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• W2080140141 abstract "Replication factor C (RF-C), a complex of five subunits, and several subassemblies of RF-C, representing intermediates along the proposed protein assembly pathway (Podust, V. N., and Fanning, E. (1997) J. Biol. Chem.272, 6303–6310), were expressed in insect cells using baculoviruses encoding individual subunits (p140, p40, p38, p37, and p36). Purified proteins were analyzed for ATPase activity to assess the role of individual subunits in ATP hydrolysis. His-tagged p40 contained low ATPase activity, but tagged p37 and p36 did not. Complexes of p40·p37·p36 bearing a His tag on any subunit displayed DNA-stimulated ATPase activity, in agreement with a recent report (Cai, J., Gibbs, E., Uhlmann, F., Philips, B., Yao, N., O'Donnell, M., and Hurwitz, J. (1997)J. Biol. Chem.272, 18974–18981). In contrast, complex p38·p37·p36-his displayed no ATPase, suggesting that p40 is essential for ATPase activity. Although p38 was not required for ATPase activity, the activity of the p40-his·p38·p37·p36 complex was more salt-resistant than that of the p40-his·p37·p36 complex. The p140 subunit further increased the specific ATPase activity of RF-C complex by enhancing its stimulation by DNA. Taken together, the data indicate that all five RF-C subunits constitute ATPase activity, although the contributions of the individual subunits differ. Predicted ATP-binding domains of all five subunits were mutated to assess the importance of multiple ATP-binding sites of RF-C. In each case, the Lys of the conserved P-loop motif was replaced by Glu. The ATP-binding domain of p38 was found to be dispensable for the activity of the five-subunit RF-C in polymerase δ DNA synthesis. In contrast, mutation of the ATP-binding domains in other RF-C subunits impaired RF-C assembly, function, or both. Replication factor C (RF-C), a complex of five subunits, and several subassemblies of RF-C, representing intermediates along the proposed protein assembly pathway (Podust, V. N., and Fanning, E. (1997) J. Biol. Chem.272, 6303–6310), were expressed in insect cells using baculoviruses encoding individual subunits (p140, p40, p38, p37, and p36). Purified proteins were analyzed for ATPase activity to assess the role of individual subunits in ATP hydrolysis. His-tagged p40 contained low ATPase activity, but tagged p37 and p36 did not. Complexes of p40·p37·p36 bearing a His tag on any subunit displayed DNA-stimulated ATPase activity, in agreement with a recent report (Cai, J., Gibbs, E., Uhlmann, F., Philips, B., Yao, N., O'Donnell, M., and Hurwitz, J. (1997)J. Biol. Chem.272, 18974–18981). In contrast, complex p38·p37·p36-his displayed no ATPase, suggesting that p40 is essential for ATPase activity. Although p38 was not required for ATPase activity, the activity of the p40-his·p38·p37·p36 complex was more salt-resistant than that of the p40-his·p37·p36 complex. The p140 subunit further increased the specific ATPase activity of RF-C complex by enhancing its stimulation by DNA. Taken together, the data indicate that all five RF-C subunits constitute ATPase activity, although the contributions of the individual subunits differ. Predicted ATP-binding domains of all five subunits were mutated to assess the importance of multiple ATP-binding sites of RF-C. In each case, the Lys of the conserved P-loop motif was replaced by Glu. The ATP-binding domain of p38 was found to be dispensable for the activity of the five-subunit RF-C in polymerase δ DNA synthesis. In contrast, mutation of the ATP-binding domains in other RF-C subunits impaired RF-C assembly, function, or both. DNA polymerase auxiliary factors PCNA 1The abbreviations used are: PCNA, proliferating cell nuclear antigen; RF-C, replication factor C; pol, DNA polymerase; ssDNA, single-stranded DNA; DTT, dithiothreitol; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; Ni-NTA, nickel-nitrilotriacetic acid; p140, p40, p38, p37, and p36, recombinant human RFC140, RFC40, RFC38, RFC37, and RFC36 subunit, respectively; v140, v40, v38, v37, and v36, recombinant baculovirus encoding p140, p40, p38, p37, and p36 subunit, respectively; -his, His-tag N-terminal fusion; WT, wild type; ATPγS, adenosine 5′-O-(thiotriphosphate). and RF-C are essential proteins required for both replicative and repair DNA synthesis (1Waga S. Bauer G. Stillman B. J. Biol. Chem. 1994; 269: 10923-10934Abstract Full Text PDF PubMed Google Scholar, 2Aboussekhra A. Biggerstaff M. Shivji M.K.K. Vilpo J.A. Moncollin V. Podust V.N. Protic M. Hübscher U. Egly J.-M. Wood R.D. Cell. 1995; 80: 859-868Abstract Full Text PDF PubMed Scopus (753) Google Scholar). PCNA is composed of three identical subunits, which form a torus-like structure with a central cavity sufficient to accommodate double-stranded DNA (3Krishna T.S.R. Kong X.-P. Gary S. Burgers P.M. Kuriyan J. Cell. 1994; 79: 1233-1243Abstract Full Text PDF PubMed Scopus (759) Google Scholar, 4Gulbis J.M. Kelman Z. Hurwitz J. O'Donnell M. Kuriyan J. Cell. 1997; 87: 297-306Abstract Full Text Full Text PDF Scopus (651) Google Scholar). RF-C loads trimeric PCNA molecules onto DNA, thereby forming a stable complex, called the sliding clamp. The clamp specifically interacts with pol δ or pol ε to assemble the corresponding holoenzymes functional in DNA replication (5Lee S.-H. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5672-5676Crossref PubMed Scopus (175) Google Scholar, 6Tsurimoto T. Stillman B. J. Biol. Chem. 1991; 266: 1961-1968Abstract Full Text PDF PubMed Google Scholar, 7Burgers P.M.J. J. Biol. Chem. 1991; 266: 22698-22706Abstract Full Text PDF PubMed Google Scholar, 8Podust V.N. Georgaki A. Strack B. Hübscher U. Nucleic Acids Res. 1992; 20: 4159-4165Crossref PubMed Scopus (73) Google Scholar) and DNA repair (9Shivji M.K.K. Podust V.N. Hübscher U. Wood R.D. Biochemistry. 1995; 34: 5011-5017Crossref PubMed Scopus (242) Google Scholar). The function of RF-C as a PCNA clamp loader is dependent on its interaction with ATP, which apparently occurs in a complex way. First, ATP stimulates the binding of RF-C to DNA (10Lee S.-H. Kwong A.D. Pan Z.-Q. Hurwitz J. J. Biol. Chem. 1991; 266: 594-602Abstract Full Text PDF PubMed Google Scholar, 11Tsurimoto T. Stillman B. J. Biol. Chem. 1991; 266: 1950-1960Abstract Full Text PDF PubMed Google Scholar). Second, ATP is absolutely required to form an RF-C-dependent complex of PCNA with DNA (12Podust L.M. Podust V.N. Sogo J.M. Hübscher U. Mol. Cell. Biol. 1995; 15: 3072-3081Crossref PubMed Scopus (97) Google Scholar). Third, ATP cofactor is required to form a stable salt-resistant complex of RF-C with PCNA in the absence of DNA (13Gerik K.J. Gary S.L. Burgers P.M.J. J. Biol. Chem. 1997; 272: 1256-1262Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). ATPγS, a nonhydrolyzable analog of ATP, substitutes for ATP in these reactions (10Lee S.-H. Kwong A.D. Pan Z.-Q. Hurwitz J. J. Biol. Chem. 1991; 266: 594-602Abstract Full Text PDF PubMed Google Scholar, 11Tsurimoto T. Stillman B. J. Biol. Chem. 1991; 266: 1950-1960Abstract Full Text PDF PubMed Google Scholar, 12Podust L.M. Podust V.N. Sogo J.M. Hübscher U. Mol. Cell. Biol. 1995; 15: 3072-3081Crossref PubMed Scopus (97) Google Scholar, 13Gerik K.J. Gary S.L. Burgers P.M.J. J. Biol. Chem. 1997; 272: 1256-1262Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). However, the RF-C·PCNA·DNA complex formed in the presence of ATPγS is not competent to interact efficiently with pol δ or pol ε to form the corresponding holoenzymes (5Lee S.-H. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5672-5676Crossref PubMed Scopus (175) Google Scholar, 7Burgers P.M.J. J. Biol. Chem. 1991; 266: 22698-22706Abstract Full Text PDF PubMed Google Scholar). A change in conformation or perhaps composition of the protein/DNA intermediate is apparently required to yield the stable functional clamp and depends on ATP hydrolysis. The mechanism of this conversion remains to be elucidated. RF-C is composed of five subunits, one large subunit and four small subunits. The subunits of human RF-C were named according their apparent molecular masses on SDS-PAGE: p140, p40, p38, p37, and p36. The cDNAs encoding all five subunits of human RF-C (14Chen M. Pan Z.-Q. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2516-2520Crossref PubMed Scopus (57) Google Scholar, 15Chen M. Pan Z.-Q. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5211-5215Crossref PubMed Scopus (58) Google Scholar, 16Bunz F. Kobayashi R. Stillman B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11014-11018Crossref PubMed Scopus (77) Google Scholar, 17O'Donnell M. Onrust R. Dean F.B. Hurwitz J. Nucleic Acids Res. 1993; 21: 1-3Crossref PubMed Scopus (144) Google Scholar) and yeast RF-C (18Li X. Burgers P.M.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 868-872Crossref PubMed Scopus (55) Google Scholar, 19Li X. Burgers P.M.J. J. Biol. Chem. 1994; 269: 21880-21884Abstract Full Text PDF PubMed Google Scholar, 20Noskov V. Maki S. Kawasaki Y. Leem S.-H. Ono B.-I. Araki H. Pavlov Y. Sugino A. Nucleic Acids Res. 1994; 22: 1527-1535Crossref PubMed Scopus (39) Google Scholar, 21Cullman G. Fien K. Kobayashi R. Stillman B. Mol. Cell. Biol. 1995; 15: 4661-4671Crossref PubMed Scopus (213) Google Scholar) have been cloned. Each human RF-C subunit corresponds closely to its yeast counterpart (Ref. 21Cullman G. Fien K. Kobayashi R. Stillman B. Mol. Cell. Biol. 1995; 15: 4661-4671Crossref PubMed Scopus (213) Google Scholar; reviewed in Ref.22Hübscher U. Maga G. Podust V.N. DePamphilis M.L. DNA Replication in Eukaryotic Cells. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 525-543Google Scholar). All human and yeast subunits show extensive amino acid sequence homology (16Bunz F. Kobayashi R. Stillman B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11014-11018Crossref PubMed Scopus (77) Google Scholar, 17O'Donnell M. Onrust R. Dean F.B. Hurwitz J. Nucleic Acids Res. 1993; 21: 1-3Crossref PubMed Scopus (144) Google Scholar, 19Li X. Burgers P.M.J. J. Biol. Chem. 1994; 269: 21880-21884Abstract Full Text PDF PubMed Google Scholar, 21Cullman G. Fien K. Kobayashi R. Stillman B. Mol. Cell. Biol. 1995; 15: 4661-4671Crossref PubMed Scopus (213) Google Scholar). One feature of this homology is that each RF-C subunit contains a sequence motif characteristic for NTP-binding/hydrolyzing proteins that consists of two separate units: GXXGXGKT followed by DE(A/V)D (Ref. 23Koonin E.V. Nucleic Acids Res. 1993; 21: 2541-2547Crossref PubMed Scopus (341) Google Scholar and references therein). Such a redundancy of NTP binding sites in the five-subunit RF-C complex would be consistent with several models for ATP hydrolysis by RF-C: 1) only one subunit is the putative ATPase, and the other subunits play no role in the binding/hydrolysis of ATP despite the characteristic sequence motifs; 2) each subunit is an ATPase and acts independently of the other subunits; 3) none of the subunits is able to hydrolyze ATP unless a functional subcomplex is assembled; and 4) one subunit is a minimal ATPase, but interaction of two or more subunits is required for ATP hydrolysis and the specific activity of the ATPase depends on the protein complexity. Recombinant RF-C has been successfully expressed using several approaches. Yeast RF-C has been overexpressed in yeast by using plasmids encoding all five RF-C subunits under the control of inducible promoters (13Gerik K.J. Gary S.L. Burgers P.M.J. J. Biol. Chem. 1997; 272: 1256-1262Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Human RF-C has been reconstituted using an in vitro coupled transcription/translation system (24Uhlmann F. Cai J. Flores-Rozas H. Dean F. Finkelstein J. O'Donnell M. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6521-6526Crossref PubMed Scopus (51) Google Scholar) and a baculovirus expression system (25Cai J. Uhlmann F. Gibbs E. Flores-Rozas H. Lee C.-G. Philips B. Finkelstein J. Yao N. O'Donnell M. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12896-12901Crossref PubMed Scopus (73) Google Scholar, 26Podust V.N. Fanning E. J. Biol. Chem. 1997; 272: 6303-6310Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Analysis of physical interactions among RF-C subunits suggested two models for assembly of individual subunits into the five-subunit complex (25Cai J. Uhlmann F. Gibbs E. Flores-Rozas H. Lee C.-G. Philips B. Finkelstein J. Yao N. O'Donnell M. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12896-12901Crossref PubMed Scopus (73) Google Scholar, 26Podust V.N. Fanning E. J. Biol. Chem. 1997; 272: 6303-6310Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). One of the intermediates common to the assembly of the five-subunit RF-C complex in both models, the subcomplex p40·p37·p36, has been recently characterized (27Cai J. Gibbs E. Uhlmann F. Philips B. Yao N. O'Donnell M. Hurwitz J. J. Biol. Chem. 1997; 272: 18974-18981Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The three-subunit subcomplex, like the five-subunit RF-C, contained DNA-stimulated ATPase. The p40·p37·p36 complex was unable to load PCNA onto DNA but did unload PCNA clamp from singly nicked circular DNA, although 1000-fold less efficiently than the five-subunit RF-C. The three-subunit complex was able to bind primed DNA and PCNA, but again more weakly than the five-subunit complex (27Cai J. Gibbs E. Uhlmann F. Philips B. Yao N. O'Donnell M. Hurwitz J. J. Biol. Chem. 1997; 272: 18974-18981Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Using recombinant human RF-C expressed in the baculovirus system, we have further investigated the role of individual RF-C subunits in ATP hydrolysis. The in vitro DNA-stimulated ATPase activity of various RF-C subunits and RF-C subcomplexes has been analyzed. The ability of five-subunit RF-C complexes assembled with subunits bearing mutations in the ATP-binding motifs to stimulate pol δ holoenzyme DNA synthesis has also been characterized. Calf thymus pol δ and recombinant human PCNA have been described (8Podust V.N. Georgaki A. Strack B. Hübscher U. Nucleic Acids Res. 1992; 20: 4159-4165Crossref PubMed Scopus (73) Google Scholar, 11Tsurimoto T. Stillman B. J. Biol. Chem. 1991; 266: 1950-1960Abstract Full Text PDF PubMed Google Scholar). Monoclonal antibody raised against RFC140 subunit (monoclonal antibody 19; Ref. 16Bunz F. Kobayashi R. Stillman B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11014-11018Crossref PubMed Scopus (77) Google Scholar) was kindly provided by B. Stillman (Cold Spring Harbor). Polyclonal antibody specific to the His tag sequence MRGSH6 was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Pwo DNA polymerase was from Boehringer Mannheim; restriction endonucleases and T4 DNA ligase were from Promega. Prestained molecular mass standard protein mixture (195, 112, 84, 63, 52.5, 35, and 32 kDa) was from Sigma. M13(mp19) ssDNA and singly primed ssDNA was prepared as described (26Podust V.N. Fanning E. J. Biol. Chem. 1997; 272: 6303-6310Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Recombinant human PCNA was bound to Affi-Gel 10 (Bio-Rad) as described for yeast PCNA (13Gerik K.J. Gary S.L. Burgers P.M.J. J. Biol. Chem. 1997; 272: 1256-1262Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Prepared PCNA-agarose contained 3.5 mg of bound PCNA per ml of resin. Recombinant WT baculoviruses encoding nonfused RF-C subunits (called v140, v40, v38, v37, v36) and N-terminal His tag-fused subunits (v40-his, v37-his and v36-his) have been described (26Podust V.N. Fanning E. J. Biol. Chem. 1997; 272: 6303-6310Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Control virus was prepared from unmodified baculovirus transfer vector pBacHisC and encoded a 54-amino acid peptide with a His tag at the N terminus (26Podust V.N. Fanning E. J. Biol. Chem. 1997; 272: 6303-6310Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The mutation of Lys84 to Glu84 was generated by mismatch PCR using the T7 promoter primer (Promega), the backward primer dGCTGCTGCCAAAATAGTGGATGTTTCTCCAGTTCC, and modified pET/RFC37 plasmid, encoding full-length RFC37 cDNA (26Podust V.N. Fanning E. J. Biol. Chem. 1997; 272: 6303-6310Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) as a template. The PCR product was digested by BamHI and BstXI. The fragment containing the 5′-end of RFC37(K84E) cDNA was used to replace the BamHI/BstXI fragment in the pET/RFC37. The full-length RFC37(K84E) cDNA was transferred as aBamHI/EcoRI fragment into pVL1393 (Invitrogen). The mutation of Lys82 to Glu82 was generated by mismatch PCR using the forward primer dTCAAGGATCCATATGGAGGTGGAGGCCG, the backward primer dCAGGGCCCGGGCCAAGCACAGAATGCTTGTGGTCTCGCCGGTTC, and the modified pET/RFC40 described in Ref. 26Podust V.N. Fanning E. J. Biol. Chem. 1997; 272: 6303-6310Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar as a template. The PCR product was digested by BamHI and XmaI. The fragment containing the 5′-end of RFC40(K82E) cDNA was used to replace the WT BamHI/XmaI fragment in the pBacHis/RFC40. The mutation of Lys657 to Glu657 was generated by mismatch PCR using the forward primer dGAATCTCAGCAACATTCC, the backward primer dGACACACCAGGGAAGCTGTGGTGGTTTCGCCAAC, and pSK/RFC140 as a template. The PCR product was digested by SacI and XcmI. The resulting fragment was used to replace theSacI/XcmI fragment in a pKS plasmid carrying theSacI/PstI fragment of RFC140 cDNA. The new plasmid carrying the SacI/PstI fragment mutated at Lys657 was digested with SacI andEcoRI. The corresponding fragment was used to replace the WTSacI/EcoRI fragment in pVL1393/RFC140. The mutation of Lys66 to Glu66 was generated by PCR overlap extension (28Vallejo A.N. Pogulis R.J. Pease L.R. Dieffenbach C.W. Dveksler G.S. PCR Primer: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1995: 603-612Google Scholar) using the primers dCCGGATTATTCATACCGTCC (first forward primer), dGGTAGATGTCTCGCCTGTCC (first backward), dGGACAGGCGAGACATCTACC (second forward), and dGATCGGTCCTCGAATGATGTC (second backward) and pVL1393/RFC36 as a template. The PCR product was digested first withBamHI and then with NcoI. The resulting fragment was used to replace the WT BamHI/NcoI fragment in the pVL1393/RFC36. The mutation of Lys48 to Glu48 was generated by PCR overlap extension (28Vallejo A.N. Pogulis R.J. Pease L.R. Dieffenbach C.W. Dveksler G.S. PCR Primer: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1995: 603-612Google Scholar) using T7 promoter primer (first forward primer), dCTTGTCTTTTCTCCAGCAC (first backward), dGTGCTGGAGAAAAGACAAG (second forward), and dGATGTAGAATTGCAGCAC (second backward) and pET19b/His38 as a template. The PCR product was digested with BamHI and NcoI. The resulting fragment was used to replace the WTBamHI/NcoI fragment in the pVL1393/RFC38. Growth and maintenance of Sf9 and High Five insect cells (ITC Biotechnology GmbH) in adherent cultures, preparation of recombinant baculoviruses, and infection of the cells were performed as described (26Podust V.N. Fanning E. J. Biol. Chem. 1997; 272: 6303-6310Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Infected cells were incubated for 48 h prior to harvesting the recombinant proteins. 7.6 × 107 cells infected by the corresponding baculoviruses were lysed in 3 ml of buffer A (20 mm Tris-HCl (pH 7.5), 0.2% (v/v) Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml concentration each of aprotinin, leupeptin, and pepstatin) containing 0.1m NaCl. Cell debris was removed by centrifugation. The supernatant was passed over 0.8 ml of DEAE-Sephacel (Amersham Pharmacia Biotech) equilibrated in lysis buffer, and the column was then washed with 1 ml of the same buffer. The flow through and wash were combined and gently mixed with 50 μl of Ni-NTA resin (QIAGEN) for 2 h at 4 °C. The resin was washed three times in batch with 0.5 ml of buffer B (20 mm Tris-HCl (pH 7.5), 2 mmimidazole-HCl, 300 mm NaCl, 0.02% (v/v) Nonidet P-40), and proteins were eluted with 200 μl of buffer C (300 mmimidazole-HCl (pH 7.2), 0.3 m NaCl, 10% (v/v) glycerol). To prepare p40-his·p38·p37·p36 and p40-his·p37·p36 complexes, 3 × 108 insect cells infected by corresponding viruses were lysed in 10 ml of buffer A, 0.1 m NaCl. Cell debris was removed by centrifugation, and the supernatant was mixed with 200 μl of Ni-NTA resin. The suspension was mixed for 2 h at 4 °C, and the resin was pelleted by centrifugation, packed into a column, and washed with 4 ml of buffer B. The proteins were eluted from the resin with 2 ml of buffer C. The eluate was diluted 5-fold with buffer Q (20 mm Tris-HCl (pH 8.0), 10% glycerol, 1 mm DTT, 0.5 mm EDTA, 0.01% Nonidet P-40) and loaded onto a 1-ml Mono Q column (Amersham Pharmacia Biotech). Proteins were eluted with a 20-ml gradient of NaCl from 0 to 350 mm in buffer Q and collected in 0.4-ml fractions. p40·p37-his·p36 and p40·p37·p36-his were expressed using the corresponding viruses and purified analogously to p40-his·p37·p36. Regardless of which of the three subunits was His-tagged, the trimeric complex eluted from the Mono Q column in the same fractions (fractions 33 and 34). To prepare the p38·p37·p36-his complex, 1.5 × 108insect cells infected by v38, v37, and v36-his were lysed in 5 ml of buffer A, 0.1 m NaCl, and proteins were bound to 100 μl of Ni-NTA resin. The resin was washed three times in batch with 1 ml of buffer B, and proteins were eluted with 150 μl of buffer C. 4.2 × 108 insect cells infected with viruses encoding all five RF-C subunits (v140, v40-his, v38, v37, and v36-his) were lysed in 12 ml of buffer A, 0.35 m NaCl. Cell debris was removed by centrifugation, and the supernatant fraction bound to 300 μl of Ni-NTA resin. The suspension was mixed for 2 h at 4 °C, and the resin was pelleted by centrifugation, packed into a column, and washed with 4 ml of buffer B. The proteins were eluted from the resin with 2 ml of buffer C. The eluate was diluted with an equal volume of buffer D (50 mm Tris-HCl (pH 7.5), 10% glycerol, 1 mmDTT). MgCl2 and ATP were added to final concentrations of 10 mm and 1 mm, respectively. The solution was mixed with 200 μl of PCNA-agarose for 2 h at 4 °C. The resin was then packed into a column and washed with 4 ml of buffer D containing 10 mm MgCl2, 1 mm ATP and 300 mm NaCl. Protein was eluted with buffer containing 30 mm Tris-HCl (pH 7.5), 10% glycerol, 400 mmNaCl, 1 mm DTT, and 2 mm EDTA. Reaction mixtures (final volume of 25 μl) contained 40 mm Tris-HCl (pH 7.5); 0.2 mg/ml bovine serum albumin; 1 mm DTT; 10 mm MgCl2; 1 mm ATP; a 50 μm concentration each of dATP, dGTP, and dTTP; 20 μm [α-32P]dCTP (500 cpm/pmol); 100 ng of primed ssDNA; 100 ng of PCNA; 1.2 μg ofEscherichia coli single-stranded DNA-binding protein; 0.25 units of pol δ; and RF-C as indicated in the figures. Samples were incubated for 30 min at 37 °C, the reactions were terminated by adding 1 ml of ice-cold 10% (w/v) trichloroacetic acid, and acid-insoluble material was analyzed by scintillation counting. One unit of RF-C activity was defined as the incorporation of 1 nmol of total dNMP into singly primed ssDNA in the presence of pol δ, PCNA, and E. coli single-stranded DNA-binding protein in 30 min at 37 °C. For product length analysis, the reactions were terminated by treating them with proteinase K (60 μg/ml) for 30 min at 37 °C in the presence of 1% (w/v) SDS and 20 mm EDTA (pH 8.0). The DNA was then precipitated with ethanol, and the products analyzed on an alkaline 1.5% agarose gel as described (29Podust V.N. Podust L.M. Müller F. Hübscher U. Biochemistry. 1995; 34: 5003-5010Crossref PubMed Scopus (43) Google Scholar). Reaction mixtures (final volume of 10 μl) contained 20 mm Tris-HCl (pH 7.5), 0.1 mg/ml bovine serum albumin, 0.5 mm DTT, 10 mm MgCl2, 50 μm [γ-32P]ATP (1 Ci/mmol), 200 ng of M13(mp19) ssDNA, and protein to be tested. Samples were incubated for 10 min at 37 °C, and reactions were terminated with 10 μl of ice-cold 50 mm EDTA. 2 μl of reaction mixtures were spotted on a polyethyleneimine-cellulose thin layer plate, which was developed in 1 m LiCl, 0.5 m formic acid. The amounts of [γ-32P]-ATP hydrolyzed to [32P]orthophosphate were quantified using a PhosphorImager (Molecular Dynamics, Inc.). The rates of ATP hydrolysis were determined in the linear range of reaction time and protein concentration dependence. To test the stability of the three- and four-subunit RF-C subcomplexes under the conditions employed to measure the ATPase activity, 10–20 μg of Mono Q-purified p40-his·p37·p36 or p40-his·p38·p37·p36 complexes were bound to 25 μl of Ni-NTA resin. Beads were washed with 1 ml of buffer E (20 mmTris-HCl (pH 7.5), 0.02% (v/v) Nonidet P-40) containing 300 mm NaCl and then resuspended in 1 ml of buffer E and divided into two equal parts. Supernatant was removed by aspiration, and the beads were resuspended in 200 μl of ATPase reaction mixture (DTT was omitted; ATP was nonradioactive). The suspensions were incubated for 10 min on ice or at 37 °C with periodical gentle agitation. Then the resin was pelleted by centrifugation at 0 or 37 °C, respectively, the supernatant was quickly aspirated, and the beads were resuspended in 30 μl of elution buffer C. Eluted proteins were analyzed by SDS-PAGE followed by Coomassie staining. To test the stability of the five-subunit RF-C, 3.8 × 107 insect cells were infected by the corresponding baculoviruses, and expressed proteins were bound to 25 μl of Ni-NTA resin. The beads were washed with buffer B, resuspended in buffer E, and divided into two equal parts. The resin with bound proteins was incubated for 10 min at 0 or 37 °C with 200 μl of mixture containing 40 mm Tris-HCl (pH 7.5), 0.2 mg/ml bovine serum albumin, 10 mm MgCl2, 1 mm ATP, and 4 μg/ml PCNA. Then the resin was pelleted by centrifugation at 0 or 37 °C, respectively, the supernatant was aspirated, and the beads were resuspended in 30 μl of elution buffer C. Eluted proteins were analyzed by SDS-PAGE. The lower half of the gel was stained with Coomassie to visualize small RF-C subunits. The upper half of the gel was blotted to nitrocellulose membrane and analyzed by immunoblotting with antibody against p140 (monoclonal antibody 19). Western blot analysis was performed as described (26Podust V.N. Fanning E. J. Biol. Chem. 1997; 272: 6303-6310Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Protein concentration was determined by densitometric scanning of Coomassie-stained protein bands in denaturing polyacrylamide gels using Image Store 7500 (Ultra-Violet Products, Inc.). As protein standards, known amounts of bovine serum albumin were loaded onto the same gel. We have recently analyzed the pathway of assembly of RF-C from individual subunits and proposed that in the last step, the large subunit p140 binds to a preformed complex of four small subunits (26Podust V.N. Fanning E. J. Biol. Chem. 1997; 272: 6303-6310Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Characterization of this four-subunit complex showed that it lacked most of the properties of RF-C. It was inactive in the PCNA loading and pol δ DNA synthesis reactions. Like the three-subunit complex p40·p37·p36 (27Cai J. Gibbs E. Uhlmann F. Philips B. Yao N. O'Donnell M. Hurwitz J. J. Biol. Chem. 1997; 272: 18974-18981Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), the four-subunit complex interacted very weakly with DNA and PCNA (data not shown). The results suggest that p140 is required for stable interaction of RF-C with DNA and PCNA. Like the three-subunit complex (27Cai J. Gibbs E. Uhlmann F. Philips B. Yao N. O'Donnell M. Hurwitz J. J. Biol. Chem. 1997; 272: 18974-18981Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), the four-subunit complex was found to possess DNA-stimulated ATPase activity (see below). To learn which RF-C subunits contribute to this activity, we tested the ATPase activities of individual RF-C subunits and various subcomplexes purified from baculovirus infected insect cells (26Podust V.N. Fanning E. J. Biol. Chem. 1997; 272: 6303-6310Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). To analyze the DNA-dependence of the ATPase activity, heteropolymeric M13 ssDNA was chosen, since the latter was found to be the most effective cofactor for ATPase activity of natural RF-C (8Podust V.N. Georgaki A. Strack B. Hübscher U. Nucleic Acids Res. 1992; 20: 4159-4165Crossref PubMed Scopus (73) Google Scholar, 10Lee S.-H. Kwong A.D. Pan Z.-Q. Hurwitz J. J. Biol. Chem. 1991; 266: 594-602Abstract Full Text PDF PubMed Google Scholar). His-tagged small RF-C subunits p40-his, p37-his, and p36-his can be expressed as soluble proteins and purified in ample amounts (26Podust V.N. Fanning E. J. Biol. Chem. 1997; 272: 6303-6310Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) (Fig.1 A). As a control to distinguish the putative ATPase activity of expressed RF-C subunits from possible host protein contaminants, the same amount of cells used to prepare individual RF-C subunits was infected with control virus, and cellular extract was purified according to the protocol for p40-his, p37-his, and p36-his (Fig. 1 A, control preparation lane). Analysis of individual subunit preparations for ATPase activity showed that only p40-his displayed very weak ATPase activity (0.017 mol ATP hydrolyzed per mol of p40-his in 1 min), and this activity was not dependent on ssDNA (TableI).Table IComparison of ATPase activities of RF-C subunits and their complexesProtein complexATP hydrolysis without DNAATP hydrolysis with DNADNA stimulation of ATP hydrolysisDNA-stimulated ATPase activity in comparison with p40-hismol/min/molmol/min/molp36-hisNT1-aNT, not tested.ND1-bND, not detected." @default.
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