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

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 50, Issue of December 12, pp. 50744–50753, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Replication Factor C Clamp Loader Subunit Arrangement within the Circular Pentamer and Its Attachment Points to Proliferating Cell Nuclear Antigen*

Received for publication, August 19, 2003, and in revised form, October 1, 2003 Published, JBC Papers in Press, October 6, 2003, DOI 10.1074/jbc.M309206200

Nina Yao‡, Lee Coryell§, Dan Zhang‡, Roxana E. Georgescu‡, Jeff Finkelstein¶, Maria M. Coman§, Manju M. Hingorani§, and Mike O’Donnell‡¶ʈ From ‡The Rockefeller University and ¶The Laboratory of DNA Replication, Howard Hughes Medical Institute, New York, New York 10021 and §Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, Connecticut 06459

Replication factor C (RFC) is a heteropentameric AAA؉ between the two (4, 5). These ring-shaped proteins require an

protein clamp loader of the proliferating cell nuclear an- ATP-fueled multiprotein clamp loader for assembly onto Downloaded from tigen (PCNA) processivity factor. The prokaryotic homo- primed DNA. logue, ␥ complex, is also a heteropentamer, and structural The eukaryotic clamp loader is the heteropentameric repli- studies show the subunits are arranged in a circle. In this cation factor C (RFC). The five subunits of RFC are each report, Saccharomyces cerevisiae RFC protomers are ex- different proteins, but they are homologous to one another (6, amined for their interaction with each other and PCNA. 7) and are members of the AAAϩ family of ATPases (8). The The data lead to a model of subunit order around the recent crystal structure of E. coli ␥ complex, the prokaryotic http://www.jbc.org/ circle. A characteristic of AAA؉ oligomers is the use of counterpart of RFC, has facilitated detailed hypothesis regard- bipartite ATP sites in which one subunit supplies a cata- ing RFC structure and mechanism (9, 10). The ␥ complex lytic arginine residue for hydrolysis of ATP bound to the (␥ ␦␦Ј␹␺) consists of a minimal core of five proteins (␥ ␦␦Ј), neighboring subunit. We find that the RFC(3/4) complex is 3 3 a DNA-dependent ATPase, and we use this activity to which contain the clamp loading activity (11). The remaining ␹ ␺ determine that RFC3 supplies a catalytic arginine to the two subunits, and , are involved in recruiting an RNA at Rockefeller University Library on August 2, 2015 ATP site of RFC4. This information, combined with the primed DNA site from the primase, and they bind single- subunit arrangement, defines the composition of the re- stranded DNA-binding protein (SSB) to assist polymerase elon- maining ATP sites. Furthermore, the RFC(2/3) and gation but are not essential to the clamp loading activity of ␥ RFC(3/4) subassemblies bind stably to PCNA, yet neither complex (12–14). Biochemical studies of ␥ complex (15–17), ␥ ␦␦Ј ␦ ␤ RFC2 nor RFC4 bind tightly to PCNA, indicating that combined with crystal structures of 3 (10) and - 1 complex ␥ ␦␦Ј RFC3 forms a strong contact point to PCNA. The RFC1 (18, 19), reveal a highly detailed view of 3 clamp loader subunit also binds PCNA tightly, and we identify two ␥ ␦␦Ј form and function. The five subunits of the 3 complex are hydrophobic residues in RFC1 that are important for this ␥ arranged in a circular formation (see Fig. 1). Like RFC, the 3, interaction. Therefore, at least two subunits in RFC make ␦, and ␦Ј subunits are members of the AAAϩ family; they share ␥ strong contacts with PCNA, unlike the Escherichia coli a characteristic chain-folding pattern consisting of three do- complex in which only one subunit makes strong contact mains each. The main intersubunit contacts in the heteropen- with the ␤ clamp. Multiple strong contact points to PCNA tamer are made via the C-terminal domains, which form a tight may reflect the extra demands of loading the PCNA trim- circular collar (Fig. 1A, top view). The N-terminal domains eric ring onto DNA compared with the dimeric ␤ ring. contain the ATP binding sites, and there is a gap between the N-terminal domains of the ␦ and ␦Ј subunits (Fig. 1A, front ␥ Replicases of cellular chromosomes utilize a circular sliding view). Only the subunits contain ATP binding and hydrolysis clamp protein that encircles DNA and tethers the polymerase activity and therefore serve as the motor of this machine; both ␦ ␦Ј to the template for high processivity in DNA synthesis (1). An and lack a consensus ATP binding sequence, and neither of ␥ ␦Ј example of this protein class is the Escherichia coli ␤ subunit, them bind ATP. The and subunits contain an SRC motif ␥ which confers high processivity onto the chromosomal repli- that is highly conserved from complex to RFC (20). The case, DNA polymerase III holoenzyme (2, 3). The eukaryotic arginine residue (Arg finger) within the SRC motif is positioned equivalent is the PCNA1 ring, which has essentially the same such that it may participate in hydrolysis of ATP bound to the shape and chain fold as ␤ despite lack of sequence similarity neighboring subunit (Fig. 1A, back view). Hence, the Arg finger ␦Ј ␥ within the SRC motif functions with ATP bound to 1 (site 1), the ␥ SRC functions with ATP bound to ␥ (site 2), and the ␥ * This work was supported in part by Howard Hughes Medical Insti- 1 2 2 ␥ tute and National Institutes of Health Grants GM38839 (to M. O’D.) SRC functions with ATP bound to 3 (site 3) (see Fig. 1B). and GM64514–01 (to M. M. H.). The costs of publication of this article Biochemical studies confirm that these conserved Arg residues were defrayed in part by the payment of page charges. This article must are catalytic and suggest that ATP must first be hydrolyzed in therefore be hereby marked “advertisement” in accordance with 18 sites 2 and/or 3 before ATP in site 1 is hydrolyzed (21). U.S.C. Section 1734 solely to indicate this fact. ʈ To whom correspondence should be addressed: The Rockefeller Uni- Study of ␥ complex and its subunits shows that the ␦ subunit versity, 1230 York Ave., Box 228, New York, NY 10021. Tel.: 212-327- forms the strongest attachment to the ␤ clamp, and in fact can 7255; Fax: 212-327-7253; E-mail: [email protected]. open the ring by itself, leading to unloading of ␤ clamps from 1 The abbreviations used are: PCNA, proliferating cell nuclear anti- closed circular DNA (19). The ␥ trimer can also bind ␤ and gen; RFC, replication factor C; SSB, single-stranded DNA-binding protein; DTT, dithiothreitol; wt, wild-type; mut, mutant; ssDNA, single- unload it, but it is feeble in these actions compared with ␦ (15). ␦ ␦ ␦ ␤ ␥ ␤ stranded DNA; Pol , DNA polymerase . Although binds 2 tightly, the complex does not bind 2 in

50744 This paper is available on line at http://www.jbc.org Analysis of RFC Form and Function 50745

appears to be a rigid protein and has been termed the stator, the stationary part of a machine upon which other pieces move (10, 20). Results of mutational studies are consistent with the idea that the ATP-induced conformation change of ␥ complex requires ␦Ј and that it may serve as a “backboard” to direct the ATP-induced changes in ␥ complex (23). The similarities in RFC and ␥ complex subunit sequences and their common function in loading circular clamps onto DNA suggest that the RFC subunits may also be arranged in a ␥ ␦␦Ј circular fashion like 3 (10). Electron microscopy and atomic force microscopy studies of RFC are consistent with a circular arrangement of RFC subunits (9, 24). The human RFC1 subunit (p140), like ␦, binds to PCNA (25), leading to the proposal that RFC1 may act to open PCNA just as the ␦ wrench opens ␤ (9, 18). RFC5, like ␦Ј, 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 ␦Ј ␥ ␦␦Ј Downloaded from may play a similar role as the stator. On the basis of the 3 structure, it is proposed that the RFC1 (wrench) and RFC5 (stator) subunits may bracket the RFC2, 3, and 4, subunits, ␥ which, like 3, contain both ATP binding and SRC motifs and thus may act as the motor of the RFC clamp loader (9). How- ever, the order of the RFC2, 3, and 4 subunits within the http://www.jbc.org/ 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 at Rockefeller University Library on August 2, 2015 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 ␦ subunit of ␥ complex and the ␤ clamp.

EXPERIMENTAL PROCEDURES Materials Radioactive nucleotides were purchased from PerkinElmer Life Sci- ences. Unlabeled deoxyribonucleoside triphosphates were supplied by Amersham Biosciences. DNA modification enzymes were supplied by New England Biolabs; DNA oligonucleotides were from Integrated DNA FIG.1.Subunit arrangement and structural features of ␥ com- Technologies. Protein concentrations were determined using the Bio- plex, the E. coli homolog of RFC. A, crystal structure of ␥ complex. Rad Protein stain and bovine serum albumin as a standard. Buffer A is Top view shows the circular pentameric arrangement of the subunits 30 mM HEPES (pH 7.5), 10% (v/v) glycerol, 0.5 mM EDTA (pH 7.5), 1 mM looking down (i.e. perpendicular to) the plane defined by the C-terminal DTT, and 0.04% Bio-Lyte 3/10 ampholyte (Bio-Rad). Buffer B is 20 mM ␦ domains. Side view shows the helix (gray) of the subunit that interacts HEPES (pH 7.4), 2 mM DTT, 10% glycerol, and 200 mM NaCl. Buffer C with the ␤ clamp. The C-terminal domains of the subunits are located is 20 mM Tris-HCl (pH 7.5), 0.5 mM DTT, 10 mM magnesium acetate, at the top, and the N-terminal domains are at the bottom of this view. and 60 mM NaCl. Buffer D is 20 mM Tris-HCl (pH 7.5), 5 mM DTT, 0.1 Back view is a 180° pivot around the vertical axis of the side view. This M EDTA, 40 ␮g/ml bovine serum albumin, 8 mM MgCl , 4% glycerol, and view shows the P-loops (yellow) and arginine fingers (white) located at 2 the interface between subunits. The elements that are labeled in the 0.5 mM ATP. figure are those of ␥ (Arg finger) and ␥ (P-loop) defining ATP site 2. 1 2 Plasmids The same elements are also present in ATP sites 1 and 3. B, schematic diagram illustrating the polarity of the P-loops and arginine fingers The Saccharomyces cerevisiae RFC genes were cloned into either pET forming the three ATPase sites in E. coli ␥ complex. This diagram (Novagen) or pLANT (26) vectors. The plasmids containing single genes corresponds to the top view looking down onto the C-terminal face of ␥ include pET(11a)-RFC(1), pET(11a)-RFC(2), pLANT (2)-RFC(2), pET(11a)- complex. In this view, the Arg finger of one subunit points clockwise RFC(3), pET(11a)-RFC(4), and pET(11a)-RFC(5). Expression plasmids con- toward the P-loop of the adjacent subunit to form a hydrolysis-compe- taining two or more genes include pET(11a)-RFC[3ϩ4], pLANT (2)- tent ATP site. RFC[1ϩ5], pET(11a)-RFC[2ϩ3ϩ4], and pET(11a)-RFC[1ϩ2ϩ3ϩ4ϩ5]. Mutations in the RFC1 Gene—Codons TAT and TTC in pET(11a)- the absence of ATP, indicating that one or more subunits of ␥ RFC(1) corresponding to residues Tyr-404 and Phe-405 in RFC1 were ␦ ␤ ␥ mutated to GCT and GCC, respectively, by Commonwealth Biotechnol- complex block the -to- 2 interaction (22). ATP binding to the ␥ ␤ ogies, changing both hydrophobic residues to alanine. The mutated subunits promotes tight interaction between complex and , ϩ ␥ RFC(1) gene was then cloned into the pLANT (2)-RFC[1 5] to replace implying that ATP binding induces a conformation change in the wild-type copy of the RFC(1) gene. complex that exposes ␦, and presumably ␥ subunits as well, for Mutations in the RFC3 and RFC4 Genes—Mutations were intro- ␤ ␤ ␦Ј interaction with and opening of the 2 ring. The subunit duced into the RFC(3) and RFC(4) genes using the QuikChange method 50746 Analysis of RFC Form and Function

(Stratagene). RFC[3SAC] was generated in pET(11a)-RFC(3) using the 100 mM NaCl before being further purified over a 6-ml Q-Sepharose following oligonucleotides: 5Ј-CAT AAA CTT ACA CT GCG TTA TTG Fast Flow column with a 60-ml gradient of 200–400 mM NaCl in Buffer AGC GCT TGC ACG AGA TTC AGA TTT CAG CCC TTG-3Ј and 5Ј-CAA A. The purified protein was dialyzed against Buffer A containing 100 GGG CTG AAA TCT GAA TCT CGT GCA AGC GCT CAA TAA CGC mM NaCl before being stored at Ϫ70 °C. The yield was ϳ1 mg RFC(2/3) AGG TGT AAG TTT ATG-3Ј. RFC[4SAC] was generated in pET(11a)- per liter of cell culture. RFC(4) using the following oligonucleotides: 5Ј-CAA GAT CAT TGA Purification of S. cerevisiae RFC(2/5) Subcomplex—The RFC(2/5) GCC GCT GCA AAG CGC TTG TGC GAT TTT GAG GTA TTC TAA subcomplex was coexpressed upon co-transformation of pLANT (2)- AC-3Ј and 5Ј-GTT TAG AAT ACC TCA AAA TCG CAC AAG CG TTT RFC(2) and pET(11a)-RFC(5). The purification protocol for this subcom- GCA GCG GCT CAA TGA TCT TG-3Ј. The entire open reading frames plex is the same as for the RFC(2/3) complex (refer to above), except that were then confirmed by DNA sequencing. a 150–600 mM NaCl gradient in Buffer A was used with the SP- Mutant genes were cloned into pET(11a)-RFC[3ϩ4] as follows: the Sepharose Fast Flow column, and a 100–450 mM NaCl gradient in RFC[3SAC] gene was exchanged for the wild-type RFC(3) gene in the Buffer A was used with the Q-Sepharose Fast Flow column. Yield was pET(11a)-RFC[3ϩ4] plasmid using KpnI. Recombinants were screened also ϳ1 mg of RFC(2/5) per liter of cell culture. with Afe and MfeI/ApaI restriction digest was performed to confirm the Purification of S. cerevisiae RFC2 Protein—The individual subunit, proper orientation of the insert. The RFC[4SAC] gene was exchanged RFC2, was expressed from the pET(11a)-RFC2 plasmid. The cells were for the wild-type RFC(4) gene in the pET(11a)-RFC[3ϩ4] plasmid using lysed and then the lysate was clarified and diluted to 180 mM NaCl as AflII/GstBI; recombinants were screened using AfeI. described above for RFC complex purification. The protein was first fractionated over a 100-ml SP-Sepharose Fast Flow column with a Over-expression of S. cerevisiae RFC Proteins in E. coli 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 The pET(11a)-RFC derived expression plasmids were transformed Buffer A to ϳ200 mM NaCl, before being applied to a 50-ml Q-Sepharose

into BL21(DE3) codon plus (Stratagene) E. coli cells, which contain a Downloaded from Fast Flow column. The flow through fraction containing RFC2 was secondary plasmid encoding genes for rare transfer RNAs, as well as precipitated by addition of ammonium sulfate (0.5 g/ml). After centrif- chloramphenicol resistance. Transformants were selected using ampi- ugation, the pellet was resuspended in Buffer A and dialyzed against cillin (100 ␮g/ml) and chloramphenicol (25 ␮g/ml). Co-transformations Buffer A containing 250 mM NaCl at 4 °C overnight. The yield of protein involving pLANT (2) and pET(11a)–derived plasmids were performed was ϳ4 mg of RFC2 per liter of cell culture. Trace contaminants con- with BLR(DE3) (Novagen) E. coli-competent cells. These transformants taining ATPase activity were separated away from the RFC(2) protein were selected using ampicillin (100 ␮g/ml) and kanamycin (50 ␮g/ml). by gel filtration. Approximately, 700 ␮g of RFC2 protein was applied to Fresh transformants were grown in 12–24 liters of LB media containing http://www.jbc.org/ a 24-ml Superdex-75 (Amersham Biosciences) column equilibrated with the appropriate antibiotics at 30 °C until reaching an A value of 0.6. 600 Buffer A containing 200 mM NaCl. After collecting 240 drops (6.1 ml), The cultures were then briefly chilled on ice before adding 1 mM iso- 5-drop (120 ␮l) fractions were collected (79 fractions). Aliquots (3 ␮l) propyl-1-thio-␤-D-galactopyranoside and then were incubated at 15 °C from every other fraction were assayed for ATPase activity (refer to for ϳ18 h. For RFC(2/3) and RFC(2/5), induction was at 37 °Cfor3h. protocol below), and 16-␮l aliquots from the same fractions were ana- The cells were harvested by centrifugation. lyzed on a 10% SDS-polyacrylamide gel. ATPase activity was detected in fractions 17–27, whereas RFC2 protein was present in fractions Purification of S. cerevisiae RFC Complexes, 25–37. RFC2 free from contaminating ATPase activity (fractions 29– at Rockefeller University Library on August 2, 2015 Subcomplexes, and Individual Subunits 37) was used in the ATPase assays reported in this study. Purification of S. cerevisiae RFC1mut and RFCwt Complex—RFCwt was Purification of S. cerevisiae RFC4 Protein—Expression of RFC(3/4) overexpressed from the single plasmid, pET(11a)-RFC[1ϩ2ϩ3ϩ4ϩ5]. The subcomplex from the pET(11a)-RFC[3ϩ4] plasmid results in the pro- RFC1mut was expressed by co-transformation of pLANT (2)-RFC[1mutϩ5] duction of excess soluble RFC4 protein. Clarified cell lysate was diluted and pET(11a)-RFC[2ϩ3ϩ4]. The harvested cells (from 24 liters of culture) to ϳ100 mM NaCl with Buffer A before being fractionated over an 18-ml were resuspended in ϳ200 ml of Buffer A containing 1 M NaCl and then SP-Sepharose Fast Flow column with a 180-ml gradient of 100–500 mM lysed using a French press at 22,000 p.s.i. The cell lysate was clarified by NaCl in Buffer A. Fractions containing both RFC(3/4) and excess RFC4 centrifugation, diluted with Buffer A to ϳ150 mM NaCl, and then applied to were pooled and dialyzed for 2 h against Buffer A until the conductivity a 30-ml SP-Sepharose Fast Flow (Amersham Biosciences) column equili- was approximately equal to 50 mM NaCl. The protein was then applied brated with Buffer A containing 150 mM NaCl. The column was eluted with to a 7-ml Q-Sepharose Fast Flow column and washed with 50 ml of a 300-ml gradient of 150–600 mM NaCl in Buffer A. Presence of proteins Buffer A. Pure RFC4 eluted in the wash. Yield was ϳ1 mg RFC4 per was followed in 10% SDS-polyacrylamide gels stained with Coomassie Blue. liter of cell culture. The peak of RFC (which eluted at ϳ365 mM NaCl) was pooled and diluted with Buffer A to ϳ150 mM NaCl. The protein was then applied to a 40 ml ATPase Assays Q-Sepharose Fast Flow (Amersham Biosciences) column equilibrated with Wild-type and mutant RFC(3/4) subcomplexes were tested for Buffer A containing 150 mM NaCl and eluted with a 400-ml gradient of ATPase activity in the presence or absence of a synthetic primed tem- 150–600 mM NaCl in Buffer A. The fractions containing RFC complex plate. The primed template was formed by mixing the following two (which eluted at ϳ300 mM NaCl) were saved individually and stored at oligonucleotides: 79-mer, 5Ј-GGG TAG CAT ATG CTT CCC GAA TTC Ϫ70 °C. Protein concentration was measured by Bradford assay, and a ACT GGC CGT CGT TTT ACA ACG TCG TGA CTG GGA AAA CCC typical final yield was ϳ1 mg purified RFC complex/1 liter of culture. TGG CGT TAC CCA ACT T-3Ј; and 45-mer, 5Ј-GGG TTT TCC CAG Purification of S. cerevisiae Wild-type and Mutant RFC(3/4) Subcom- TCA CGA CGT TGT AAA ACG ACG GCC AGT GAA TTC-3Ј. The plexes—RFC(3/4) subcomplexes (wild-type or SAC mutants) were ex- 79-mer (800 pmols) and 45-mer (2.4 nmol) were mixed in 100 ␮lof5mM pressed using the pET(11a)-RFC[3ϩ4] plasmid containing either wild- Tris-HCl, 150 mM NaCl, and 15 mM sodium citrate (final pH 8.5). The type or mutant genes. The cell pellet (harvested from 12 liters of mixture was brought to 95 °C and then cooled to room temperature over culture) was resuspended in 200 ml of Buffer A containing 800 mM a 30-min interval. The ATPase reactions contained 2 ␮M RFC(3/4), 2 mM NaCl, lysed with a French press, and clarified by centrifugation. The [␣-32P]ATP, 500 nM primed template (when present), and 2 ␮M RFC2 clarified cell lysate was diluted with Buffer A to ϳ180 mM NaCl before (when present) in a final volume of 70 ␮l of Buffer C. Reactions were being applied to a 100-ml SP-Sepharose column and eluted with a incubated at 30 °C, and 10-␮l aliquots were removed at intervals (0 to 1-liter gradient of 150–600 mM NaCl in Buffer A. The peak of RFC(3/4) 4 min) and quenched with an equal volume of 5% SDS/40 mM EDTA (which eluted at about 350 mM NaCl) was pooled, diluted with Buffer A mixture. One microliter of each quenched reaction was spotted on a to ϳ150 mM NaCl, and then applied to a 50-ml Heparin-agarose (Am- polyethyleneimine cellulose TLC sheet (EM Science) and developed in 1 ersham Biosciences) column followed by a 500-ml elution gradient of M formic acid/0.5 M lithium chloride buffer. After the TLC sheet was 150–600 mM NaCl in Buffer A. The peak fractions of RFC(3/4) subcom- dried, [␣-32P]ATP and [␣-32P]ADP were quantitated using a Phospho- plex eluted at ϳ300 mM NaCl and were stored at Ϫ70 °C. The final rImager (Amersham Biosciences) and the ImageQuant software (Mo- yields for RFC(3/4) were ϳ4 mg of RFC(3/4)wt, 4 mg of RFC(3/4SAC), and lecular Dynamics). 15 mg of RFC(3SAC/4) per liter of cell culture. Purification of S. cerevisiae RFC(2/3) Subcomplex—The RFC(2/3) Gel Filtration Analysis protein complex was expressed by co-transformation of pLANT (2)- RFC(2) and pET(11a)-RFC(3). The clarified cell lysate was diluted to Gel filtration was performed at 4 °C using a 24-ml Superdex-200 150 mM NaCl with Buffer A and then purified over a 20-ml SP-Sepha- column (Amersham Biosciences) equilibrated in Buffer B. Subunit mix- rose Fast Flow column with a 200-ml gradient of 300–700 mM NaCl in tures (protein concentrations indicated in the figure legends) were Buffer A. The peak of RFC(2/3) was pooled and diluted with Buffer A to incubated at 16 °C for 15 min in 250 ␮l Buffer B and, when present, Analysis of RFC Form and Function 50747 ␮ ␮ included 1 M ATP, 8 mM MgCl2, and 6.5 M PCNA (as trimer). Subunit mixtures were applied to the column, and after collecting the first 5.6 ml (void volume), fractions of 180 ␮l were collected. Column fractions were analyzed in 7.5% SDS-polyacrylamide gels stained with Coomas- sie 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 Measurements Steady-state fluorescence intensity measurements were performed using a PTI spectrofluorimeter (Photon Technology International). Flu- orescence 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 MgCl2,1mM 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 ␮ wt nM, 200 nM,or1 M PCNA3 and increasing amounts of RFC or 1mut RFC . FIG.2. RFC complex, RFC subassemblies, and isolated RFC subunits used in this study. RFC complexes and individual subunits Downloaded from Replication Assays were expressed and purified as described under “Experimental Proce- ϳ ␮ The primer-template was prepared by annealing a synthetic DNA dures.” Gel analysis of the purified proteins was as follows: 0.5–1.0 g 30-mer oligonucleotide (M13mp18 map position 6815–6847) to of each preparation was analyzed in a 7.5% SDS-polyacrylamide gel M13mp18 ssDNA as described (2). The large subunit of Pol ␦ used in stained with Coomassie Blue. The position of each RFC subunit is identified on the right of the gel. this assay was a truncated version of the full-length protein; it con-

tained the N-terminal 807 amino acids (from a total of 1098 amino acids http://www.jbc.org/ in the full-length Pol ␦) and an additional 14 amino acids (LRDPLIIS- which contains a p15A origin and a kanamycin resistance gene. PKRNHV). The gene for this subunit of Pol ␦ was cloned into a pET(11a) The pLANT vector used in this study includes genes encoding vector, along with the other two subunits of Pol ␦ holoenzyme. Induced tRNAs that are prevalent in yeast but underrepresented in cell lysate was fractionated over a Q-Sepharose Fast Flow column. The E. coli (codons AUA (Ile), CUA (Leu), AGG (Arg), and AGA enzyme activity required the presence of both PCNA and yeast RFC and (Arg)) and utilizes the same inducible T7 promoter and cloning had a specific activity of ϳ1.0 fmol dNTP incorporated per ␮g of protein sites of the pET11a vector. The two subunit combinations for

per min. Assays contained 14.1 fmol of primed ssDNA, 3.55 pmol of E. at Rockefeller University Library on August 2, 2015 coli SSB, 60 ␮M deoxyribonucleoside triphosphates (dGTP, dCTP, the five subunits of RFC were tested by placing one gene in dATP), and 20 ␮M [␣-32P]TTP in 12.5 ␮l of Buffer E. First, 50 fmol of pLANT and another in pET. Cells carrying both plasmids were RFC and PCNA (ranging from 25 fmol to 2.5 pmol) were incubated then induced, and the cell lysate was examined for the presence together at 30 °C for 5 min and then 1 ␮l of recombinant yeast Pol ␦ was of soluble heterodimer. Using this approach three combinations added to initiate replication. After 5 min at 30 °C, the reaction was provided soluble proteins, RFC(2/3), RFC(2/5), and RFC(3/4). quenched with an equal volume of 40 mM EDTA/1% SDS and then These findings are supported by an earlier observation that spotted onto DE81 paper followed by quantitation of DNA synthesis by scintillation counting as described (2). coexpression in E. coli of RFC2 and 3 and of RFC3 and 4 appeared to increase their solubility (27). In theory, for a cir- RESULTS cular heteropentamer, there should be five combinations of two We have reported previously a compatible two-vector expres- subunits that are adjacent to one another. Unfortunately, we sion plasmid system for coexpression of multiple proteins in E. observed no soluble complexes containing RFC1, which may coli (26). In that study we described the expression and purifi- account for the two complexes that could not be obtained in cation of five-subunit yeast RFC in which an unessential N- soluble form (i.e. RFC1 with either of its two neighboring terminal region of RFC1 is deleted. In the current study we subunits). express and purify RFC in which all subunits are full-length Next we asked whether the three apparently soluble RFC and unmodified. The earlier study indicated that expression of heterodimers can be purified intact, and if so, whether they individual RFC subunits provided only insoluble protein. In the remain stably associated with each other during analysis in a current study we coexpress two subunits at a time and find that gel filtration sizing column. For each heterodimer, induced three heterodimeric RFC subcomplexes are soluble and can be cells were grown and lysed, and the cell lysate was fractionated purified. In fact, during preparation of an RFC(3/4) complex, using an SP-Sepharose cation exchange column. After this isolated RFC4 is also obtained from one of the purification step, methods for the three preparations diverged, as explained steps. Moreover, we find that RFC2 expression results in some under “Experimental Procedures.” However, we succeeded in RFC2 that remains soluble and can be purified as an isolated obtaining each of the RFC heterodimers in pure form as illus- subunit. In the experiments to follow, these subunit prepara- trated in the SDS-PAGE analysis of Fig. 2 (RFC(2/3), RFC(2/5), tions are used to examine the architecture of RFC and its and RFC(3/4), lanes 3, 4, and 5, respectively). interactions with PCNA. The RFC heterodimers were tested for true stable complex Subunit-Subunit Connections in RFC—Based on the E. coli ␥ formation by asking whether they remain together during complex circular pentamer, the five subunits of RFC are likely analysis in a gel filtration sizing column (Fig. 3). The individual arranged in a similar circular fashion. In the case of a circular RFC2 and RFC4 subunits were also examined by this ap- heteropentamer one may expect that neighboring subunits, proach. Individual RFC2 and RFC4 eluted in fractions 54–58 when expressed together in the absence of other subunits, may and 56–58, respectively, slightly later than the 66-kDa size form heterodimers and thereby identify which subunits are marker, indicating that these subunits are monomeric and next to one another. To test this approach we used two com- consistent with the presence of only one of each these proteins patible T7-based expression vectors described in our earlier in the RFC complex (Fig. 3, A and B). Next the RFC(3/4) study (26). One vector is the commercially available pET11a preparation was analyzed (Fig. 3C). The RFC3 and RFC4 sub- plasmid, which uses a ColE1 origin and contains an ampicillin units comigrated with one another and eluted earlier than resistance gene for selection. The other is the pLANT vector, either RFC2 or RFC4 alone, indicating that the RFC(3/4) sub- 50748 Analysis of RFC Form and Function

to test this hypothesis by co-expression of RFC1 with each of the other subunits, but in no case could we obtain a soluble complex. Nor could we obtain RFC1 alone. We also tested N- and C-terminal truncated versions of RFC1 for solubility and also constructed glutathione S-transferase and maltose-binding protein fusions of RFC1 (and various truncated RFC1 fusion subunits), but what little soluble protein was obtained behaved as an aggregate in gel filtration and did not bind the other subunit preparations. We also tried immobilizing the RFC1 fusion proteins on microtiter plates and on beads but still could not obtain reliable information pertaining to which subunit(s) it binds to. Hence, we were unable to demonstrate directly that RFC1 is adjacent to RFC5 and RFC4. However, protein-protein interaction studies of human RFC have demonstrated that RFC1 (p140) binds to both RFC4 (p40) and

FIG.3.Gel filtration analysis of RFC heterodimers and indi- RFC5 (p38) subunits (28, 29) thus supporting the above posi- vidual subunits. Approximately 12.5 nmol of protein (ϳ500 ␮g of each tion assigned to RFC1. subunit) was analyzed by gel filtration on a Superdex-200 column as ATP Site Polarity within the RFC Pentamer—Given the sub- Downloaded from described under “Experimental Procedures.” Fraction numbers are in- unit arrangement described above for the RFC pentamer, it dicated above the top gel, and each RFC protein is identified on the left of each gel. Panel A, RFC2; panel B, RFC4; panel C, RFC(3/4); panel D, may exist in either of two orientations as illustrated in the RFC(2/3); panel E, RFC(2/5). diagrams of Fig. 4E. These schemes reflect the view looking down the C-terminal plane of the pentamer and result in two very different consequences for how the complex may utilize units indeed exist as a complex. The two subunits stain with

ATP. The location of ATP sites in AAAϩ multimeric machines http://www.jbc.org/ similar intensity suggesting that the complex probably con- are at subunit interfaces and require an Arg residue from the tains one of each subunit. The RFC(2/3) preparation also be- adjacent subunit. In the ␥ complex pentamer, this Arg finger is haved as an equimolar complex except the complex eluted contained within a highly conserved SRC motif in RFC box VII slightly earlier than RFC(3/4) (Fig. 3D). Likewise the RFC(2/5) (10). Only four RFC subunits contain the SRC motif, RFC2, 3, preparation coeluted earlier than either RFC2 or RFC4 indi- 4, and 5 (6). Also, only four subunits contain consensus Walker cating that they form a stable complex, although the two sub- A motifs for ATP hydrolysis, RFC1, 2, 3, and 4. In the subunit at Rockefeller University Library on August 2, 2015 units did not resolve well from one another in the polyacryl- polarity shown in Model 1 of Fig. 4E, the placement of these amide gel because of their very similar mass (Fig. 3E). motifs is maximized so that each ATP binding subunit is placed A Model for Subunit Arrangement in RFC—The fact that clockwise next to an SRC-containing subunit. This arrange- RFC(2/3) and RFC(3/4) form complexes indicates that RFC3 ment could provide four competent ATP hydrolysis sites. The binds to both RFC2 and RFC4. If RFC is a circular pentamer in other subunit polarity, in Model 2 of Fig. 4E, produces only which each subunit has only two neighbors, then the results three competent ATP sites. indicate a subunit order RFC2:RFC3:RFC4 where RFC3 serves as a bridge between RFC2 and RFC4. This arrangement makes To determine the polarity of the RFC pentamer we first two predictions: first that RFC2 will not form a complex with focused on the RFC(3/4) complex and then extended the study RFC4, and second, that RFC2 and RFC(3/4) will form a RFC2/ to the RFC2/(3/4) complex. We find that the RFC(3/4) complex (3/4) heterotrimer. The test of whether RFC2 and RFC4 inter- is an ssDNA stimulated ATPase, as illustrated in Fig. 5A.In act is shown in Fig. 4A. Although the two subunits appear to the experiments to follow, we used this activity to examine the coelute, their elution position in fractions 54–58 is similar to polarity of these two subunits. Two mutant RFC(3/4) complexes their individual elution positions (compare Fig. 3, A and B) were constructed, containing a mutated SRC motif in either indicating that they do not form a complex. Had they bound to RFC3 or RFC4 (R replaced by A and referred to here as SAC). one another the complex should have eluted earlier, as a larger The mutant complexes were then analyzed for ssDNA-depend- molecular weight complex. Next, the mixture of RFC2 with ent ATPase activity. If the subunit polarity is as shown in SAC RFC(3/4) was examined for ability to form an RFC2/(3/4) com- Model 1 of Fig. 4E, the RFC(3 /4) complex should be inactive, SAC plex. The results, in Fig. 4B, show that the RFC2 subunit whereas the RFC(3/4 ) mutant should remain active. If the coelutes with RFC(3/4) several fractions earlier than RFC2 subunits are arranged in the opposite polarity, as in Model 2, alone, indicating formation of the RFC2/(3/4) complex. Hence, then the opposite result should be obtained. The results, in Fig. the predictions listed above hold true and indicate that the 5, B and C, are consistent with the polarity of Model 1 in which subunit order of these three proteins is RFC2:RFC3:RFC4. RFC3 contributes a catalytic Arg to the ATP site of RFC4. The RFC(2/5) complex, taken together with the formation of In Fig. 6 we examine RFC2 for ATPase activity and its affect RFC(2/3) complex, suggests that RFC2 binds to both RFC5 and on the RFC(3/4) complex. We find that RFC2 alone lacks RFC3. Thus RFC4, which is positioned on the other side of ATPase activity Ϯ DNA (data not shown) but provides about RFC3 from RFC2, should not form a complex with RFC(2/5). 4-fold stimulation to the RFC(3/4) ATPase (see Fig. 6A). The The test of this prediction, in Fig. 4C, demonstrates that RFC4 subunit arrangement and polarity of RFC, depicted in Model 1 does not coelute with the RFC(2/5) complex. Taken together of Fig. 4E, makes the prediction that RFC2 contributes a cat- with the formation of a RFC2/(3/4) complex described above, alytic Arg residue to the ATP binding site in RFC3, which may the subunit order of the four RFC subunits is RFC5:RFC2: explain how RFC2 stimulates the RFC(3/4) ATPase activity. RFC3:RFC4. Consistent with this assignment, when the Namely, RFC(3/4) has only one complete ATP site (site 3), and RFC(2/5) and RFC(3/4) complexes are mixed they coelute at a RFC2 generates a second complete ATP site (site 2). In this much earlier position than either of the subcomplexes alone. case, the RFC(3/4SAC) mutant should still be stimulated by Given the arrangement of the RFC5/2/3/4 subunits, and the RFC2 to the same extent as wild-type RFC complex. If RFC2 presumed circular arrangement of the RFC pentamer, the were to bind RFC4, instead of RFC3, addition of RFC2 would RFC1 subunit probably fits between RFC5 and RFC4. We tried not stimulate the activity of the resulting RFC(2/3/4SAC) com- Analysis of RFC Form and Function 50749

FIG.4. Interaction between RFC subunits and subcomplexes. Panels A–D are protein-protein interaction studies of RFC subunit mixtures analyzed by gel filtration. Approximately 1.75 nmol of each subunit or heterodimer were incubated together and then analyzed on a Superdex-200 gel filtration column. Col- umn fraction numbers are indicated above the top gel, and RFC subunits are identified on the left of each gel. E, two possible models of RFC subunit arrangement in the RFC pentamer, clockwise or

counter clockwise, and the corresponding Downloaded from implications for placement of the Arg finger and P-loop, which form the ATPase site at the interface between adjacent subunits. Both diagrams depict the top view looking down onto the C-terminal face of the pentamer (e.g. see Fig. 1A). http://www.jbc.org/ at Rockefeller University Library on August 2, 2015 plex mutant. The results (in Fig. 6B) show that RFC2 indeed stimulates the ATPase activity of the RFC(3/4SAC) mutant, supporting the arrangement of subunits in Model 1. Moreover, if RFC2 provides an arginine finger to the ATP site in RFC3, the inactive RFC(3SAC/4) mutant complex should become ATPase-competent upon addition of RFC2. On the contrary, if the polarity of subunits is as depicted in Model 2, the RFC(3SAC/4) mutant complex should gain no ATPase activity upon addition of RFC2. The results in Fig. 6C uphold the predictions of subunit polarity in Model 1. The level of ATPase in panel C is less than in panels A and B, consistent with only one competent ATP site in the resulting RFC(2/3SAC/4) complex. RFC(3/4) and RFC(2/3) Bind PCNA—RFC, like prokaryotic ␥ complex, binds the clamp in an ATP-dependent fashion (30). ATP binding is sufficient to promote the RFC⅐PCNA complex, hydrolysis is not required. We examined the individual RFC subunits and heterodimers for ability to form a stable complex with PCNA and tested whether the interaction requires ATP. The RFC-PCNA interaction study of Fig. 7 uses gel filtration analysis, which is a non-equilibrium technique, and only the tightest protein complexes will be observed. PCNA was mixed with the various RFC subunit preparations in the presence of ATP and then the mixture was assayed for complex formation. The result shows an interaction between RFC(3/4) and PCNA, and RFC(2/3) and PCNA, but no interaction between PCNA and RFC(2/5), RFC2, or RFC4. It is interesting to note that neither RFC4 nor RFC2 stay attached to PCNA, yet PCNA stably binds to both the RFC(3/4) and RFC(2/3) complexes. Thus RFC3 may form the main contribution between PCNA FIG.5. Identification of the Arg finger and P-loop of the and these heterodimeric complexes. This prediction that RFC3 RFC(3/4) ATPase. The ATPase activity was as follows: panel A, wild- SAC SAC can bind PCNA is supported by studies in the human system type RFC(3/4); panel B, RFC(3 /4); panel C, RFC(3/4 ). ATPase indicating that the p36 subunit (yeast RFC3 homolog) of hu- assays were performed in the presence (closed triangles) and absence (open squares) of a synthetic primed template. The scheme at the top man RFC binds PCNA (31). It is still possible that other RFC illustrates the orientation of the Arg finger and P-loop of the RFC(3/4) subunits bind PCNA but do not form a complex with sufficient ATPase based on the data. 50750 Analysis of RFC Form and Function

OG 1mut OG app ϭ ␮ PCNA reveal that RFC binds PCNA (Kd 3.1 M) app ϭ ␮ about 10-fold weaker than wild-type RFC (Kd 0.34 M), demonstrating that the RFC1 subunit is involved in binding to PCNA. A second experiment was performed to test the above conclusion based on the PCNA-stimulated activity of Pol ␦.In Fig. 8, bottom panel, RFC1mut is examined for activity with PCNA on a singly primed M13mp18 ssDNA template using a recombinant derivative of Pol ␦. The results show that the RFC1mut is significantly less active than wild-type RFC, consistent with RFC1 forming an important contact site between RFC and PCNA.

DISCUSSION RFC Subunit Arrangement and ATP Site Architecture—This study examines the subunit arrangement of the heteropentameric RFC clamp loader using recombinant heterodimeric subassemblies of RFC and individual RFC subunits to obtain information about subunit-subunit interactions within RFC. These protein reagents also made possible the reconstitution of Downloaded from three- and four-subunit RFC subassemblies. The results are all consistent with the presumed circular subunit arrangement, as no one subunit interacted with more than two other subunits. The five RFC subunits have been proposed to be arranged in a circle by analogy to the crystal structure of the circular penta- ␥ ␦␦Ј http://www.jbc.org/ mer E. coli 3 clamp loader (9, 10) and is consistent with electron microscopy and atomic force microscopy studies of FIG.6. RFC2 contributes an Arg finger to the ATP site in RFC (24). The final arrangement of RFC, illustrated in Fig. 9, RFC3. The DNA-stimulated ATPase activity was as follows: panel A, confirms earlier proposals based on homologies between RFC SAC SAC wild-type RFC(3/4); panel B, RFC(3/4 ); and panel C, RFC(3 /4). and ␥ complex (9, 33) and on previous protein-protein interac- ATPase activity was assayed in the presence (closed circles) and absence (closed triangles) of RFC2. Open squares represent ATPase reaction studies of human RFC (28, 29). at Rockefeller University Library on August 2, 2015 tions of RFC(3/4) in the absence of both RFC2 and synthetic primed The ATP sites of AAAϩ multimers are located at subunit template. The scheme at the top of the figure illustrates the conclusion interfaces, and the adjacent subunit contributes a catalytic Arg regarding the orientation of the Arg finger and P-loop of RFC2, RFC3, residue to facilitate ATP hydrolysis. With this in mind, there and RFC4. are two ways to order the five RFC subunits in a circle, clockwise and counter clockwise (i.e. Models 1 and 2 in Fig. 4E). The stability to be isolated by this nonequilibrium technique. two arrangements result in RFC pentamers having either four Study of these same protein mixtures in the absence of ATP competent ATP sites (Model 1) or three competent ATP sites revealed no new complexes with PCNA, and in fact the RFC(3/ (Model 2). The correct arrangement of subunits around the ring 4)⅐PCNA complex was no longer observed (Fig. 7A). Possible was determined by exploiting a finding herein showing that the explanations underlying the ATP dependence of the RFC(3/ RFC(3/4) complex contains DNA-dependent ATPase activity, 4)⅐PCNA complex and ATP-independent RFC(2/3)⅐PCNA com- and this ATPase is stimulated further by RFC2. The catalytic plex are presented under “Discussion.” We lack the RFC1 sub- Arg residue of the SRC motif in either RFC3 or RFC4 was unit in isolation or as a heterodimer with another RFC subunit, mutated to alanine, and the effect of these mutations on the but we presume it also forms a major contact to PCNA based on ATPase activity of the RFC(3/4) complex was determined. The studies of human RFC (25, 32). The next few experiments take clockwise or counter clockwise arrangements of RFC predict a different approach to assess the importance of RFC1 in opposite results, and only one of the two results was experi- RFC⅐PCNA complex formation. mentally observed (i.e. RFC2 contributes an Arg finger to the RFC1 Contributes to PCNA Interaction—We have no subas- ATP site of RFC3, and RFC3 correspondingly contributes an sembly of RFC that contains RFC1. Therefore, to address Arg finger to the ATP site of RFC4). The experimentally deter- whether it contributes to PCNA binding we mutated two hy- mined polarity of the RFC2/3/4 subunits and knowledge of the drophobic residues of RFC1 (Tyr-404 and Phe-405) to alanine positions of RFC subunits clearly revealed Model 1 to be the and expressed it, along with the other subunits, to produce correct structure of RFC complex (i.e. Model 1 of Fig. 4, and as RFC1mut. These two residues are located between the P-loop illustrated in Fig. 9). (Walker A) and DEXX (Walker B) motifs and have been hy- Interaction between RFC and PCNA—RFC1 is involved in a pothesized to form the main contact between RFC1 and PCNA main connection to PCNA based on previous studies in the based upon the crystal structure of prokaryotic ␦⅐␤ complex human system (25). The current study confirms this interac- (18). Like RFC1, the ␦ subunit of ␥ complex is a member of the tion in the yeast system and also demonstrates that the Tyr- AAAϩ family, and it binds to ␤ via two hydrophobic residues 404 and Phe-405 residues of RFC1 are involved in PCNA bind- that are located in the same relative position in ␦ as residues ing. The position of these two residues in RFC1 is analogous to Tyr-404 and Phe-405 of RFC1. Previous studies of ␥ complex that of the two hydrophobic residues in E. coli ␦ that bind the mutated in these two residues showed that the ␥ (␦mut) complex ␤ clamp and thus were hypothesized to be important to RFC1- is less active than wild-type ␥ complex in replication assays, PCNA interaction (18). This report also documents that and its interaction with ␤ is compromised (23). RFC(3/4) and RFC(2/3) also form a main attachment site be- To determine whether RFC1mut binds PCNA with lower af- tween RFC and PCNA. Because neither RFC2 nor RFC4 bind finity than wild-type RFC we developed a fluorescence assay by stably to PCNA it is possible that RFC3 alone forms the main labeling PCNA at its two exposed Cys residues using the Ore- contact to PCNA within these complexes (illustrated in Fig. 9). gon Green fluorophore. Titrations of RFC and RFC1mut into We observe here that the RFC(3/4) heterodimer displays ATP- Analysis of RFC Form and Function 50751

FIG.7.Interactions between PCNA and RFC subunits and heterodimers. Interactions between PCNA and the various subcomplexes of RFC were analyzed by gel filtration on a Superdex-200 column as described under “Experimental Downloaded from Procedures.” Column fractions were analyzed by SDS-PAGE. Column fraction numbers are indicated above the top gel, and the positions of RFC subunits are identified on the left of the gel. Panel A, RFC(3/4) Ϯ PCNA Ϯ ATP. Analysis of PCNA alone is also shown. Panel B, http://www.jbc.org/ RFC(2/3) Ϯ PCNA Ϯ ATP. Panel C, RFC(2/5) Ϯ PCNA Ϯ ATP. Panel D, RFC2 Ϯ PCNA Ϯ ATP. Panel E, RFC4 Ϯ PCNA Ϯ ATP. at Rockefeller University Library on August 2, 2015

dependent behavior in binding to PCNA, whereas the RFC(2/3) so only with very weak affinity, and there is no detectable heterodimer binds PCNA in the absence of ATP. We propose interaction thus far between ␦Ј and ␤ (15). Consistent with ␦ ␤ ␥ ␦Ј␹␺ that the PCNA binding site(s) within the RFC(3/4) complex is forming the main contact to , the 3 subassembly lacking occluded in this heterodimer, and ATP results in a conforma- the ␦ subunit does not form a stable complex with ␤ (22). The tion change that makes these sites more accessible to PCNA. fact that more than one RFC subunit binds PCNA tightly may The ATP-independent interaction of RFC(2/3) with PCNA in- underlie a divergence in mechanism between prokaryotic and dicates that the PCNA binding site(s) of this heterodimer are eukaryotic clamp loaders. This difference may simply be a more exposed to PCNA and thus do not require ATP to power consequence of clamp loaders that function with either a dimer a conformation change for PCNA interaction. However, the (␤) or trimer (PCNA) ring. For example, PCNA dissociates into intact 5-subunit RFC requires ATP to observe PCNA binding monomers at low concentrations (34), and RFC may need mul- (29), and thus the PCNA binding site(s) of the RFC(2/3) het- tiple contact points with the PCNA trimer to prevent it from erodimer must be obscured in the intact RFC, presumably by falling apart when it opens the ring at one interface. one or more of the other three subunits, and that ATP is The ␥ trimer has been referred to as the “motor” of ␥ complex, required to expose them for PCNA interaction. The studies of as these subunits are the only ones that bind ATP (9, 10, 35). this report do not rule out contact between PCNA and RFC2 or The RFC(2/3/4) subassembly has been proposed to act like the ␥ 5, as only strongly interacting complexes are observed by the 3 motor subassembly as they are the only RFC subunits that non-equilibrium gel filtration technique used here. Indeed it contain both the ATP site and the SRC arginine finger motifs, has been suggested that all five RFC subunits contact PCNA in like ␥ (9, 35). Results of this study are consistent with this the human system (32). assignment as they demonstrate that RFC2, 3, and 4 bind one Comparison of RFC with ␥ Complex—The E. coli ␥ complex, another and contain an inherent DNA-stimulated ATPase ac- like RFC, contains five AAAϩ subunits that are required for tivity. Likewise, the analogous human RFC p40/p37/p36 com- clamp loading activity (10). However, unlike RFC, ␥ complex plex contains DNA-dependent ATPase activity (28, 29). More- has only one strong subunit contact to the ␤ clamp, mediated by over, the current study also confirms the hypothesis that these the ␦ subunit (22). Although the ␥ subunits contact ␤, they do RFC subunits require the SRC arginine finger for ATP hydro- 50752 Analysis of RFC Form and Function Downloaded from http://www.jbc.org/

FIG.9.RFC architecture and contacts to PCNA. A, cartoon of the subunit arrangement in the RFC pentamer looking down the C-terminal plane corresponding to the top view of ␥ complex in Fig. 1. ATP sites are located at subunit interfaces. B, cartoon of RFC side views. ATP sites of RFC and major subunit contacts to PCNA are shown in the two side views. The two side views illustrate the identity of each subunit that contributes an Arg finger to the four different ATP sites. at Rockefeller University Library on August 2, 2015

necessary for ␤ to bind ␥ complex (9, 10). It is not clear at this time whether RFC5 is rigid, and thus assigning a stator role for this subunit is still preliminary. It has been proposed that RFC1 acts like the ␦ wrench to open the ring, although no clear evidence for this exists at the present time (9, 18). This speculation derives from the fact that RFC1, like ␦, is the most divergent in sequence from the other subunits, lacks an SRC motif, and binds directly to the PCNA ring. Furthermore, as ␦ is positioned between the stator and a motor subunit in ␥ complex, so is RFC1 positioned in RFC, based upon the results in this report. If RFC1 is the wrench, one may predict that there will be a gap between the N-terminal domains of RFC1 (wrench) and RFC5 subunits, by analogy FIG.8.RFC complex containing mutant RFC1 is defective in to the gap between the ␦ and ␦Ј subunits of E. coli ␥ ␦␦Ј (see Fig. 1mut 3 function with PCNA. Top panel, wild-type RFC or RFC was 9). Unlike ␦, RFC1 contains an ATP site, but mutational anal- titrated into PCNA labeled with a fluorescent reporter. Open circles represent reactions with wild-type RFC, and closed triangles represent ysis demonstrates that this site is not required for clamp load- reactions with RFC1mut. Bottom panel, PCNA is titrated into replication ing function and thus may be involved in some other activity assays containing a recombinant Pol ␦ derivative and either wild-type (36). 1mut RFC or RFC . RFC and PCNA were preincubated with an SSB- It has been noted previously (18) that RFC1 contains two coated singly primed M13mp18 ssDNA template coated with SSB before initiating replication with recombinant yeast Pol ␦ as described adjacent hydrophobic residues that are located in the same under “Experimental Procedures.” Open squares represent reactions position as the two hydrophobic residues of ␦ involved in bind- containing wild-type RFC, and open triangles represent RFC1mut. ing to ␤. Studies in this report show that RFC mutated in these Closed circles are reactions in which no RFC was added. residues of RFC1 binds less tightly to the clamp and that the RFC1mut is impaired in replication activity, similar to E. coli ␥ lytic activity, as has been shown previously for the ␥ subunits complex containing a ␦ subunit that is mutated in the analo- of ␥ complex (21). gous hydrophobic residues (23). However, it also remains pos- The E. coli ␦Ј stator contains an SRC motif that donates a sible that the mutations in RFC1 examined here causes some ␥ catalytic arginine to the ATP site of 1 (21) but lacks the ATP alteration of the RFC structure that causes indirect effects that binding site consensus sequence. Similarly, RFC5 contains an change PCNA interactions. SRC motif and has a modified ATP binding site sequence sug- The apparent similarity between RFC1 and ␦ may imply that gesting that even if it binds ATP it may not easily hydrolyze it RFC1 opens the clamp like ␦, but until this is demonstrated, (6). According to the subunit arrangement determined here, other possibilities also exist. For example, it has been pointed RFC5 contributes a catalytic arginine to the ATP site of RFC2. out that RFC3 and RFC5 also contain two adjacent hydropho- The ␦Ј stator is thought to be a rigid subunit, perhaps acting as bic residues in a similar position as ␦ and RFC1 (33). Therefore a backboard to direct the ATP-induced changes in ␥ subunits it is also possible that either of these other subunits acts like ␦. Analysis of RFC Form and Function 50753

More importantly, RFC may simply function quite differently 14. Yuzhakov, A., Kelman, Z., and O’Donnell, M. (1999) Cell 96, 153–163 ␥ 15. Leu, F. P., and O’Donnell, M. (2001) J. Biol. Chem. 276, 47185–47194 than complex, although the fact that both clamp loaders are 16. Hingorani, M. M., and O’Donnell, M. (1998) J. Biol. Chem. 273, 24550–24563 composed of five AAAϩ proteins suggests that many aspects of 17. Stewart, J., Hingorani, M. M., Kelman, Z., and O’Donnell, M. (2001) J. Biol. ␥ Chem. 276, 19182–19189 their mechanism will be similar. For example, both RFC and 18. Jeruzalmi, D., Yurieva, O., Zhao, Y., Young, M., Stewart, J., Hingorani, M., complex bind ATP to associate with their respective clamps. O’Donnell, M., and Kuriyan, J. (2001) Cell 106, 417–428 However, the different oligomeric structures of their rings may 19. Turner, J., Hingorani, M. M., Kelman, Z., and O’Donnell, M. (1999) EMBO J. 18, 771–783 require different mechanistic approaches to the problem of 20. Guenther, B., Onrust, R., Sali, A., O’Donnell, M., and Kuriyan, J. (1997) Cell binding, opening and closing the rings around DNA. A detailed 91, 335–345 21. Johnson, A., and O’Donnell, M. (2003) J. Biol. Chem. 278, 14406–14413 comparison between the prokaryotic and eukaryotic clamp 22. Naktinis, V., Onrust, R., Fang, L., and O’Donnell, M. (1995) J. Biol. Chem. 270, loaders awaits crystal structure studies of RFC. 13358–13365 23. Indiani, C., and O’Donnell, M. (2003) J. Biol. Chem. In press Acknowledgments—We thank Max Stadler, Monett Librizzi, and 24. Shiomi, Y., Usukura, J., Masamura, Y., Takeyasu, K., Nakayama, Y., Obuse, Olga Yurieva for technical assistance. We are also grateful to C., Yoshikawa, H., and Tsurimoto, T. (2000) Proc. Natl. Acad. Sci. U. S. A. Dr. Greg Bowman of the Kuriyan Laboratory (University of California, 97, 14127–14132 25. Fotedar, R., Mossi, R., Fitzgerald, P., Rousselle, T., Maga, G., Brickner, H., Berkeley, CA) for helpful advice and critical reading of the manuscript. Messier, H., Kasibhatla, S., Hubscher, U., and Fotedar, A. (1996) EMBO J. 15, 4423–4433 REFERENCES 26. Finkelstein, J., Antony, E., Hingorani, M. M., and O’Donnell, M. (2003) Anal. 1. Kelman, Z., and O’Donnell, M. (1995) Annu. Rev. Biochem. 64, 171–200 Biochem. 319, 78–87 2. Stukenberg, P. T., Studwell-Vaughan, P. S., and O’Donnell, M. (1991) J. Biol. 27. Gomes, X. V., Gary, S. L., and Burgers, P. M. (2000) J. Biol. Chem. 275, Chem. 266, 11328–11334 14541–14549

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Nina Yao, Lee Coryell, Dan Zhang, Roxana E. Downloaded from Georgescu, Jeff Finkelstein, Maria M. Coman, Manju M. Hingorani and Mike O'Donnell J. Biol. Chem. 2003, 278:50744-50753. doi: 10.1074/jbc.M309206200 originally published online October 6, 2003 http://www.jbc.org/

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