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

2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês

10.1074/jbc.m309206200

ISSN

1083-351X

Autores

Nina Y. Yao, Lee Coryell, Dan Zhang, Roxana E. Georgescu, Jeff Finkelstein, Maria Magdalena Coman, Manju Hingorani, Mike O’Donnell,

Tópico(s)

RNA and protein synthesis mechanisms

Resumo

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 Heparin-agarose (Amersham Biosciences) column followed by a 500-ml elution gradient of 150–600 mm NaCl in Buffer A. The peak fractions of RFC(3/4) subcomplex eluted at ∼300 mm NaCl and were stored at -70 °C. The final yields for RFC(3/4) were ∼4 mg of RFC(3/4)wt, 4 mg of RFC(3/4SAC), and 15 mg of RFC(3SAC/4) per liter of cell culture. Purification of S. cerevisiae RFC(2/3) Subcomplex—The RFC(2/3) protein complex was expressed by co-transformation of pLANT (2)RFC(2) and pET(11a)-RFC(3). The clarified cell lysate was diluted to 150 mm NaCl with Buffer A and then purified over a 20-ml SP-Sepharose Fast Flow column with a 200-ml gradient of 300–700 mm NaCl in Buffer A. The peak of RFC(2/3) was pooled and diluted with Buffer A to 100 mm NaCl before being further purified over a 6-ml Q-Sepharose Fast Flow column with a 60-ml gradient of 200–400 mm NaCl in Buffer A. The purified protein was dialyzed against Buffer A containing 100 mm NaCl before being stored at -70 °C. The yield was ∼1 mg RFC(2/3) per liter of cell culture. Purification of S. cerevisiae RFC(2/5) Subcomplex—The RFC(2/5) subcomplex was coexpressed upon co-transformation of pLANT (2)RFC(2) and pET(11a)-RFC(5). The purification protocol for this subcomplex is the same as for the RFC(2/3) complex (refer to above), except that a 150–600 mm NaCl gradient in Buffer A was used with the SPSepharose Fast Flow column, and a 100–450 mm NaCl gradient in Buffer A was used with the Q-Sepharose Fast Flow column. Yield was also ∼1 mg of RFC(2/5) per liter of cell culture. Purification of S. cerevisiae RFC2 Protein—The individual subunit, RFC2, was expressed from the pET(11a)-RFC2 plasmid. The cells were lysed and then the lysate was clarified and diluted to 180 mm NaCl as described above for RFC complex purification. The protein was first fractionated over 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 300 mm NaCl) was pooled and diluted with Buffer A to ∼200 mm NaCl, before being applied to a 50-ml Q-Sepharose Fast Flow column. The flow through fraction containing RFC2 was precipitated by addition of ammonium sulfate (0.5 g/ml). After centrifugation, the pellet was resuspended in Buffer A and dialyzed against Buffer A containing 250 mm NaCl at 4 °C overnight. The yield of protein was ∼4 mg of RFC2 per liter of cell culture. Trace contaminants containing ATPase activity were separated away from the RFC(2) protein by gel filtration. Approximately, 700 μg of RFC2 protein was applied to a 24-ml Superdex-75 (Amersham Biosciences) column equilibrated with Buffer A containing 200 mm NaCl. After collecting 240 drops (6.1 ml), 5-drop (120 μl) fractions were collected (79 fractions). Aliquots (3 μl) from every other fraction were assayed for ATPase activity (refer to protocol below), and 16-μl aliquots from the same fractions were analyzed on a 10% SDS-polyacrylamide gel. ATPase activity was detected in fractions 17–27, whereas RFC2 protein was present in fractions 25–37. RFC2 free from contaminating ATPase activity (fractions 29–37) was used in the ATPase assays reported in this study. Purification of S. cerevisiae RFC4 Protein—Expression of RFC(3/4) subcomplex from the pET(11a)-RFC[3+4] plasmid results in the production of excess soluble RFC4 protein. Clarified cell lysate was diluted to ∼100 mm NaCl with Buffer A before being fractionated over an 18-ml SP-Sepharose Fast Flow column with a 180-ml gradient of 100–500 mm NaCl in Buffer A. Fractions containing both RFC(3/4) and excess RFC4 were pooled and dialyzed for 2 h against Buffer A until the conductivity was approximately equal to 50 mm NaCl. The protein was then applied to a 7-ml Q-Sepharose Fast Flow column and washed with 50 ml of Buffer A. Pure RFC4 eluted in the wash. Yield was ∼1 mg RFC4 per liter of cell culture. Wild-type and mutant RFC(3/4) subcomplexes were tested for ATPase activity in the presence or absence of a synthetic primed template. The primed template was formed by mixing the following two oligonucleotides: 79-mer, 5′-GGG TAG CAT ATG CTT CCC GAA TTC ACT GGC CGT CGT TTT ACA ACG TCG TGA CTG GGA AAA CCC TGG CGT TAC CCA ACT T-3′; and 45-mer, 5′-GGG TTT TCC CAG TCA CGA CGT TGT AAA ACG ACG GCC AGT GAA TTC-3′. The 79-mer (800 pmols) and 45-mer (2.4 nmol) were mixed in 100 μlof5mm Tris-HCl, 150 mm NaCl, and 15 mm sodium citrate (final pH 8.5). The mixture was brought to 95 °C and then cooled to room temperature over a 30-min interval. The ATPase reactions contained 2 μm RFC(3/4), 2 mm [α-32P]ATP, 500 nm primed template (when present), and 2 μm RFC2 (when present) in a final volume of 70 μl of Buffer C. Reactions were incubated at 30 °C, and 10-μl aliquots were removed at intervals (0 to 4 min) and quenched with an equal volume of 5% SDS/40 mm EDTA mixture. One microliter of each quenched reaction was spotted on a polyethyleneimine cellulose TLC sheet (EM Science) and developed in 1 m formic acid/0.5 m lithium chloride buffer. After the TLC sheet was dried, [α-32P]ATP and [α-32P]ADP were quantitated using a PhosphorImager (Amersham Biosciences) and the ImageQuant software (Molecular Dynamics). Gel filtration was performed at 4 °C using a 24-ml Superdex-200 column (Amersham Biosciences) equilibrated in Buffer B. Subunit mixtures (protein concentrations indicated in the figure legends) were incubated at 16 °C for 15 min in 250 μl Buffer B and, when present, included 1 μm ATP, 8 mm MgCl2, and 6.5 μm PCNA (as trimer). Subunit mixtures were applied to the column, and after collecting the first 5.6 ml (void volume), fractions of 180 μl were collected. Column fractions were analyzed in 7.5% SDS-polyacrylamide gels stained with Coomassie Blue. Bovine serum albumin (Sigma) (66 kDa) was added to the protein mixtures to serve as an internal molecular mass marker for each gel filtration analysis. The fraction numbers were normalized to the 66-kDa standard, which was set at peak fraction 49. Steady-state fluorescence intensity measurements were performed using a PTI spectrofluorimeter (Photon Technology International). Fluorescence emission spectra were obtained from 500 to 600 nm using an excitation wavelength of 490 nm. The band-pass for excitation and emission was 2 and 4, respectively. All samples 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 indicated. Fluorescent measurements utilized PCNA labeled at its two exposed Cys residues (22 and 62) with Oregon Green 488 maleimide (Molecular Probes). Measurements were performed at 100 nm, 200 nm, or 1 μm PCNA3 and increasing amounts of RFCwt or RFC1mut. The primer-template was prepared by annealing a synthetic DNA 30-mer oligonucleotide (M13mp18 map position 6815–6847) to M13mp18 ssDNA as described (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 large subunit of Pol δ used in this assay was a truncated version of the full-length protein; it contained the N-terminal 807 amino acids (from a total of 1098 amino acids in the full-length Pol δ) and an additional 14 amino acids (LRDPLIISPKRNHV). The gene for this subunit of Pol δ was cloned into a pET(11a) vector, along with the other two subunits of Pol δ holoenzyme. Induced cell lysate was fractionated over a Q-Sepharose Fast Flow column. The enzyme activity required the presence of both PCNA and yeast RFC and had a specific activity of ∼1.0 fmol dNTP incorporated per μg of protein per min. Assays contained 14.1 fmol of primed ssDNA, 3.55 pmol of E. coli SSB, 60 μm deoxyribonucleoside triphosphates (dGTP, dCTP, dATP), and 20 μm [α-32P]TTP in 12.5 μl of Buffer E. First, 50 fmol of RFC and PCNA (ranging from 25 fmol to 2.5 pmol) were incubated together at 30 °C for 5 min and then 1 μl of recombinant yeast Pol δ was added to initiate replication. After 5 min at 30 °C, the reaction was quenched with an equal volume of 40 mm EDTA/1% SDS and then spotted onto DE81 paper followed by quantitation of DNA synthesis by scintillation counting as described (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 have reported previously a compatible two-vector expression plasmid system for coexpression of multiple 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 described the expression and purification of five-subunit yeast RFC in which an unessential N-terminal region of RFC1 is deleted. In the current study we express and purify RFC in which all subunits are full-length and unmodified. The earlier study indicated that expression of individual RFC subunits provided only insoluble protein. In the current study we coexpress two subunits at a time and find that three heterodimeric RFC subcomplexes are soluble and can be purified. In fact, during preparation of an RFC(3/4) complex, isolated RFC4 is also obtained from one of the purification steps. Moreover, we find that RFC2 expression results in some RFC2 that remains soluble and can be purified as an isolated subunit. In the experiments to follow, these subunit preparations are used to examine the architecture of RFC and its interactions with PCNA. Subunit-Subunit Connections in RFC—Based on the E. coli γ complex circular pentamer, the five subunits of RFC a

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