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• W2076554477 abstract "The recruitment of DNA ligase I to replication foci and the efficient joining of Okazaki fragments is dependent on the interaction between DNA ligase I and proliferating cell nuclear antigen (PCNA). Although the PCNA sliding clamp tethers DNA ligase I to nicked duplex DNA circles, the interaction does not enhance DNA joining. This suggests that other factors may be involved in the joining of Okazaki fragments. In this study, we describe an association between replication factor C (RFC), the clamp loader, and DNA ligase I in human cell extracts. Subsequently, we demonstrate that there is a direct physical interaction between these proteins that involves both the N- and C-terminal domains of DNA ligase I, the N terminus of the large RFC subunit p140, and the p36 and p38 subunits of RFC. Although RFC inhibited DNA joining by DNA ligase I, the addition of PCNA alleviated inhibition by RFC. Notably, the effect of PCNA on ligation was dependent on the PCNA-binding site of DNA ligase I. Together, these results provide a molecular explanation for the key in vivo role of the DNA ligase I/PCNA interaction and suggest that the joining of Okazaki fragments is coordinated by pairwise interactions among RFC, PCNA, and DNA ligase I. The recruitment of DNA ligase I to replication foci and the efficient joining of Okazaki fragments is dependent on the interaction between DNA ligase I and proliferating cell nuclear antigen (PCNA). Although the PCNA sliding clamp tethers DNA ligase I to nicked duplex DNA circles, the interaction does not enhance DNA joining. This suggests that other factors may be involved in the joining of Okazaki fragments. In this study, we describe an association between replication factor C (RFC), the clamp loader, and DNA ligase I in human cell extracts. Subsequently, we demonstrate that there is a direct physical interaction between these proteins that involves both the N- and C-terminal domains of DNA ligase I, the N terminus of the large RFC subunit p140, and the p36 and p38 subunits of RFC. Although RFC inhibited DNA joining by DNA ligase I, the addition of PCNA alleviated inhibition by RFC. Notably, the effect of PCNA on ligation was dependent on the PCNA-binding site of DNA ligase I. Together, these results provide a molecular explanation for the key in vivo role of the DNA ligase I/PCNA interaction and suggest that the joining of Okazaki fragments is coordinated by pairwise interactions among RFC, PCNA, and DNA ligase I. Mammalian cells contain multiple species of DNA ligase that are encoded by the LIG1, LIG3, and LIG4 genes (1Tomkinson A.E. Mackey Z.B. Mutat. Res. 1998; 407: 1-9Crossref PubMed Scopus (175) Google Scholar). There is compelling evidence linking the product of the LIG1 gene, DNA ligase I, with the joining of Okazaki fragments generated during lagging strand DNA synthesis. For example, DNA ligase I co-localizes with DNA replication foci, co-purifies with a multisubunit DNA replication complex, and efficiently joins Okazaki fragments when DNA replication is reconstituted with purified replication factors (2Waga S. Bauer G. Stillman B. J. Biol. Chem. 1994; 269: 10923-10934Abstract Full Text PDF PubMed Google Scholar, 3Li C. Goodchild J. Baril E.F. Nucleic Acids Res. 1994; 22: 632-638Crossref PubMed Scopus (33) Google Scholar, 4Lasko D.D. Tomkinson A.E. Lindahl T. J. Biol. Chem. 1990; 265: 12618-12622Abstract Full Text PDF PubMed Google Scholar). Moreover, a DNA ligase I-deficient human cell line 46BR1G1 is defective in the conversion of Okazaki fragments into larger DNA replication intermediates (5Prigent C. Satoh M.S. Daly G. Barnes D.E. Lindahl T. Mol. Cell. Biol. 1994; 14: 310-317Crossref PubMed Scopus (129) Google Scholar, 6Mackenney V.J. Barnes D.E. Lindahl T. J. Biol. Chem. 1997; 272: 11550-11556Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 7Levin D.S. McKenna A. Motycka T. Matsumoto Y. Tomkinson A.E. Curr. Biol. 2000; 10: 919-922Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 8Barnes D.E. Tomkinson A.E. Lehmann A.R. Webster A.D.B. Lindahl T. Cell. 1992; 69: 495-503Abstract Full Text PDF PubMed Scopus (232) Google Scholar). The identification and characterization of a specific interaction between DNA ligase I and the homotrimeric sliding clamp, proliferating cell nuclear antigen (PCNA), 1The abbreviations used are: PCNA, proliferating cell nuclear antigen; BSA, bovine serum albumin; FEN-1, flap endonuclease 1; GST, glutathione S-transferase; RFC, replication factor C. provided the first evidence physically linking DNA ligase I with a component of the DNA replication machinery (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, 10Jonsson Z. Hindges R. Hubscher U. EMBO J. 1998; 17: 2412-2425Crossref PubMed Scopus (236) Google Scholar). Subsequent studies revealed that the 20-amino acid sequence at the N terminus of DNA ligase I is necessary and sufficient to bind to PCNA and that this sequence is homologous to the canonical PCNA binding motif that has been found in a growing number of PCNA-interacting proteins (7Levin D.S. McKenna A. Motycka T. Matsumoto Y. Tomkinson A.E. Curr. Biol. 2000; 10: 919-922Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 11Montecucco A. Rossi R. Levin D.S. Gary R. Park M.S. Motycka T.A. Ciarrocchi G. Villa A. Biamonti G. Tomkinson A.E. EMBO J. 1998; 17: 3786-3795Crossref PubMed Scopus (174) Google Scholar, 12Warbrick E. BioEssays. 1998; 20: 195-199Crossref PubMed Scopus (315) Google Scholar). Amino acid changes that inactivate the PCNA binding site of DNA ligase I prevent the recruitment of DNA ligase I to DNA replication foci and abolish the ability of this enzyme to efficiently join Okazaki fragments (7Levin D.S. McKenna A. Motycka T. Matsumoto Y. Tomkinson A.E. Curr. Biol. 2000; 10: 919-922Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 11Montecucco A. Rossi R. Levin D.S. Gary R. Park M.S. Motycka T.A. Ciarrocchi G. Villa A. Biamonti G. Tomkinson A.E. EMBO J. 1998; 17: 3786-3795Crossref PubMed Scopus (174) Google Scholar). The interactions of PCNA with other DNA replication enzymes such as replication factor C (RFC), the heteropentameric clamp loader, the replicative DNA polymerases, polymerases δ and ϵ, and the 5′ flap endonuclease (FEN-1) suggest that this homotrimer plays a central role in coordinating and regulating the actions of these enzymes during gap-filling DNA synthesis and ligation (12Warbrick E. BioEssays. 1998; 20: 195-199Crossref PubMed Scopus (315) Google Scholar, 13Jonsson Z. Hubscher U. BioEssays. 1997; 19: 967-975Crossref PubMed Scopus (218) Google Scholar). Biochemical assays measuring DNA synthesis and ligation in reactions either reconstituted with purified replication factors or catalyzed by cell extracts have shown that the ability of DNA ligase I to bind to PCNA is critical for efficient ligation and that the extent of strand displacement DNA synthesis is limited by the functional interaction between DNA ligase I and PCNA (7Levin D.S. McKenna A. Motycka T. Matsumoto Y. Tomkinson A.E. Curr. Biol. 2000; 10: 919-922Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 14Matsumoto Y. Kim K. Hurwitz J. Gary R. Levin D.S. Tomkinson A.E. Park M. J. Biol. Chem. 1999; 274: 33703-33708Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 15Mossi R. Ferrari E. Hubscher U. J. Biol. Chem. 1998; 273: 14322-14330Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Although DNA ligase I stably associates with PCNA trimers that are topologically linked to duplex DNA circles (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), PCNA only weakly stimulates DNA joining at very high concentrations (16Tom S. Henricksen L.A. Park M.S. Bambara R.A. J. Biol. Chem. 2001; 276: 24817-24825Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). This suggests that additional replication factors may be involved in the functional interaction between DNA ligase I and PCNA. In this study, we describe a physical interaction between DNA ligase I and the large subunit of the clamp loader RFC that is conserved among eukaryotes. Furthermore, we show that RFC modulates DNA joining by DNA ligase I in a reaction that is dependent upon the interaction between DNA ligase I and PCNA. Protein Purification—Recombinant DNA ligase I was purified from Sf9 cells infected with a baculovirus expressing human DNA ligase I (17Wang Y.C. Burkhart W.A. Mackey Z.B. Moyer M.B. Ramos W. Husain I. Chen J. Besterman J.M. Tomkinson A.E. J. Biol. Chem. 1994; 269: 31923-31928Abstract Full Text PDF PubMed Google Scholar). PCNA was overexpressed in and purified from Escherichia coli as described (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, 18Fien K. Stillman B. Mol. Cell Biol. 1992; 12: 155-163Crossref PubMed Scopus (190) Google Scholar). Recombinant RFC complex containing full-length RFCp140 and a truncated version of RFCp140 lacking the N-terminal 555 residues (RFCΔNp140) were purified from baculovirus-infected insect cells (19Uhlmann F. Cai J. Gibbs E. O'Donnell M. Hurwitz J. J. Biol. Chem. 1997; 272: 10058-10064Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The intact RFC complex was also obtained from Dr. Vladimir Podust (20Podust V.N. Fanning F. J. Biol. Chem. 1997; 272: 6303-6310Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). DNA Ligase I Affinity Chromatography—Nuclear and cytoplasmic extracts were prepared from a frozen pellet of HeLa S3 cells (109 cells) as described (21Wu Y. Hickey R. Lawlor K. Wills P. Yu F. Ozer H. Starr R. Quan J.Y. Lee M. Malkas M. J. Cell. Biochem. 1994; 54: 32-46Crossref PubMed Scopus (52) Google Scholar). The nuclear extract (20 mg) was fractionated by DNA ligase I affinity chromatography as described previously (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). Fractions were analyzed for protein by immunoblotting after separation by SDS-PAGE (22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar). Purified RFC was incubated with either DNA ligase I or bovine serum albumin (BSA) beads (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) in binding buffer (50 mm Tris-HCl, pH 7.5, 100 mm KCl, 1 mm dithiothreitol, 1% Nonidet P-40, and 5 μg of BSA) for 30 min at 4 °C with constant agitation. After collection by centrifugation, the beads were washed with binding buffer, and bound proteins were eluted with SDS sample buffer (22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar). After separation by SDS-PAGE, proteins were detected by immunoblotting with antibodies against RFC p37 (GeneTex Inc.) and RFC p140 (a gift from Dr. Bruce Stillman). Immunoprecipitation—HeLa cells (8 × 106) were lysed in IP buffer (50 mm Tris-HCl, pH 7.5, 150 mm KCl, 10 mm MgCl2, 1% Nonidet P-40 (Sigma), 1 mm dithiothreitol, 50 μg/ml ethidium bromide, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 μg/ml chymostatin, 0.1 mm phenylmethanesulfonyl fluoride, 50 mm NaF, 1 mm Na3VO4). Approximately 1 mg of the clarified extract was used for each immunoprecipitation. The extracts were precleared by incubation for 1 h at 4°C with 50 μl of Protein A-Sepharose and Protein G-Sepharose beads (Amersham Biosciences) equilibrated with IP buffer, prior to the addition of antibodies against DNA ligase I, RFC p37, or Cdc25 (all from GeneTex Inc.). After incubation at 4°C for 2 h, 50 μl of Protein A-Sepharose and Protein G-Sepharose beads were added, and the incubation continued for 1 h. The beads were collected by centrifugation, washed three times with IP buffer lacking ethidium bromide, and then resuspended in SDS-sample buffer. After separation by SDS-PAGE, proteins were detected by immunoblotting. Glutathione S-Transferase (GST) Fusion Proteins—GST fusion proteins containing either the N-terminal 118 residues of DNA ligase I (GST-N Lig 1–118) or residues 479–919 of DNA ligase I (GST-C Lig1) were expressed and purified as described previously (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). Sequences encoding residues 1–584 and 479–1148 of RFC p140 were amplified from pET16a-p140 (19Uhlmann F. Cai J. Gibbs E. O'Donnell M. Hurwitz J. J. Biol. Chem. 1997; 272: 10058-10064Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) by the polymerase chain reaction and subcloned in frame with the GST open reading frame in the pGEX vector to generate pGST-Np140 and pGST-Cp140, respectively. Similarly, the open reading frame encoding Cdc9 DNA ligase was amplified from Saccharomyces cerevisiae genomic DNA by the polymerase chain reaction and subcloned in frame with the GST open reading frame in the pGSTag vector (23Ron D. Dressler H. BioTechniques. 1992; 13: 866-868PubMed Google Scholar). After expression in E. coli, GST fusion proteins were purified from cell extracts by affinity chromatography using glutathione-Sepharose beads. In Vitro Transcription and Translation—Coupled in vitro transcription and translation reactions were performed using the TNT Quick Coupled Transcription/Translation system (Promega, Madison, WI). The plasmids for the in vitro transcription and translation of human RFC subunits have been described previously (19Uhlmann F. Cai J. Gibbs E. O'Donnell M. Hurwitz J. J. Biol. Chem. 1997; 272: 10058-10064Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The open reading frame encoding Rfc1 was amplified from S. cerevisiae genomic DNA by the polymerase chain reaction and subcloned into pET-28b for coupled in vitro transcription and translation. Labeled in vitro translated polypeptides were partially purified by ammonium sulfate precipitation (24Bardwell L. Cooper A.J. Friedberg E.C. Mol. Cell. Biol. 1992; 12: 3041-3049Crossref PubMed Scopus (73) Google Scholar). Pull-down Assays—To prepare GST and GST-Np140, GST-Cp140, GST-N Lig 1–118, and GST-C Lig1 beads, 5 μg of each purified protein was incubated with a 20-μl slurry of glutathione-Sepharose beads (Amersham Biosciences) equilibrated in binding buffer for 30 min at 4 °C with constant agitation. After washing with binding buffer, the beads were resuspended in 500 μl of binding buffer containing a labeled in vitro translated polypeptide and then incubated at room temperature for 30 min with constant agitation. Next the beads were collected by centrifugation and then washed extensively in binding buffer prior to being resuspended in 20 μl of SDS-PAGE sample buffer. After separation by SDS-PAGE, labeled polypeptides were visualized by PhosphorImager analysis (Amersham Biosciences). Preparation of Biotin-labeled Linear DNA Joining Substrate—A 5′-biotinylated 90-mer oligonucleotide, Bio-5–90, with a sequence corresponding to nucleotide positions 4881 and 4971 of M13mp19 single-stranded DNA was purchased from Integrated DNA Technologies, Inc. Bio15-1 5′-TGAGGCGGTCAGTAT-3′ and Bio15-2 5′-AAGATAAAACAGAGG-3′ (Integrated Technologies, Inc.) are complementary to Bio-5-90. Bio15-1 was 5′-end-labeled with 150 μCi of [γ-32P]ATP using T4 Polynucleotide Kinase (New England Biolabs). After purification on Micro Bio-Spin 30 column (Bio-Rad), labeled Biol5-1 and Bio15-2 were annealed to Bio-5-90 to generate a partial duplex of 30 bp containing a single ligatable nick in the middle flanked by single-stranded regions of 30 nucleotides. DNA Joining Reaction with Biotin-labeled Linear Substrate— Streptavidin-agarose beads (10 μl; Pierce) were incubated with 1.6 pmol of the biotinylated linear DNA substrate in PBS for 30 min at room temperature. After washing three times in ligation buffer (50 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 1 mm dithiothreitol, 0.25 mg/ml BSA, 100 μm ATP, and 100 mm NaCl), the beads were incubated with 2 pmol of RPA/pmol of DNA in the same buffer for 15 min at room temperature. This substrate was then incubated with 2 pmol of RFC or 2 pmol of ΔNRFC in the presence and absence of PCNA (2 pmol of trimer) at 30 °C for 2 min. DNA ligase I (2 pmol), either wild type or mutant, was added, and the reaction was incubated at room temperature for 5 min. The beads were then spun down, and the reaction was terminated by the addition of 10 μl of stop mix (50% glycerol, 1% SDS, 20 mm EDTA, and 0.05% bromphenol blue). The beads were heated at 100 °C for 3 min to denature DNA. A 2-μl aliquot was mixed with 2 μl of denaturing PAGE dye (80% formamide, 0.05% bromphenol, and 0.05% xylene cyanol). The samples were electrophoresed through a 12% denaturing polyacrylamide gel. After drying, the gel was exposed to a Storage Phosphor screen and subjected to PhosphorImager analysis (Amersham Biosciences). Preparation of Circular DNA Joining Substrate—The oligonucleotides, 5′-CGTACGGGGAAGGACGTCAA-3′ and 5′-CATGAAACCAACATAAACGTTATTGCCCGG-3′ (100 pmol of each), were end-labeled with T4 polynucleotide kinase (New England Biolabs) in the presence of 3.2 μm ATP and 70 μCi of [γ-32P]ATP. After purification from free nucleotides by passage through a 1-ml G25 spin column, both of the labeled oligonucleotides were annealed to 25 μg of circular ΦX174 single-stranded DNA by heating at 100 °C for 15 min in 30 mm Tris-HCl, pH 7.5, and 300 mm NaCl (200-μl total volume), followed by slow cooling to room temperature. The labeled partial duplex circles were purified from free oligonucleotides by passage through a 5-ml Bio-Gel A15m (Bio-Rad) column. Peak fractions were ethanol-precipitated, resuspended in 10 mm Tris-HCl, pH 7.5, and 1 mm EDTA, and then run on a 1% agarose gel with ethidium bromide to verify the presence of the substrate DNA. When annealed to their complementary sequences in ΦX174 single-stranded DNA, the oligonucleotides formed a partial duplex region containing a single ligatable nick. DNA Joining Reaction with Circular Substrate—The labeled circular DNA substrate DNA (100 fmol) was incubated with RFC (0.6 pmol) in the presence or absence of PCNA (1.2 pmol of trimer) at 30 °C for 1 min in ligation buffer. Human DNA ligase I (90 fmol), either wild type or a mutant version with a disrupted PCNA binding site (7Levin D.S. McKenna A. Motycka T. Matsumoto Y. Tomkinson A.E. Curr. Biol. 2000; 10: 919-922Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar), was then added. Aliquots were taken after 0, 30, 90, and 300 s and added directly to DNA termination dye. After boiling, samples were electrophoresed through a 6% denaturing acrylamide gel. Labeled oligonucleotides in the dried gel were detected and quantitated by PhosphorImager analysis (Amersham Biosciences). Previously, we have shown that DNA ligase I forms a stable complex with PCNA molecules that are topologically linked to a nicked DNA circle (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). Since this interaction did not significantly increase the efficiency of DNA joining (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), we suspected that additional DNA replication factors may be involved in promoting DNA joining by DNA ligase I. To identify such factors, we fractionated a HeLa nuclear extract by DNA ligase I-affinity chromatography. As reported previously, PCNA was specifically retained by the DNA ligase I resin (Fig. 1A, compare lanes 3 and 4). Analysis of the same fractions by immunoblotting with antibodies specific for the p37 subunit and p140 subunit of the clamp loader RFC revealed that these proteins were also present in the 150 mm NaCl eluates from the DNA ligase I column but not in the equivalent eluates from the BSA column (Fig. 1A, compare lanes 3 and 4). PCNA and the RFC subunits, p37 and p140, were also present in the 300 mm NaCl eluate from the DNA ligase I but not from the BSA column (data not shown). Since RFC also binds to PCNA (25Zhang G. Gibbs E. Kelman Z. O'Donnell M. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1869-1874Crossref PubMed Scopus (73) Google Scholar), it is possible that DNA ligase I-bound PCNA acts as a binding site for other PCNA binding proteins. However, FEN-1, another PCNA-binding replication protein (26Li 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), was not retained by the DNA ligase I resin (Fig. 1A), indicating that the binding of PCNA to the DNA ligase I beads does not result in the nonspecific retention of other PCNA binding proteins. To provide further evidence for the specific association between RFC and DNA ligase I in HeLa cell lysates, we performed immunoprecipitations in the presence of ethidium bromide. As shown in Fig. 1B, the DNA ligase I antibody co-immunoprecipitated the p37 subunit of RFC (Fig. 1B, lane 3), and, in reciprocal experiments, the RFC p37 antibody co-immunoprecipitated DNA ligase I (Fig. 1B, lane 4). Since RFC p37 and DNA ligase I can be co-immunoprecipitated in the presence of ethidium bromide, it appears likely that the association between these factors is mediated by protein-protein interactions. To determine whether there is a direct interaction between DNA ligase I and RFC, we performed pull-down assays with DNA ligase I affinity beads and purified recombinant human RFC (20Podust V.N. Fanning F. J. Biol. Chem. 1997; 272: 6303-6310Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) (Fig. 2A, lane 2). The binding of RFC to DNA ligase I- but not BSA-beads (Fig. 2B, compare lanes 2 and 3), demonstrates that there is a direct interaction between RFC and DNA ligase I. RFC is composed of a large subunit, p140 and four smaller subunits p40, p38, p37, and p36 (27Tsurimoto T. Stillman B. J. Biol. Chem. 1991; 266: 1950-1960Abstract Full Text PDF PubMed Google Scholar, 28Lee S.H. Kwong A.D. Pan Z.Q. Hurwitz J. J. Biol. Chem. 1991; 266: 594-602Abstract Full Text PDF PubMed Google Scholar). Recently, several different variants of RFC, in which p140 has been replaced by different polypeptides, have been described (29Bermudez V.P. Maniwa Y. Tappin I. Ozato K. Yokomori K. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10237-10242Crossref PubMed Scopus (103) Google Scholar, 30Lindsey-Boltz L.A. Bermudez V.P. Hurwitz J. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11236-11241Crossref PubMed Scopus (175) Google Scholar). Since p140 is the unique component of the replicative clamp loader, we reasoned that the specific association with RFC is likely to involve an interaction with p140. To test this idea, we expressed the N-terminal domain of p140, which can be removed without loss of catalytic activity (19Uhlmann F. Cai J. Gibbs E. O'Donnell M. Hurwitz J. J. Biol. Chem. 1997; 272: 10058-10064Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), and the C-terminal domain of p140, which is required for complex formation with the four small subunits (19Uhlmann F. Cai J. Gibbs E. O'Donnell M. Hurwitz J. J. Biol. Chem. 1997; 272: 10058-10064Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), as GST fusion proteins. In pull-down assays, in vitro translated DNA ligase I was specifically retained on glutathione beads liganded by the noncatalytic N-terminal domain of p140 (Fig. 3A). If the interaction between the clamp loader and the DNA ligase plays a critical role in lagging strand DNA synthesis, then it should be conserved in other eukaryotes. This prompted us to examine whether Cdc9, the S. cerevisiae DNA ligase I homolog, interacts with the large subunit of S. cerevisiae RFC, Rfc1. As shown in Fig. 3B, in vitro translated Rfc1 was specifically retained on glutathione beads liganded by GST-Cdc9, indicating that the interaction between the large subunit of the replicative clamp loader and DNA ligase is conserved in eukaryotes. Since previous studies have shown that the N-terminal 118 amino acid residues of DNA ligase I contain the binding sites for both PCNA and DNA polymerase β (11Montecucco A. Rossi R. Levin D.S. Gary R. Park M.S. Motycka T.A. Ciarrocchi G. Villa A. Biamonti G. Tomkinson A.E. EMBO J. 1998; 17: 3786-3795Crossref PubMed Scopus (174) Google Scholar, 31Dimitriadis E.K. Prasad R. Vaske M.K. Chen L. Tomkinson A.E. Lewis M.S. Wilson S.H. J. Biol. Chem. 1998; 32: 20540-20550Abstract Full Text Full Text PDF Scopus (68) Google Scholar), we examined whether the same region was involved in the interaction with RFC p140. As shown in Fig. 4A, this fragment did bind specifically to the N-terminal domain of RFC p140. This interaction was not disrupted by the substitution of the adjacent phenylalanine residues by alanine residues in the PCNA binding site at the N terminus of DNA ligase I (data not shown). Unexpectedly, the N-terminal domain of RFC p140 interacted more efficiently with a fragment encompassing the catalytic domain of DNA ligase I (Fig. 4A). This is the first example of a protein-protein interaction involving this region of DNA ligase I. The observation that more than one region of DNA ligase I binds to the N-terminal domain of RFC p140 prompted us to examine whether DNA ligase I also interacts with the small RFC subunits. As expected, in vitro translated RFC p140 bound specifically to both the N-terminal (Fig. 4B, lane 19) and C-terminal (Fig. 4B, lane 20) fragments of DNA ligase I expressed as GST fusion proteins. Similar, albeit weaker, interactions were observed with in vitro translated p36 (Fig. 4B, lanes 3 and 4) and p38 (Fig. 4B, lanes 11 and 12). The results of our protein-protein interaction experiments described above, together with published studies, have identified a series of pairwise interactions among PCNA, RFC, and DNA ligase I (7Levin D.S. McKenna A. Motycka T. Matsumoto Y. Tomkinson A.E. Curr. Biol. 2000; 10: 919-922Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 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, 11Montecucco A. Rossi R. Levin D.S. Gary R. Park M.S. Motycka T.A. Ciarrocchi G. Villa A. Biamonti G. Tomkinson A.E. EMBO J. 1998; 17: 3786-3795Crossref PubMed Scopus (174) Google Scholar). To examine the effect of RFC on DNA ligase I catalytic activity, we constructed a partial duplex linear substrate containing a single ligatable nick (Fig. 5A). Preincubation of this substrate with RFC inhibited the extent of DNA joining by about 50% (Fig. 5B). In accord with previous studies (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), PCNA had essentially no effect on the DNA joining activity of DNA ligase I (Fig. 5B). Interestingly, when PCNA was preincubated with RFC, the inhibitory effect of RFC on DNA joining was alleviated (Fig. 5B). Since we have shown earlier that the N-terminal region of p140 is involved in the interaction with DNA ligase I (Fig. 3), we asked whether the catalytically active RFC complex containing a truncated version of p140 lacking the N-terminal region had a similar effect on DNA ligase I activity. As shown in Fig. 5C, the RFC complex containing a truncated version of p140 inhibited DNA joining by about 50%, but, as with the intact complex, this inhibition was alleviated in the presence of PCNA. Under these reaction conditions, the RFC complex containing a truncated version of p140 was still specifically retained by DNA ligase I beads (Fig. 5D), presumably because of the interactions between DNA ligase I and one or more of the small RFC subunits (Fig. 4B). The simplest explanation for the observations described above is that the ATP-dependent loading of PCNA by RFC induces the dissociation of RFC from the nicked DNA, thereby making the nick accessible for ligation. Preincubation of a circular ligatable substrate (Fig. 6A) with RFC also caused an inhibition of DNA ligase I activity (Fig. 6B). In this assay, the initial rate of DNA joining was reduced by about 4-fold, but this inhibition was alleviated by the inclusion of PCNA (Fig. 6B). However, in similar experiments with a mutant variant of DNA ligase I in which the PCNA binding site had been inactivated (7Levin D.S. McKenna A. Motycka T. Matsumoto Y. Tomkinson A.E. Curr. Biol. 2000; 10: 919-922Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar), preincubation of PCNA with RFC did not alleviate the inhibitory effect of RFC (Fig. 6C). A similar result was obtained in an assay with the linear ligatable substrate (data not shown). Thus, the ability of DNA ligase I to efficiently join DNA in the presence of RFC and PCNA is dependent upon its ability to bind to PCNA. There is substantial evidence supporting the notion that multiprotein DNA transactions such as DNA replication are coordinated by protein-protein interactions among the participating factors. Notably, PCNA, which is a homotrimeric sliding clamp, interacts with multiple DNA replication factors, indicating that it plays a central role in directing the sequential actions of these proteins (12Warbrick E. BioEssays. 1998; 20: 195-199Crossref PubMed Scopus (315) Google Scholar, 13Jonsson Z. Hubscher U. BioEssays. 1997; 19: 967-975Crossref PubMed Scopus (218) Google Scholar). In previous studies, we have shown that the interaction between DNA ligase I and PCNA is critical for the efficient joining of Okazaki fragments and the completion of the repair of DNA lesions by long patch BER (7Levin D.S. McKenna A. Motycka T. Matsumoto Y. Tomkinson A.E. Curr. Biol. 2000; 10: 919-922Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 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). Our failure to find conditions under which PCNA stimulated the catalytic activity of DNA ligase I (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) prompted us to look for additional protein factors that may contribute to DNA joining events involving DNA ligase I and PCNA. In this study, we describe a direct interaction between DNA ligase I and RFC, the clamp loader. Unlike PCNA binding, which is dependent on a 20-amino acid sequence at the N terminus of DNA ligase I (11Montecucco A. Rossi R. Levin D.S. Gary R. Park M.S. Motycka T.A. Ciarrocchi G. Villa A. Biamonti G. Tomkinson A.E. EMBO J. 1998; 17: 3786-3795Crossref PubMed Scopus (174) Google Scholar), the interaction with RFC involves residues from both the noncatalytic N-terminal and the catalytic C-terminal domains of DNA ligase I. Moreover, DNA ligase I not only interacts with the noncatalytic N-terminal region of the large subunit of p140 but also with two of the smaller RFC subunits. Together, these results indicate that there are multiple sites of contact between DNA ligase I and RFC. Previously, we had shown that PCNA tethered DNA ligase I to a nicked circular DNA duplex but did not stimulate ligation (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). This suggested that the free-sliding DNA ligase I-PCNA complex required an additional factor(s) to position it at a nick. In this regard, it is intriguing that RFC binds to the 3′-hydroxyl termini of primer-template junctions (27Tsurimoto T. Stillman B. J. Biol. Chem. 1991; 266: 1950-1960Abstract Full Text PDF PubMed Google Scholar) and so could potentially recruit DNA ligase I to nicks. However, the recent structures of prokaryotic and eukaryotic RFC complexes determined by electron microscopy and x-ray crystallography suggest that the binding of RFC would prevent DNA ligase I from gaining access to the nick (32Miyata T. Oyama Y. Mayanagi K. Ishino S. Ishino Y. Morikawa K. Nat. Struct. Cell Biol. 2004; 11: 632-636Crossref PubMed Scopus (49) Google Scholar, 33Bowman G.D. O'Donnell M. Kuriyan J. Nature. 2004; 429: 724-730Crossref PubMed Scopus (344) Google Scholar). Consistent with this prediction and a previous biochemical study (2Waga S. Bauer G. Stillman B. J. Biol. Chem. 1994; 269: 10923-10934Abstract Full Text PDF PubMed Google Scholar), RFC inhibited joining by DNA ligase I. This inhibitory effect was alleviated when PCNA was included in the reaction, but only when DNA ligase I had a functional PCNA binding site. Thus, it appears that the pairwise interactions among RFC, PCNA, and DNA ligase I coordinate the joining step that links Okazaki fragments and completes certain DNA excision repair pathways. The conservation of the interaction between the functionally homologous S. cerevisiae proteins supports the notion that the interaction between the replicative DNA ligase and the replicative clamp loader is functionally and biologically significant. Although PCNA is a ring-shaped molecule, the two faces of the ring are not equivalent. RFC loads PCNA onto DNA in a particular orientation and all of the replication proteins appear to bind to the same face of the PCNA ring (12Warbrick E. BioEssays. 1998; 20: 195-199Crossref PubMed Scopus (315) Google Scholar, 13Jonsson Z. Hubscher U. BioEssays. 1997; 19: 967-975Crossref PubMed Scopus (218) Google Scholar). Since PCNA is a homotrimer, it is possible that up to three replication factors can bind to the same PCNA trimer. Alternatively, the factors may bind sequentially with the DNA structure presumably dictating their sequential action. Only 1 molecule of DNA ligase I was bound per PCNA trimer topologically linked to DNA (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). Moreover, we have been unable to detect formation of a ternary complex of PCNA, DNA ligase I and FEN-1, 2J. Varkey and A. E. Tomkinson, unpublished results. suggesting that the binding of one molecule of DNA ligase I to a PCNA trimer occludes the other binding sites and that the binding of DNA ligase I and FEN-1 to a PCNA trimer are mutually exclusive. In Okazaki fragment maturation, the removal of flaps by FEN-1 linked to PCNA generates the nicked DNA substrate for DNA ligase I. We propose that flap removal is the signal for the dissociation of the FEN-1-PCNA complex. Since PCNA is then free to slide away from the nick, we suggest that RFC acts to maintain PCNA at the nick site by binding to both the nick and PCNA. It should be noted that, based on the structure of yeast RFC (33Bowman G.D. O'Donnell M. Kuriyan J. Nature. 2004; 429: 724-730Crossref PubMed Scopus (344) Google Scholar), it appears likely that the interaction of RFC with a 3′-OH terminus will differ, depending on whether or not there is an adjacent strand. Since there are pairwise interactions among PCNA, RFC, and DNA ligase I, we suggest that DNA ligase I initially interacts with a ternary complex of RFC and PCNA bound at a DNA nick via its interaction with the N-terminal region of the large subunit of RFC. This protein-protein interaction may induce a conformational change in RFC, enabling DNA ligase I to contact the PCNA ring and catalyze DNA ligation. Finally, we speculate that nick ligation is the signal for RFC to unload PCNA and for the dissociation of the ternary complex containing DNA ligase I, PCNA, and RFC. In summary, we have identified a conserved interaction between the replicative clamp loader and the replicative DNA ligase. Moreover, we have shown that the interaction between DNA ligase I and PCNA is required for PCNA to overcome the inhibitory effect of RFC on DNA ligation, suggesting that pair-wise physical and functional interactions among RFC, PCNA, and DNA ligase I coordinate the DNA joining step that links adjacent Okazaki fragments. Further studies are needed to delineate the functional significance of the protein-protein interaction between RFC and DNA ligase I and the molecular mechanisms involved in this key reaction in lagging strand DNA synthesis. We thank Drs. Bruce Stillman and Vladimir Podust for reagents. We thank Dr. Sean Post for help with the immunoprecipitation experiments." @default.
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• W2076554477 title "A Conserved Interaction between the Replicative Clamp Loader and DNA Ligase in Eukaryotes" @default.
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