Replication Factor C Clamp Loader Subunit Arrangement within the Circular Pentamer and Its Attachment Points to Proliferating Cell Nuclear Antigen

Abstract

Replication factor C (RFC) is a heteropentameric AAA+ protein clamp loader of the proliferating cell nuclear antigen (PCNA) processivity factor. The prokaryotic homologue, γ complex, is also a heteropentamer, and structural studies show the subunits are arranged in a circle. In this report, Saccharomyces cerevisiae RFC protomers are examined for their interaction with each other and PCNA. The data lead to a model of subunit order around the circle. A characteristic of AAA+ oligomers is the use of bipartite ATP sites in which one subunit supplies a catalytic arginine residue for hydrolysis of ATP bound to the neighboring subunit. We find that the RFC(3/4) complex is a DNA-dependent ATPase, and we use this activity to determine that RFC3 supplies a catalytic arginine to the ATP site of RFC4. This information, combined with the subunit arrangement, defines the composition of the remaining ATP sites. Furthermore, the RFC(2/3) and RFC(3/4) subassemblies bind stably to PCNA, yet neither RFC2 nor RFC4 bind tightly to PCNA, indicating that RFC3 forms a strong contact point to PCNA. The RFC1 subunit also binds PCNA tightly, and we identify two hydrophobic residues in RFC1 that are important for this interaction. Therefore, at least two subunits in RFC make strong contacts with PCNA, unlike the Escherichia coli γ complex in which only one subunit makes strong contact with the β clamp. Multiple strong contact points to PCNA may reflect the extra demands of loading the PCNA trimeric ring onto DNA compared with the dimeric β ring. Replication factor C (RFC) is a heteropentameric AAA+ protein clamp loader of the proliferating cell nuclear antigen (PCNA) processivity factor. The prokaryotic homologue, γ complex, is also a heteropentamer, and structural studies show the subunits are arranged in a circle. In this report, Saccharomyces cerevisiae RFC protomers are examined for their interaction with each other and PCNA. The data lead to a model of subunit order around the circle. A characteristic of AAA+ oligomers is the use of bipartite ATP sites in which one subunit supplies a catalytic arginine residue for hydrolysis of ATP bound to the neighboring subunit. We find that the RFC(3/4) complex is a DNA-dependent ATPase, and we use this activity to determine that RFC3 supplies a catalytic arginine to the ATP site of RFC4. This information, combined with the subunit arrangement, defines the composition of the remaining ATP sites. Furthermore, the RFC(2/3) and RFC(3/4) subassemblies bind stably to PCNA, yet neither RFC2 nor RFC4 bind tightly to PCNA, indicating that RFC3 forms a strong contact point to PCNA. The RFC1 subunit also binds PCNA tightly, and we identify two hydrophobic residues in RFC1 that are important for this interaction. Therefore, at least two subunits in RFC make strong contacts with PCNA, unlike the Escherichia coli γ complex in which only one subunit makes strong contact with the β clamp. Multiple strong contact points to PCNA may reflect the extra demands of loading the PCNA trimeric ring onto DNA compared with the dimeric β ring. Replicases of cellular chromosomes utilize a circular sliding clamp protein that encircles DNA and tethers the polymerase to the template for high processivity in DNA synthesis (1.Kelman Z. O'Donnell M. Annu. Rev. Biochem. 1995; 64: 171-200Crossref PubMed Scopus (360) Google Scholar). An example of this protein class is the Escherichia coli β subunit, which confers high processivity onto the chromosomal replicase, DNA polymerase III holoenzyme (2.Stukenberg P.T. Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1991; 266: 11328-11334Abstract Full Text PDF PubMed Google Scholar, 3.Kong X.P. Onrust R. O'Donnell M. Kuriyan J. Cell. 1992; 69: 425-437Abstract Full Text PDF PubMed Scopus (629) Google Scholar). The eukaryotic equivalent is the PCNA 1The abbreviations used are: PCNAproliferating cell nuclear antigenRFCreplication factor CSSBsingle-stranded DNA-binding proteinDTTdithiothreitolwtwild-typemutmutantssDNAsingle-stranded DNAPol δDNA polymerase δ.1The abbreviations used are: PCNAproliferating cell nuclear antigenRFCreplication factor CSSBsingle-stranded DNA-binding proteinDTTdithiothreitolwtwild-typemutmutantssDNAsingle-stranded DNAPol δDNA polymerase δ. ring, which has essentially the same shape and chain fold as β despite lack of sequence similarity between the two (4.Gulbis J.M. Kelman Z. Hurwitz J. O'Donnell M. Kuriyan J. Cell. 1996; 87: 297-306Abstract Full Text Full Text PDF PubMed Scopus (632) Google Scholar, 5.Krishna T.S. Kong X.P. Gary S. Burgers P.M. Kuriyan J. Cell. 1994; 79: 1233-1243Abstract Full Text PDF PubMed Scopus (744) Google Scholar). These ring-shaped proteins require an ATP-fueled multiprotein clamp loader for assembly onto primed DNA. proliferating cell nuclear antigen replication factor C single-stranded DNA-binding protein dithiothreitol wild-type mutant single-stranded DNA DNA polymerase δ. proliferating cell nuclear antigen replication factor C single-stranded DNA-binding protein dithiothreitol wild-type mutant single-stranded DNA DNA polymerase δ. The eukaryotic clamp loader is the heteropentameric replication factor C (RFC). The five subunits of RFC are each different proteins, but they are homologous to one another (6.Cullmann G. Fien K. Kobayashi R. Stillman B. Mol. Cell. Biol. 1995; 15: 4661-4671Crossref PubMed Scopus (210) Google Scholar, 7.O'Donnell M. Onrust R. Dean F.B. Chen M. Hurwitz J. Nucleic Acids Res. 1993; 21: 1-3Crossref PubMed Scopus (142) Google Scholar) and are members of the AAA+ family of ATPases (8.Neuwald A.F. Aravind L. Spouge J.L. Koonin E.V. Genome Res. 1999; 9: 27-43Crossref PubMed Google Scholar). The recent crystal structure of E. coli γ complex, the prokaryotic counterpart of RFC, has facilitated detailed hypothesis regarding RFC structure and mechanism (9.O'Donnell M. Jeruzalmi D. Kuriyan J. Curr. Biol. 2001; 11: R935-R946Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 10.Jeruzalmi D. O'Donnell M. Kuriyan J. Cell. 2001; 106: 429-441Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). The γ complex (γ3δδ′χΨ) consists of a minimal core of five proteins (γ3δδ′), which contain the clamp loading activity (11.Onrust R. O'Donnell M. J. Biol. Chem. 1993; 268: 11766-11772Abstract Full Text PDF PubMed Google Scholar). The remaining two subunits, χ and Ψ, are involved in recruiting an RNA primed DNA site from the primase, and they bind single-stranded DNA-binding protein (SSB) to assist polymerase elongation but are not essential to the clamp loading activity of γ complex (12.Glover B.P. McHenry C.S. J. Biol. Chem. 1998; 273: 23476-23484Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 13.Kelman Z. Yuzhakov A. Andjelkovic J. O'Donnell M. EMBO J. 1998; 17: 2436-2449Crossref PubMed Scopus (154) Google Scholar, 14.Yuzhakov A. Kelman Z. O'Donnell M. Cell. 1999; 96: 153-163Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Biochemical studies of γ complex (15.Leu F.P. O'Donnell M. J. Biol. Chem. 2001; 276: 47185-47194Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 16.Hingorani M.M. O'Donnell M. J. Biol. Chem. 1998; 273: 24550-24563Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 17.Stewart J. Hingorani M.M. Kelman Z. O'Donnell M. J. Biol. Chem. 2001; 276: 19182-19189Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), combined with crystal structures of γ3δδ′ (10.Jeruzalmi D. O'Donnell M. Kuriyan J. Cell. 2001; 106: 429-441Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar) and δ-β1 complex (18.Jeruzalmi D. Yurieva O. Zhao Y. Young M. Stewart J. Hingorani M. O'Donnell M. Kuriyan J. Cell. 2001; 106: 417-428Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 19.Turner J. Hingorani M.M. Kelman Z. O'Donnell M. EMBO J. 1999; 18: 771-783Crossref PubMed Scopus (154) Google Scholar), reveal a highly detailed view of γ3δδ′ clamp loader form and function. The five subunits of the γ3δδ′ complex are arranged in a circular formation (see Fig. 1). Like RFC, the γ3, δ, and δ′ subunits are members of the AAA+ family; they share a characteristic chain-folding pattern consisting of three domains each. The main intersubunit contacts in the heteropentamer are made via the C-terminal domains, which form a tight circular collar (Fig. 1A, top view). The N-terminal domains contain the ATP binding sites, and there is a gap between the N-terminal domains of the δ and δ′ subunits (Fig. 1A, front view). Only the γ subunits contain ATP binding and hydrolysis activity and therefore serve as the motor of this machine; both δ and δ′ lack a consensus ATP binding sequence, and neither of them bind ATP. The γ and δ′ subunits contain an SRC motif that is highly conserved from γ complex to RFC (20.Guenther B. Onrust R. Sali A. O'Donnell M. Kuriyan J. Cell. 1997; 91: 335-345Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). The arginine residue (Arg finger) within the SRC motif is positioned such that it may participate in hydrolysis of ATP bound to the neighboring subunit (Fig. 1A, back view). Hence, the Arg finger within the δ′ SRC motif functions with ATP bound to γ1 (site 1), the γ1 SRC functions with ATP bound to γ2 (site 2), and the γ2 SRC functions with ATP bound to γ3 (site 3) (see Fig. 1B). Biochemical studies confirm that these conserved Arg residues are catalytic and suggest that ATP must first be hydrolyzed in sites 2 and/or 3 before ATP in site 1 is hydrolyzed (21.Johnson A. O'Donnell M. J. Biol. Chem. 2003; 278: 14406-14413Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Study of γ complex and its subunits shows that the δ subunit forms the strongest attachment to the β clamp, and in fact can open the ring by itself, leading to unloading of β clamps from closed circular DNA (19.Turner J. Hingorani M.M. Kelman Z. O'Donnell M. EMBO J. 1999; 18: 771-783Crossref PubMed Scopus (154) Google Scholar). The γ trimer can also bind β and unload it, but it is feeble in these actions compared with δ (15.Leu F.P. O'Donnell M. J. Biol. Chem. 2001; 276: 47185-47194Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Although δ binds β2 tightly, the γ complex does not bind β2 in the absence of ATP, indicating that one or more subunits of γ complex block the δ-to-β2 interaction (22.Naktinis V. Onrust R. Fang L. O'Donnell M. J. Biol. Chem. 1995; 270: 13358-13365Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). ATP binding to the γ subunits promotes tight interaction between γ complex and β, implying that ATP binding induces a conformation change in γ complex that exposes δ, and presumably γ subunits as well, for interaction with β and opening of the β2 ring. The δ′ subunit appears to be a rigid protein and has been termed the stator, the stationary part of a machine upon which other pieces move (10.Jeruzalmi D. O'Donnell M. Kuriyan J. Cell. 2001; 106: 429-441Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 20.Guenther B. Onrust R. Sali A. O'Donnell M. Kuriyan J. Cell. 1997; 91: 335-345Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Results of mutational studies are consistent with the idea that the ATP-induced conformation change of γ complex requires δ′ and that it may serve as a "backboard" to direct the ATP-induced changes in γ complex (23.Indiani C. O'Donnell M. J. Biol. Chem. 2003; (In press)Google Scholar). The similarities in RFC and γ complex subunit sequences and their common function in loading circular clamps onto DNA suggest that the RFC subunits may also be arranged in a circular fashion like γ3δδ′ (10.Jeruzalmi D. O'Donnell M. Kuriyan J. Cell. 2001; 106: 429-441Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Electron microscopy and atomic force microscopy studies of RFC are consistent with a circular arrangement of RFC subunits (9.O'Donnell M. Jeruzalmi D. Kuriyan J. Curr. Biol. 2001; 11: R935-R946Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 24.Shiomi Y. Usukura J. Masamura Y. Takeyasu K. Nakayama Y. Obuse C. Yoshikawa H. Tsurimoto T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14127-14132Crossref PubMed Scopus (66) Google Scholar). The human RFC1 subunit (p140), like δ, binds to PCNA (25.Fotedar R. Mossi R. Fitzgerald P. Rousselle T. Maga G. Brickner H. Messier H. Kasibhatla S. Hubscher U. Fotedar A. EMBO J. 1996; 15: 4423-4433Crossref PubMed Scopus (89) Google Scholar), leading to the proposal that RFC1 may act to open PCNA just as the δ wrench opens β (9.O'Donnell M. Jeruzalmi D. Kuriyan J. Curr. Biol. 2001; 11: R935-R946Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 18.Jeruzalmi D. Yurieva O. Zhao Y. Young M. Stewart J. Hingorani M. O'Donnell M. Kuriyan J. Cell. 2001; 106: 417-428Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). RFC5, like δ′, contains an SRC motif, and the putative ATP site deviates from the consensus sequence (GKKT instead of GKT) suggesting that if it can bind ATP, the ATP may not hydrolyze efficiently. These similarities suggest that RFC5 may play a similar role as the δ′ stator. On the basis of the γ3δδ′ structure, it is proposed that the RFC1 (wrench) and RFC5 (stator) subunits may bracket the RFC2, 3, and 4, subunits, which, like γ3, contain both ATP binding and SRC motifs and thus may act as the motor of the RFC clamp loader (9.O'Donnell M. Jeruzalmi D. Kuriyan J. Curr. Biol. 2001; 11: R935-R946Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). However, the order of the RFC2, 3, and 4 subunits within the pentamer and the identity of the subunits that bind RFC5 and RFC1 are not certain. The aim of this study is to define the arrangement of the subunits within the RFC complex and determine which subunits form the major contact(s) to PCNA. The results indicate the arrangement RFC5:RFC2:RFC3 RFC4:RFC1, and in an orientation looking down the C-terminal plane, RFC5 contributes an arginine finger (SRC) to ATP in RFC2 (site 1), RFC2 contributes an arginine finger to the RFC3 ATP site (site 2), RFC3 contributes an arginine finger to the ATP site in RFC4 (site 3), and RFC4 contributes an arginine finger to the RFC1 ATP site (site 4). Moreover, we find that the RFC(3/4) complex, RFC(2/3) complex, and RFC1 subunit form major contacts to PCNA, unlike the case of the single major contact between δ subunit of γ complex and the β clamp. Radioactive nucleotides were purchased from PerkinElmer Life Sciences. Unlabeled deoxyribonucleoside triphosphates were supplied by Amersham Biosciences. DNA modification enzymes were supplied by New England Biolabs; DNA oligonucleotides were from Integrated DNA Technologies. Protein concentrations were determined using the Bio-Rad Protein stain and bovine serum albumin as a standard. Buffer A is 30 mm HEPES (pH 7.5), 10% (v/v) glycerol, 0.5 mm EDTA (pH 7.5), 1 mm DTT, and 0.04% Bio-Lyte 3/10 ampholyte (Bio-Rad). Buffer B is 20 mm HEPES (pH 7.4), 2 mm DTT, 10% glycerol, and 200 mm NaCl. Buffer C is 20 mm Tris-HCl (pH 7.5), 0.5 mm DTT, 10 mm magnesium acetate, and 60 mm NaCl. Buffer D is 20 mm Tris-HCl (pH 7.5), 5 mm DTT, 0.1 m EDTA, 40 μg/ml bovine serum albumin, 8 mm MgCl2, 4% glycerol, and 0.5 mm ATP. The Saccharomyces cerevisiae RFC genes were cloned into either pET (Novagen) or pLANT (26.Finkelstein J. Antony E. Hingorani M.M. O'Donnell M. Anal. Biochem. 2003; 319: 78-87Crossref PubMed Scopus (59) Google Scholar) vectors. The plasmids containing single genes include pET(11a)-RFC(1), pET(11a)-RFC(2), pLANT (2)-RFC(2), pET(11a)RFC(3), pET(11a)-RFC(4), and pET(11a)-RFC(5). Expression plasmids containing two or more genes include pET(11a)-RFC[3+4], pLANT (2)RFC[1+5], pET(11a)-RFC[2+3+4], and pET(11a)-RFC[1+2+3+4+5]. Mutations in the RFC1 Gene—Codons TAT and TTC in pET(11a)RFC(1) corresponding to residues Tyr-404 and Phe-405 in RFC1 were mutated to GCT and GCC, respectively, by Commonwealth Biotechnologies, changing both hydrophobic residues to alanine. The mutated RFC(1) gene was then cloned into the pLANT (2)-RFC[1+5] to replace the wild-type copy of the RFC(1) gene Mutations in the RFC3 and RFC4 Genes—Mutations were introduced into the RFC(3) and RFC(4) genes using the QuikChange method (Stratagene). RFC[3SAC] was generated in pET(11a)-RFC(3) using the following oligonucleotides: 5′-CAT AAA CTT ACA CT GCG TTA TTG AGC GCT TGC ACG AGA TTC AGA TTT CAG CCC TTG-3′ and 5′-CAA GGG CTG AAA TCT GAA TCT CGT GCA AGC GCT CAA TAA CGC AGG TGT AAG TTT ATG-3′. RFC[4SAC] was generated in pET(11a)RFC(4) using the following oligonucleotides: 5′-CAA GAT CAT TGA GCC GCT GCA AAG CGC TTG TGC GAT TTT GAG GTA TTC TAA AC-3′ and 5′-GTT TAG AAT ACC TCA AAA TCG CAC AAG CG TTT GCA GCG GCT CAA TGA TCT TG-3′. The entire open reading frames were then confirmed by DNA sequencing. Mutant genes were cloned into pET(11a)-RFC[3+4] as follows: the RFC[3SAC] gene was exchanged for the wild-type RFC(3) gene in the pET(11a)-RFC[3+4] plasmid using KpnI. Recombinants were screened with Afe and MfeI/ApaI restriction digest was performed to confirm the proper orientation of the insert. The RFC[4SAC] gene was exchanged for the wild-type RFC(4) gene in the pET(11a)-RFC[3+4] plasmid using AflII/GstBI; recombinants were screened using AfeI. The pET(11a)-RFC derived expression plasmids were transformed into BL21(DE3) codon plus (Stratagene) E. coli cells, which contain a secondary plasmid encoding genes for rare transfer RNAs, as well as chloramphenicol resistance. Transformants were selected using ampicillin (100 μg/ml) and chloramphenicol (25 μg/ml). Co-transformations involving pLANT (2.Stukenberg P.T. Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1991; 266: 11328-11334Abstract Full Text PDF PubMed Google Scholar) and pET(11a)–derived plasmids were performed with BLR(DE3) (Novagen) E. coli-competent cells. These transformants were selected using ampicillin (100 μg/ml) and kanamycin (50 μg/ml). Fresh transformants were grown in 12–24 liters of LB media containing the appropriate antibiotics at 30 °C until reaching an A600 value of 0.6. The cultures were then briefly chilled on ice before adding 1 mm isopropyl-1-thio-β-d-galactopyranoside and then were incubated at 15 °C for ∼18 h. For RFC(2/3) and RFC(2/5), induction was at 37 °C for 3 h. The cells were harvested by centrifugation. Purification of S. cerevisiae RFC1mut and RFCwt Complex—RFCwt was overexpressed from the single plasmid, pET(11a)-RFC[1+2+3+4+5]. The RFC1mut was expressed by co-transformation of pLANT (2)-RFC[1mut+5] and pET(11a)-RFC[2+3+4]. The harvested cells (from 24 liters of culture) were resuspended in ∼200 ml of Buffer A containing 1 m NaCl and then lysed using a French press at 22,000 p.s.i. The cell lysate was clarified by centrifugation, diluted with Buffer A to ∼150 mm NaCl, and then applied to a 30-ml SP-Sepharose Fast Flow (Amersham Biosciences) column equilibrated with Buffer A containing 150 mm NaCl. The column was eluted with a 300-ml gradient of 150–600 mm NaCl in Buffer A. Presence of proteins was followed in 10% SDS-polyacrylamide gels stained with Coomassie Blue. The peak of RFC (which eluted at ∼365 mm NaCl) was pooled and diluted with Buffer A to ∼150 mm NaCl. The protein was then applied to a 40 ml Q-Sepharose Fast Flow (Amersham Biosciences) column equilibrated with Buffer A containing 150 mm NaCl and eluted with a 400-ml gradient of 150–600 mm NaCl in Buffer A. The fractions containing RFC complex (which eluted at ∼300 mm NaCl) were saved individually and stored at -70 °C. Protein concentration was measured by Bradford assay, and a typical final yield was ∼1 mg purified RFC complex/1 liter of culture. Purification of S. cerevisiae Wild-type and Mutant RFC(3/4) Subcomplexes—RFC(3/4) subcomplexes (wild-type or SAC mutants) were expressed using the pET(11a)-RFC[3+4] plasmid containing either wild-type or mutant genes. The cell pellet (harvested from 12 liters of culture) was resuspended in 200 ml of Buffer A containing 800 mm NaCl, lysed with a French press, and clarified by centrifugation. The clarified cell lysate was diluted with Buffer A to ∼180 mm NaCl before being applied to a 100-ml SP-Sepharose column and eluted with a 1-liter gradient of 150–600 mm NaCl in Buffer A. The peak of RFC(3/4) (which eluted at about 350 mm NaCl) was pooled, diluted with Buffer A to ∼150 mm NaCl, and then applied to a 50-ml (Amersham Biosciences) column followed by a gradient of 150–600 mm NaCl in Buffer A. The peak fractions of RFC(3/4) eluted at ∼300 mm NaCl and were stored at -70 °C. The final for RFC(3/4) were mg of 4 mg of and 15 mg of liter of cell culture. Purification of S. cerevisiae RFC(2/3) RFC(2/3) protein complex was expressed by co-transformation of pLANT and The clarified cell lysate was diluted to 150 mm NaCl with Buffer A and then purified a SP-Sepharose Fast Flow column with a gradient of mm NaCl in Buffer A. The peak of RFC(2/3) was pooled and diluted with Buffer A to mm NaCl before being purified a Q-Sepharose Fast Flow column with a gradient of mm NaCl in Buffer A. The purified protein was Buffer A containing mm NaCl before being stored at -70 °C. The yield was ∼1 mg RFC(2/3) liter of cell culture. Purification of S. cerevisiae was upon co-transformation of pLANT and pET(11a)-RFC(5). The for this is the same as for the RFC(2/3) complex to that a 150–600 mm NaCl gradient in Buffer A was used with the Fast Flow and a mm NaCl gradient in Buffer A was used with the Q-Sepharose Fast Flow was also ∼1 mg of liter of cell culture. Purification of S. cerevisiae RFC2 subunit, RFC2, was expressed from the The cells were lysed and then the lysate was clarified and diluted to mm NaCl as for RFC complex The protein was first a 100-ml SP-Sepharose Fast Flow column with a 1-liter gradient of 150–600 mm NaCl in Buffer A. The peak of RFC2 (which eluted at about mm NaCl) was pooled and diluted with Buffer A to ∼200 mm NaCl, before being applied to a 50-ml Q-Sepharose Fast Flow The containing RFC2 was by of centrifugation, the pellet was resuspended in Buffer A and Buffer A containing mm NaCl at 4 °C The yield of protein was mg of RFC2 liter of cell culture. containing activity were from the protein by of RFC2 protein was applied to a (Amersham Biosciences) column equilibrated with Buffer A containing 200 mm NaCl. fractions were from other were for activity to and from the same fractions were on a 10% SDS-polyacrylamide activity was in fractions RFC2 protein was in fractions RFC2 from activity was used in the in this Purification of S. cerevisiae RFC4 of RFC(3/4) from the pET(11a)-RFC[3+4] plasmid results in the of RFC4 cell lysate was diluted to mm NaCl with Buffer A before being an SP-Sepharose Fast Flow column with a gradient of mm NaCl in Buffer A. containing both RFC(3/4) and RFC4 were pooled and for 2 Buffer A until the was to mm NaCl. The protein was then applied to a Q-Sepharose Fast Flow column and with ml of Buffer A. RFC4 eluted in the was ∼1 mg RFC4 liter of cell culture. Wild-type and mutant RFC(3/4) subcomplexes were for activity in the or absence of a primed The primed template was by the following two oligonucleotides: TAG CAT CTT CCC GAA TTC CGT CGT TTT ACA ACG TCG TGA CTG AAA CCC CGT and TTT CAG TCA CGT TGT AAA ACG ACG GCC GAA The and were in 150 mm NaCl, and 15 mm The was to °C and then to a The 2 2 mm primed template and 2 RFC2 in a final of of Buffer C. were incubated at 30 and were at to 4 and with an of mm EDTA of each was on a and in 1 m m the was and were using a (Amersham Biosciences) and the was performed at 4 °C using a column (Amersham Biosciences) equilibrated in Buffer B. concentrations in the were incubated at °C for 15 in Buffer B 1 ATP, 8 mm MgCl2, and PCNA were applied to the and the first ml fractions of were fractions were in SDS-polyacrylamide gels stained with Coomassie Blue. serum albumin was to the protein to serve as an for each The were to the which was at peak were performed using a were from to using an of The for and was 2 and 4, were in 10 mm Tris-HCl (pH 7.5), 5 mm DTT, 1 mm EDTA, 8 mm MgCl2, 1 mm ATP, and 150 mm NaCl, as PCNA at its two residues and with were performed at 200 or 1 and of RFCwt or The was by a DNA to as (2.Stukenberg P.T. Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1991; 266: 11328-11334Abstract Full Text PDF PubMed Google Scholar). The subunit of δ used in this was a of the it the N-terminal (from a of in the and an The gene for this subunit of δ was cloned into a with the other two subunits of δ cell lysate was a Q-Sepharose Fast Flow The activity the of both PCNA and RFC and a activity of of protein of primed of E. coli 60 deoxyribonucleoside triphosphates and 20 in of Buffer E. of RFC and PCNA from to were incubated at 30 °C for 5 and then 1 of δ was to 5 at 30 the was with an of 40 mm and then onto followed by of DNA synthesis by as (2.Stukenberg P.T. Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1991; 266: 11328-11334Abstract Full Text PDF PubMed Google Scholar). We a expression plasmid for of proteins in E. coli (26.Finkelstein J. Antony E. Hingorani M.M. O'Donnell M. Anal. Biochem. 2003; 319: 78-87Crossref PubMed Scopus (59) Google Scholar). In that study we the expression and of RFC in which an N-terminal of RFC1 is In the study we and RFC in which subunits are and The study that expression of RFC subunits only In the study we two subunits at a and find that three RFC subcomplexes are and can be In of an RFC(3/4) complex, RFC4 is also from one of the Moreover, we find that RFC2 expression results in RFC2 that and can be purified as an subunit. In the to these subunit are used to the of RFC and its with PCNA. in on the E. coli γ complex circular the five subunits of RFC a