and hydrophilic linker so that GFP cannot interfere the formation of a functional PCNA homotrimer ring (24.Leonhardt H. Rahn H.P. Weinzierl P. Sporbert A. Cremer T. Zink D. Cardoso M.C. J. Cell Biol. 2000; 149: 271-280Crossref PubMed Scopus (458) Google Scholar). The pEGFPCNAL2 construct (a kind gift of Dr. C. Cardoso) is identical to pEGFPCNA except that it contains a nuclear localization sequence. Further manipulations were carried out in our laboratory to generate several deletion and point mutants using appropriate oligoprimers and PCR. Cross-linking by Formaldehyde and Two-dimensional PAGE—Crosslinking was carried out by incubating intact cells, total cell extracts, or purified proteins in PBS containing 1.0–1.5% formaldehyde at room temperature. The reaction was stopped by adding LDS buffer (final concentration, 2% LDS, 60 mm Tris, pH 6.8, 10% glycerol, 0.1 m dithiothreitol, 0.001% bromphenol blue), or by adding glycine at a final concentration of 0.15 m. Protein analysis by two-dimensional PAGE and Western blot analysis was carried out as described previously (25.Naryzhny S.N. Lee H. Proteomics. 2003; 3: 930-936Crossref PubMed Scopus (25) Google Scholar). PCNA Purification by Gel Filtration and SDS-PAGE—Cell extracts were prepared from bacteria (BL21 (DE3)) transformed with pT7hPCNA (23.Fien K. Stillman B. Mol. Cell. Biol. 1992; 12: 155-163Crossref PubMed Scopus (190) Google Scholar) by sonication in buffer containing 25 mm Tris, pH 7.4, 25 mm NaCl, 1 mm EDTA, 10% glycerol, 0.01% Triton X-100, and protease inhibitor mixture. The extracts were “cleared” by centrifugation and applied to a Sephacryl S200, 16/60 gel-filtration column (Amersham Biosciences). The main PCNA peak was collected and subjected to another round of purification using the same Sephacryl filtration system. PCNA eluted in PBS was subjected to UnoQ1 (Bio-Rad) liquid chromatography. PCNA was then further purified using the Superdex 200 10/300 GL gel-filtration system (separation range, molecular weight 10,000–600,000). In some cases, PCNA was further purified using Phenyl-Sepharose Fast Flow (Amersham Biosciences) chromatography prior to the gel filtration by the Superdex 200 10/300 GL system. However, without this extra step, the purity of PCNA estimated by SDS-PAGE, and silver staining was usually more than 95%. The isolation of the PCNA monomer by SDS-PAGE was carried out as described previously (26.Naryzhny S.N. Anal. Biochem. 1996; 238: 50-53Crossref PubMed Scopus (17) Google Scholar). Renaturation of these PCNA molecules was achieved by sequential dialysis in the following order: 1) buffer containing 4 m urea, 0.1% Triton X-100, 1 mm EDTA, 20 mm Tris-HCl, pH 7.2, 2) 0.5% Triton X-100, 20 mm Tris-HCl, pH 7.2, 3) PBS containing 20% glycerol and 1.0 mm EDTA, and 4) PBS containing 50% glycerol. Immunoprecipitation and Immunostaining—For double immunoprecipitation, CHO cells transfected with pEGFPCNAL2 were treated with cell extraction buffer (10 mm Tris, pH 6.8, 100 mm NaCl, 3 mm MgCl2, 1 mm EDTA, 10% glycerol, 0.5% Triton X-100, 1.0 mm phenylmethylsulfonyl fluoride, and protease inhibitor mixture). The extracts (1 mg/ml protein) were then incubated at 4 °C overnight with agarose-conjugated PC10 monoclonal anti-PCNA antibody. The immunoprecipitate was washed four times with PBS after which PCNA complexes were dissociated from antibodies by treatment with elution buffer (0.1 m glycine, 0.125 m NaCl, pH 2.8). The pH of the supernatant was immediately adjusted to 7.0 using 1.0 m Tris, followed by a second immunoprecipitation with a monoclonal antibody generated against the 125-kDa catalytic subunit of pol δ (C-2, Santa Cruz Biotechnology). Subsequently, the immunoprecipitate was washed four times with PBS, eluted with LDS sample buffer, and boiled for 30 s. The proteins were then analyzed by SDS-PAGE and Western blot analysis. For a negative control, normal mouse IgG (Santa Cruz) was used. For the co-immunoprecipitation and subsequent Western blot analysis described in Fig. 4, D–F, total cell extracts of CHO cells transfected with either wild or the PCNA construct were prepared as described previously (25.Naryzhny S.N. Lee H. Proteomics. 2003; 3: 930-936Crossref PubMed Scopus (25) Google Scholar). cell extracts prepared in of buffer 0.5% 0.1% and the of protease were at for The supernatant was collected and the was in of DNA buffer mm Tris, pH 7.4, mm and treated with (10 for at 4 The DNA was stopped by adding 100 of m and 2% The supernatant was collected after the was for at The and the second extracts were then mg/ml protein) and subjected to immunoprecipitation, using of a monoclonal antibody against the subunit of CAF-1 followed by overnight with of protein (Santa Cruz Biotechnology). The immunoprecipitate was washed four times with PBS and boiled for in of LDS sample The protein complexes were then subjected to SDS-PAGE and Western blot analysis using a antibody Santa Cruz a mouse monoclonal δ or a PC10 anti-PCNA antibody. of PCNA were by at Cell extracts prepared using buffer mm 1.0 mm mm mm NaCl, pH were incubated with or 1.0 mg/ml for on followed by by treatment with formaldehyde for at room temperature. PCNA following were used to PCNA by the of and PCNA a the of PCNA to form complexes in cells, we analyzed PCNA by were these However, cells were treated with formaldehyde for 30 to proteins prior to SDS-PAGE, PCNA was at the of and the was to all of the PCNA molecules in the cells a protein complex Further of the to 3 not the of complex 200 that the complex in a and a The of cell extracts, which was more than that of cells, the same the complexes other we carried out two-dimensional in Fig. and proteins were cell extracts were for 20 all of the PCNA molecules complexes cell extracts were for the and complexes the same as the PCNA monomer it is that PCNA molecular the and complexes are a and a of PCNA the double trimer may a it also be that was required to all of the that are to protein purification and subsequent gel and the PCNA double trimer complex may exist as a complex. be the a PCNA double trimer not by 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, 8.Krishna T.S. Kong X.P. Gary S. Burgers P.M. Kuriyan J. Cell. 1994; 79: 1233-1243Abstract Full Text PDF PubMed Scopus (755) Google this of and interactions may be in the regulation of many different cellular functions. our we not between PCNA and other may be all of the PCNA molecules exist in the cells as homotrimer as a and of the or residues in PCNA and proteins present at to be It be that proteins are associated with PCNA through its and the of which contains (1.Tsurimoto T. Front Biosci. 1999; 4: 849-858Crossref PubMed Google Scholar). we not that PCNA be with other known PCNA-associating proteins by form a complex with PCNA in further that the PCNA complex not any other we the using purified PCNA as purity PCNA was achieved using a of Sephacryl S200, and the Superdex 200 10/300 GL as described Cross-linking of purified PCNA in its form for using formaldehyde in the formation of the trimer and double trimer complexes 4, and In to PCNA molecules purified by gel by SDS-PAGE double Furthermore, many complexes the in Fig. It also be in to the PCNA molecules purified by gel from SDS-PAGE of are that it is for PCNA to to its it is and other are not for the Furthermore, it that the protein is for the formation of the PCNA double trimer complex. The Arg-5 and Lys-110 for further the double trimer complex in cells, we analyzed proteins in CHO cells transfected with pEGFPCNA (24.Leonhardt H. Rahn H.P. Weinzierl P. Sporbert A. Cremer T. Zink D. Cardoso M.C. J. Cell Biol. 2000; 149: 271-280Crossref PubMed Scopus (458) Google Scholar). It was previously that the GFP of this protein not interfere with the formation of a functional PCNA trimer (24.Leonhardt H. Rahn H.P. Weinzierl P. Sporbert A. Cremer T. Zink D. Cardoso M.C. J. Cell Biol. 2000; 149: 271-280Crossref PubMed Scopus (458) Google Scholar). Consistent with this the PCNA protein in CHO cells to PCNA following and of PCNA is than that of the PCNA protein in this previously (24.Leonhardt H. Rahn H.P. Weinzierl P. Sporbert A. Cremer T. Zink D. Cardoso M.C. J. Cell Biol. 2000; 149: 271-280Crossref PubMed Scopus (458) Google Scholar), the proteins to be in the replication foci further the protein, extracts prepared from CHO cells transfected with pEGFPCNA were subjected to two-dimensional PAGE using a pH gel and subsequent using previously (25.Naryzhny S.N. Lee H. Proteomics. 2003; 3: 930-936Crossref PubMed Scopus (25) Google Scholar, S.N. Lee H. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), three of PCNA were and are for the main and The protein also three as and that are identical with the three except molecular and and were The in and were of the of a of the cell extracts in the formation of trimer and double trimer complexes by PCNA and the protein also trimer and double trimer complexes and Consistent with the data in Fig. the monomer and complexes the same that no other proteins are in the trimer or double trimer complexes by the the contact points between the two trimer of the we analyzed complex formation by several pEGFPCNA point and deletion mutants on the surface of the PCNA and PC10 antibodies were used for to the protein and The following mutants not the formation of either trimer or double and not It be that and are the PCNA trimer ring, and are on the surface of the front side. The of the sites are on the back side of the PCNA trimer Further analysis Arg-5 and Lys-110 as two residues for double trimer formation by and We also the of synthetic peptides to inhibit PCNA double trimer formation. in Fig. synthetic peptides encompassing Arg-5 and Lys-110 formation of the double trimer a to the at the C on the front of these peptides the formation of the PCNA homotrimer complex Consistent with the data by analysis these that the Arg-5 and Lys-110 residues are essential contact points between the two homotrimers of a PCNA doublet. It is to that the of double trimer formation by the two peptides was with cell extracts using buffer containing mm and was not in the cells with buffer containing mm not suggests that PCNA exists as a double trimer complex in the complex may be and it cannot be by short synthetic peptides The of a PCNA May for and in the of double trimer in cells, we transfected CHO cells with wild or the double that the of hamster and human PCNA proteins are identical except for three We found that and of cells transfected by wild and the a 4, and the of cells were and for cells transfected by wild and Flow that the cells by were in and a However, of the cells in the were normal cells and not We also found that a or to the double not with in Fig. that Arg-5 and Lys-110 are essential for a double trimer formation and cell and PCNA but a DNA δ and front side and outer surface of the PCNA trimer can bind to many different including pol δ and CAF-1 (1.Tsurimoto T. Front Biosci. 1999; 4: 849-858Crossref PubMed Google Scholar, 4.Jonsson Z.O. Hubscher U. BioEssays. 1997; 19: 967-975Crossref PubMed Scopus (218) Google Scholar, 5.Maga G. Hubscher U. J. Cell Sci. 2003; 116: 3051-3060Crossref PubMed Scopus (877) Google Scholar, 10.Loor G. Zhang S.J. Zhang P. Toomey N.L. Lee M.Y. Nucleic Acids Res. 1997; 25: 5041-5046Crossref PubMed Scopus (77) Google Scholar, 11.Warbrick E. BioEssays. 2000; 22: 997-1006Crossref PubMed Scopus (353) Google Scholar, 12.Paunesku T. Mittal S. Protic M. Oryhon J. Korolev S.V. Joachimiak A. Woloschak G.E. Int. J. Radiat. Biol. 2001; 77: 1007-1021Crossref PubMed Scopus (290) Google Scholar, 13.Ohta S. Shiomi Y. Sugimoto K. Obuse C. Tsurimoto T. J. Biol. Chem. 2002; 277: 40362-40367Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 19.Moggs J.G. 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). It was previously suggested that the back side be involved in the regulation of PCNA functions by with other proteins (6.Fukuda K. Morioka H. Imajou S. Ikeda S. Ohtsuka E. Tsurimoto T. J. Biol. Chem. 1995; 270: 22527-22534Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). However, there is no direct evidence that any protein binds to the back side of the PCNA back-to-back double trimer model can no proteins bind to the back side of the PCNA trimer In the of two front sides in the trimer to protein binding it for one PCNA complex to simultaneously functions such as DNA replication, repair, and cell-cycle G. Hubscher U. J. Cell Sci. 2003; 116: 3051-3060Crossref PubMed Scopus (877) Google Scholar). this we used a chromatin immunoprecipitation to the of PCNA on the chromatin to simultaneously with pol δ and We found that PCNA, CAF-1, and pol δ were present on chromatin in not PCNA can bind to pol δ and CAF-1 simultaneously, extracts prepared from cells transfected by PCNA were subjected to and with The PCNA complexes in the were dissociated from the antibody and then were subjected to a second immunoprecipitation with δ antibody. The final which was to be a δ was subjected to SDS-PAGE The proteins on the were then analyzed by sequential without the antibodies with δ and PC10 3) data that the δ complex also CAF-1 that pol δ and CAF-1 are simultaneously associated with further this we the binding of CAF-1 and pol δ between wild and The CAF-1 complexes were with a monoclonal antibody from cells transfected with either the wild or the The CAF-1 complexes were then analyzed by blot analysis using antibodies against pol δ CAF-1 or PCNA The of the these proteins in the was by with the of prepared from total protein extracts 4, D–F, and and 4, and the subunit of CAF-1 was present in the total extracts and the immunoprecipitate prepared from the cells transfected by the wild or the construct the CAF-1 complex from the cells transfected with PCNA also pol δ In the CAF-1 complex from cells transfected by the construct not pol δ wild and the binding for CAF-1 and It is clear that PCNA homotrimers can bind either pol δ or CAF-1 but not the PCNA can form a back-to-back double trimer it can accommodate pol δ and However, the cannot form a double trimer complex and, cannot simultaneously accommodate pol δ and of with Fig. that PCNA, but not the can simultaneously accommodate CAF-1 and pol δ through PCNA double trimer formation. a PCNA trimer contains three identical to accommodate proteins with the binding to a protein to in a at the of other proteins (6.Fukuda K. Morioka H. Imajou S. Ikeda S. Ohtsuka E. Tsurimoto T. J. Biol. Chem. 1995; 270: 22527-22534Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 7.Gulbis 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, 19.Moggs J.G. 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, S. S. Y. K. 2002; PubMed Scopus (58) Google Scholar, G. 1997; 277: PubMed Scopus Google Scholar). is one PCNA-associating protein can of the PCNA homotrimer surface as in the of the between a PCNA homotrimer and DNA 1 J.M. T. Nature. 2004; PubMed Scopus Google Scholar). It be that pol δ and CAF-1 are and at four p60, and and three p60, and one PCNA homotrimer not with the pol δ and CAF-1 of a that from the PCNA homotrimer back side T.S. Kong X.P. Gary S. Burgers P.M. Kuriyan J. Cell. 1994; 79: 1233-1243Abstract Full Text PDF PubMed Scopus (755) Google Scholar). it for Arg-5 on one trimer to with Lys-110 on the other trimer. Furthermore, formaldehyde is a that than a between the two residues V. Biochem. Sci. 2000; 25: Full Text Full Text PDF PubMed Scopus Google Scholar). However, it may be that between Arg-5 and Lys-110 is achieved by a of the two PCNA through and Consistent with this idea, analysis using the that the can the two so that the of one trimer a and of the second trimer this Arg-5 and Lys-110 be in to be by 2000; Google Scholar) that which was from PCNA two PCNA trimer a back-to-back complex. our data direct evidence to back-to-back double trimer model that many cellular functions such as control, DNA and are tightly coupled with DNA replication the PCNA complex. B. Stillman and C. Cardoso are for of pT7hPCNA and We also K. for and Dr. R. for reading the

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