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J. Biol. Chem., Vol. 281, Issue 25, 17528-17539, June 23, 2006

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Mechanism of Proliferating Cell Nuclear Antigen Clamp Opening by Replication Factor C^*

Nina Y. Yao, Aaron Johnson, Greg D. Bowman¹, John Kuriyan^¶||, and Mike O'Donnell^¶2

From the Rockefeller University and the ^¶Howard Hughes Medical Institute, New York, New York 10021 and the ^||Departments of Molecular and Cell Biology and Chemistry, Howard Hughes Medical Institute, University of California, and the Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720

Received for publication, February 9, 2006 , and in revised form, April 5, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The eukaryotic replication factor C (RFC) clamp loader is an AAA+ spiral-shaped heteropentamer that opens and closes the circular proliferating cell nuclear antigen (PCNA) clamp processivity factor on DNA. In this study, we examined the roles of individual RFC subunits in opening the PCNA clamp. Interestingly, Rfc1, which occupies the position analogous to the clamp-opening subunit in the Escherichia coli clamp loader, is not required to open PCNA. The Rfc5 subunit is required to open PCNA. Consistent with this result, Rfc2·3·4·5 and Rfc2·5 subassemblies are capable of opening and unloading PCNA from circular DNA. Rfc5 is positioned opposite the PCNA interface from Rfc1, and therefore, its action with Rfc2 in opening PCNA indicates that PCNA is opened from the opposite side of the interface that the E. coli wrench acts upon. This marks a significant departure in the mechanism of eukaryotic and prokaryotic clamp loaders. Interestingly, the Rad·RFC DNA damage checkpoint clamp loader unloads PCNA clamps from DNA. We propose that Rad·RFC may clear PCNA from DNA to facilitate shutdown of replication in the face of DNA damage.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The proliferating cell nuclear antigen (PCNA)³ DNA sliding clamp is a ring-shaped homotrimer that is opened and closed around DNA by the replication factor C (RFC) clamp loader (1-4). The PCNA clamp then binds numerous different proteins, including the cellular replicases DNA polymerase and DNA polymerase (5). The RFC clamp loader was initially identified as a factor required for SV40 DNA replication in vitro (2). Subsequent characterization of RFC showed it to consist of five different subunits in both yeast and human cells (6). The five subunits are homologous to one another and are members of the AAA+ family of ATPases (7). The structures of yeast RFC and the Escherichia coli clamp loader complex reveal that the subunits are arranged in a similar spiral fashion (8, 9). To avoid confusion over the different nomenclatures used for the different systems, the positions of the various clamp loader subunits are designated A-E as listed in Table 1. The position of each subunit in the complex is indicated in parentheses with a letter when comparisons are made between systems.

RFC shares striking structural and functional similarity with the E. coli clamp loader complex (1, 8, 9, 13, 14). Both RFC and the E. coli complex require the binding of ATP or ATPS to induce a conformational change that allows interaction with the clamp and subsequent ring opening (15-19). The nature of this conformational change is unclear, but ATP binding is sufficient, and hydrolysis is not needed. The complex consists of five clamp loader subunits, three , one , and one ', each of which are composed of three domains (9). Two small subunits of the complex, and , are not required for the clamp-loading operation. The first two N-terminal domains of the , , and ' subunits consist of the AAA+ region of homology. The crystal structure reveals that the C-terminal domain of each subunit mediates oligomerization into a spiral heteropentamer and that a gap exists between the AAA+ regions of the (A) and '(E) subunits (9). The subunit binds tightly and can open the clamp by itself (20). The crystal structure of the "wrench" bound to reveals that binds the N-terminal AAA+ domain of the subunit (21). Superposition of · onto ₃' indicates that likely docks onto the AAA+ domains of all five clamp loader subunits (9).

The structure of RFC in complex with PCNA and ATPS shows that the RFC subunits have a similar domain arrangement and circular architecture to the complex (8). RFC retains the pronounced gap between the AAA+ domains of the Rfc1(A) and Rfc5(E) subunits, which correspond spatially to the (A) and '(E) subunits in the complex. This gap serves the purpose of allowing DNA to enter the central chamber in the clamp loader and thereby positions DNA into the opened clamp docked underneath the clamp loader. The C-terminal extension of Rfc1 is positioned within a portion of this gap.

Rfc1(A) is located in the position analogous to the (A) wrench in ₃'. However, the RFC·PCNA·ATPS structure reveals a closed PCNA ring despite interaction with Rfc1. This closed conformation of PCNA may possibly be due to specific mutations that were made in RFC to prevent nucleotide hydrolysis during crystal growth. The Rfc3 and Rfc4 subunits also bind PCNA. Due to the spiral structure of RFC in complex with a flat planar PCNA ring, each adjacent RFC subunit is poised further away from the PCNA ring. Thus, in going around the RFC pentamer, Rfc1(A) forms the most extensive contact with PCNA; Rfc4(B) is positioned over an interface and contacts PCNA in a minimal fashion; then Rfc3(C) binds the second protomer of PCNA, but buries less surface area than the Rfc1-PCNA contact; and finally, Rfc2(D) and Rfc5(E) are suspended above PCNA and do not bind the ring at all. Like Rfc4, Rfc2 is positioned above a PCNA interface. Rfc5 is positioned above the remaining hydrophobic site in the third PCNA protomer.

The PCNA ring is closed in the RFC·PCNA·ATPS structure (9), yet ATPS enables the RFC·PCNA complex to bind DNA and therefore presumably promotes PCNA opening (17, 18). It is possible that Rfc1 normally opens PCNA but that the ring is closed in the structure due to mutations that were made to prevent ATPS hydrolysis during crystal growth. Alternatively, Rfc5 and/or Rfc2 must bind PCNA for the clamp to open, consistent with their lack of contact with PCNA in the structure.

In this study, we examined RFC subunits for the ability to open PCNA, which was observed experimentally by the unloading of PCNA from circular DNA. This work demonstrates that Rfc1 is not required for PCNA opening. In fact, the Rfc2·3·4·5 subcomplex of the four small RFC subunits can open PCNA clamps and clear them from DNA. This study further demonstrates that Rfc2 and Rfc5 bind directly to PCNA and that Rfc5 is required for PCNA opening by RFC. In fact, an Rfc2·5 subcomplex can open and unload PCNA from DNA. Rfc5(E) is positioned opposite the PCNA interface from Rfc1(A), and therefore, its action with Rfc2 in opening PCNA indicates that the PCNA clamp is opened from the opposite side of the interface that the E. coli wrench acts upon. These internal workings mark a significant departure in the underlying mechanism of clamp opening by eukaryotic and prokaryotic clamp openers.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials
Radioactive nucleotides were purchased from PerkinElmer Life Sciences. DNA modification enzymes were from New England Biolabs, and DNA oligonucleotides were from Integrated DNA Technologies. RFC concentrations were determined using the Bio-Rad protein stain and bovine serum albumin as a standard. PCNA concentration was determined by absorbance at 280 nm using a molar extinction coefficient of 6420 M^-1 cm^-1. Buffer A contained 30 mM HEPES (pH 7.5), 10% (v/v) glycerol, 0.5 mM EDTA (pH 7.5), 1 mM dithiothreitol (DTT), and 0.04% Bio-Lyte 3/10 (Bio-Rad). Buffer B was composed of 20 mM Tris-HCl (pH 7.5), 2 mM DTT, 0.5 mM EDTA, and 10% glycerol. Buffer C was composed of 20 mM Tris-Cl (pH 7.9), 10% sucrose, 500 mM NaCl, and 4% glycerol. Buffer D contained of 20 mM Tris-HCl (pH 7.9), 0.04% Bio-Lyte 3/10, and 4% glycerol. Buffer E contained 20 mM Tris-HCl (pH 7.9), 5 mM imidazole, and 4% glycerol. Surface plasmon resonance buffer was composed of 10 mM HEPES (pH 7.5), 150 mM NaCl, 3.4 mM EDTA, and 0.005% surfactant P2. Clamp loading buffer contained 20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 5 mM DTT, 40 µg/ml bovine serum albumin, and 5% glycerol. Gel filtration buffer was composed of 20 mM Tris-HCl (pH 7.5), 2 mM DTT, 0.5 mM EDTA, 100 µg/ml bovine serum albumin, 8 mM MgCl₂, 4% glycerol, and 150 mM NaCl.

DNA Substrates
Singly nicked pBluescript was prepared as described (22) using M13 gpII endonuclease and supercoiled pBluescript SK⁺ plasmid (Stratagene, La Jolla, CA) until 50% of the replicative form I DNA was converted to replicative form II DNA and then was purified by phenol extraction. Singly primed single-stranded M13 DNA was prepared by annealing the synthetic DNA primer 5'-cga cgt tgt aaa acg acg gcc agt gcc aag ctt gca tgc ctg cag gtc gac tct aga gga ggg tag cat atg ctt ccc-3' to purified single-stranded circular M13EBNA1 DNA (refer to Ref. 23 for both a description of the template and the procedure for annealing). The 3'-end of this primer contains an 18-nucleotide EBNA1-binding sequence that hybridizes to the complementary sequence on the M13EBNA1 template.

Cloning of Mutant RFC Genes
The Saccharomyces cerevisiae RFC genes were cloned into the pET11a (Novagen), pLANT2/RIL (24), or pCDFduet (Novagen) vector. The plasmids containing single genes include pET11a-RFC1, pET11a-RFC2, pLANT2/RIL-RFC2, pET11a-RFC3, pET11a-RFC4, and pET11a-RFC5. The expression plasmids containing two or more genes include pET11a-RFC3+4, pLANT2/RIL-RFC1+5, and pET11a-RFC2+3+4.

RFC DNA-binding Mutant
Mutations were introduced into the RFC2, RFC3, and RFC4 genes using the QuikChange method (Stratagene). Arg¹⁰¹, Arg¹⁰⁷, and Arg¹⁷⁵ of Rfc2 (RFC2(DNA-bind mut)); Arg⁸⁸, Arg⁹⁴, and Lys¹⁵² of Rfc3 (RFC3(DNA-bind mut)); and Arg⁸⁴, Arg⁹⁰, and Lys¹⁴⁹ of Rfc4 (RFC4(DNA-bind mut)) were all mutated to alanine.

RFC2(DNA-bind mut) was generated in pET11a-RFC2 using the following oligonucleotides: 5'-gag ttg aac gct tct gac gaa gct ggt atc tct att gtg gcc gaa aag gta aaa aat ttt gc-3' (to introduce Rfc2(R101A/R107A)) and 5'-c tgt ttg att tgt aat tat gtg acg gcc att att gac ccg cta gcg tcc cg-3' (to introduce Rfc2(R175A)). RFC3(DNA-bind mut) was generated in pET11a-RFC3+4 using the following primers: 5'-gag ctg aat gca tcc gat gac gca ggt att gat gtg gtc gcg aat caa att aaa gac ttt gc-3' (to introduce Rfc3(R88A/R94A)) and 5'-gta ttg gcc aat tat gcg cat gca ctt aca cct gcg tta ttg ag-3' (to introduce Rfc3(K152A)). RFC4(DNA-bind mut) was generated in pET11a-RFC4 using the following primers: 5'-gag ttg aac gct tca gat gac gca ggt att gat gtc gtc gcg aac caa ata aaa cat ttt gcc-3' (to introduce Rfc4(R84A/R90A)) and 5'-ct tgt aat caa tca aac gcg atc att gag ccg ctg cag agt aga tgt gcg att ttg-3' (to introduce Rfc4(K149A)). The open reading frames of the mutated genes were confirmed by DNA sequencing.

To construct pET-RFC(2DNA-bind mut+3DNA-bind mut+4DNA-bind mut), plasmids pET11a-RFC4(DNA-bind mut) and pET11a-RFC(3DNA-bind mut+4) were digested with SacI/AflI. The fragment containing RFC4(DNA-bind mut) was ligated into the fragment containing pET11a-RFC3(DNA-bind mut) to yield pET11a-RFC(3DNA-bind mut+4DNA-bind mut). In addition, pET11a-RFC2(DNA-bind mut) was digested with BglII/HindIII and blunt-filled with Klenow fragment; the small fragment was gel-purified and ligated into pET-RFC(3SAC+4SAC) (which was cut with SphI, blunted with Klenow fragment, and treated with alkaline phosphatase; the large fragment was gel purified). The screen for orientation showed that RFC2 was facing in the opposite orientation from RFC3 and RFC4. Finally, both pET-RFC(3DNA-bind mut+4DNA-bind mut) and pET-RFC(2DNA-bind mut+3SAC+4SAC) were digested with MluI/SgrAI; the large fragment from the former and the small fragment from the latter were ligated together to form pET-RFC(2DNA-bind mut+3DNA-bind mut+ 4DNA-bind mut).

Truncation Mutations in the RFC1 and RFC5 Genes
RCF(Rfc1 AAA+)—Rfc1 lacking the N-terminal ligase region has four remaining domains. The AAA+ region of homology is contained in the first two domains (domains 1 and 2), and domain 3 interacts with the other RFC subunits to form the pentameric collar; domain 4 is a C-terminal extension that is positioned within the gap between Rfc1 and Rfc5 in the crystal structure (9). Domain 3 of Rfc1 (Leu⁵⁴⁹-Thr⁶⁶⁷) was isolated from pET11a-RFC1 by PCR using the following primers: 5'-ggg aaa aga atc ata tgc taa aac cct ttg aca ttg ccc-3' and 5'-ccg gat cct cat gtg aaa ttg att ctt ccc gcc atg tgg ccg gca act ttt gat gc-3'. This fragment, lacking domains 1, 2, and 4 (i.e. Rfc1 AAA+), was cloned into the NdeI and BamHI sites of the pCDFduet vector to make pCDF-duet-RFC1 AAA+. The open reading frame was confirmed by DNA sequencing.

RCF(Rfc5 AAA+)—Rfc5(domain 3), which interacts with the other subunits to form the collar of pentameric contacts, corresponds to Pro²⁵⁷-Asp³⁵⁴. The vector for expression of this domain was generated using the same protocol as described above, except that the template DNA used was pET-RFC5. The PCR primers were 5'-ccc tat aca tat gcc aga ttg gat tat agt gat cca taa att aac gag g-3' and 5'-g cgt taa gga tcc gct ttt tgt gtg act tat ac-3'. The DNA fragment encoding Rfc5 Pro²⁵⁷-Asp³⁵⁴ was then cloned into the NdeI and BamHI sites of pCDFduet to yield pCDFduet-RFC5 AAA+.

Rfc5(domain 1)—The same protocol as described above was performed using primers 5'-c agc aca cat atg tca ttg tgg gta gat aaa tac aga cc-3' and 5'-ttt cgg atc ctc atg gtg cag gac aac gaa tca aca gac-3' to amplify a fragment containing the isolated domain 1 of Rfc5 (Trp⁴-Pro¹⁹³, corresponding to the PCNA- and ATP-binding region) by PCR. This fragment was then cloned into HK-pET (gene reference) at the NdeI and BamHI sites to produce HK-pET-RFC5(domain 1).

Overexpression of S. cerevisiae RFC Proteins in E. coli
Cotransformation reactions utilizing pET-, pLANT/RIL-, and pCD-Fduet-derived RFC subunit expression plasmids were performed using the BL21(DE3) Star strain of E. coli competent cells (Invitrogen). Transformants were selected on LB plates containing ampicillin (100 mg/ml), kanamycin (50 µg/ml), and streptomycin (50 mg/ml). Fresh transformants were grown in 12-24 liters of Terrific broth containing the appropriate antibiotics at 30 °C until cell growth reached A₆₀₀ = 0.6. Cultures were then brought to 15 °C by chilling on ice before addition of 1 mM isopropyl -D-thiogalactopyranoside, followed by incubation at 15 °C for 18 h. Cells were harvested by centrifugation.

Purification of RFC Complexes and Individual Subunits
For purification of the RFC DNA-binding mutant, the five-subunit RFC containing DNA-binding mutations in Rfc2-4 was expressed by cotransformation of pLANT2/RIL-RFC1+5 and pET11a-RFC(2DNA-bind mut+3DNA-bind mut+4DNA-bind mut). Cells harvested from 12 liters of culture were resuspended in 300 ml of Buffer A containing 150 mM NaCl and then lysed using a French press at 22,000 p.s.i. The cell lysate was clarified by centrifugation and applied to a 100-ml SP-Sepharose Fast Flow column (Amersham Biosciences) equilibrated with Buffer A containing 150 mM NaCl. The column was eluted with a 1-liter gradient of 150–600 mM NaCl in Buffer A. Column fractions were analyzed on 10% SDS-polyacrylamide gels stained with Coomassie Blue. The peak of RFC (which eluted at 480 mM NaCl) was pooled and diluted with Buffer B to 150 mM NaCl. Protein was then applied to a 20-ml Q-Sepharose Fast Flow column (Amersham Biosciences) equilibrated with Buffer B containing 90 mM NaCl and eluted with a 200-ml gradient of 90-500 mM NaCl in Buffer B. Fractions containing the RFC complex (which eluted at 300 mM NaCl) were stored at -80 °C. The final yield was 40 mg of purified RFC DNA-binding mutant/12 liters of culture.

Purification of RFC Complexes Containing Either Truncated Rfc1 or Rfc5
RFC(Rfc1 AAA+) containing truncated Rfc1 (domain 3 only) was expressed by cotransformation of pLANT2/RIL-RFC5, pET11a-RFC2+3+4, and pCDFduet-RFC1 AAA+. Purification of the RFC mutant followed the same procedure as described for the RFC DNA-binding mutant, except that a 100-ml Q-Sepharose Fast Flow column was used with a 1-liter gradient of 90-500 mM NaCl in Buffer B. The final yield was 49 mg of purified RFC(Rfc1 AAA+)/12 liters of culture.

Expression of RFC containing truncated Rfc5 (domain 3 only) was performed by cotransformation of pLANT2/RIL-RFC1, pET11a-RFC2+3+4, and pCDFduet-RFC5 AAA+. The RFC mutant was purified following the same procedure as described above, except that a 100-ml gradient of 90-500 mM NaCl in Buffer B was used to elute protein from a 20-ml Q-Sepharose Fast Flow column. The final yield was 22 mg of RFC(Rfc5 AAA+)/12 liters of culture.

Purification of Rfc5(domain 1)
HK-pET-RFC5(domain 1) was transformed and expressed in BL21-(DE3) Star competent cells. The cell pellet harvested from 12 liters of culture was resuspended in 180 ml of Buffer C. After lysis using a French press and clarification by centrifugation, the supernatant from the crude lysate was diluted with Buffer D to a conductivity equal to 150 mM NaCl. At this conductivity, the Rfc5(domain 1) protein flowed through both an SP-Sepharose and a Q-Sepharose Fast Flow column. The protein was then precipitated using 70% ammonium sulfate and resuspended in 60 ml of Buffer E. The protein was dialyzed against Buffer E containing 500 mM NaCl and then applied to a 10-ml nickel-charged chelating Sepharose column and eluted using a 100-ml gradient of 5-1000 mM imidazole in Buffer E. Column fractions were analyzed by 10% SDS-PAGE, and fractions containing Rfc5(domain 1) were pooled and dialyzed against Buffer B. Protein was loaded onto a 1-ml Mono Q column and eluted using a 20-ml gradient of 25-500 mM NaCl in Buffer B. Contaminants bound to this column, but Rfc5(domain 1) flowed through. The final yield was 10 mg of Rfc5(domain 1)/12 liters of culture.

Purification of Wild-type RFC, Subcomplexes, and Individual Subunits
Wild-type RFC with either full-length Rfc1 or Rfc1 lacking the N-terminal ligase domain (referred to here as RFC_1N) was purified as described (25). Rfc2, Rfc3·4, and Rfc2·5 were also purified as described (25). The four-subunit Rfc2·3·4·5 complex was reconstituted by mixing equimolar Rfc2·5 and Rfc3·4, followed by incubation at 16 °C for 10 min.

View larger version (24K): [in this window] [in a new window]   FIGURE 1. RFC1N can open PCNA and unload it from DNA. RFC1N (A), which lacks the N-terminal ligase homology domain of Rfc1, and wild-type RFC (RFCwt; B) were tested for 32P-PCNA unloading activity using circular nicked DNA to which 32P-PCNA was attached in an earlier reaction as described under "Experimental Procedures." The 32P-PCNA unloading assays were quantitated at different concentrations of RFC1N or wild-type RFC (C).

Surface Plasmon Resonance
Rfc5(domain 1) was immobilized on a CM5 sensor chip using carbodiimide chemistry in 10 mM sodium acetate (pH 4.5) to yield a final value of 4000 response units of immobilized Rfc5(domain 1). 1 µM protein kinase-tagged PCNA in surface plasmon resonance buffer containing 8 mM MgCl₂ was passed over immobilized Rfc5(domain 1) at a flow rate of 6 µl/min for 5 min, after which buffer lacking protein was injected over the chip. This protocol was repeated using immobilized Rfc2 protein (13,700 response units immobilized).

View larger version (34K): [in this window] [in a new window]   FIGURE 2. Rfc5, but not Rfc1, is required for PCNA clamp opening (i.e. unloading) activity. Two different RFC mutants were prepared. One retained the Rfc1-4 subunits and only the C-terminal collar domain of Rfc5 (RFC(Rfc5 AAA+]). The other retained the Rfc2-5 subunits and only the C-terminal collar domain of Rfc1 (RFC(Rfc1 AAA+)). The mutants were tested for 32P-PCNA clamp unloading (i.e. clamp opening) activity off circular nicked DNA. Unloading assays were performed using either 25 nM RFC(Rfc1 AAA+)(A)or 25 nM RFC(Rfc5 AAA+) (B).

1N

Fluorescent DNA Binding Assays
RFC binding to DNA was measured using a fluorescent primed DNA template. The fluorescent 30-mer oligonucleotide primer contained a Rhodamine Red-X fluorophore group (Integrated DNA Technologies) linked to the 3'-OH by a six-carbon spacer. The primed DNA template was prepared by annealing 1.0 nmol of 3'-Rhodamine Red-X-conjugated 30-mer with 1.2 nmol of unlabeled 66-mer in 100 µl of 5 mM Tris-HCl, 150 mM NaCl, and 15 mM sodium citrate (final pH 8.5) as described above for unlabeled templates. RFC complexes were titrated from 0 to 120 nM into 60-µl reactions containing 20 mM Tris (pH 7.5), 125 mM NaCl, 8 mM MgCl₂, 2 mM DTT, 0.04 mg/ml bovine serum albumin, 0.5 mM EDTA, 100 µM ATPS, and 10 nM rhodamine-labeled primer·template. The fluorophore was excited at 570 nm, and emission was monitored over 580-680 nm. The relative intensity of the peak at 588 nm was plotted versus RFC concentration. The apparent K_d for wild-type RFC binding to DNA was determined using the model A + B AB and the curve-fitting software KaleidaGraph (Synergy Software).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Clamp Unloading as a Means for Measuring PCNA Opening—One of the primary steps in RFC assembly of PCNA on DNA is the opening of the clamp. Clamp loaders (both prokaryotic and eukaryotic) can also unload clamps from DNA (3, 28-30). ATP is required in both directions (28), and therefore, the forward (loading) and reverse (unloading) reactions do not constitute a true equilibrium. Opening of the clamp is required for both reactions, but loading clamps onto DNA has the additional requirements that DNA be positioned into the clamp and that the clamp be reclosed. In contrast, the unloading reaction requires only that the clamp be opened, allowing DNA to depart. Hence, clamp unloading from DNA provides a convenient method of assaying the PCNA-opening event. In the unloading assay, PCNA is labeled with ³²P (via a six-residue N-terminal kinase tag) and loaded onto DNA by the clamp loader. Then, the ³²P-PCNA·DNA complex is purified from ATP, clamp loader, and any ³²P-PCNA free in solution by gel filtration. The pure ³²P-PCNA·DNA complex can then be treated with wild-type or mutant RFC and examined for clamp unloading and thus opening by gel filtration analysis, which resolves the unloaded ³²P-PCNA from the ³²P-PCNA·DNA complex.

Clamp unloading by the E. coli complex and human RFC has been observed previously using purified ³²P-clamp·DNA complex as a substrate, which allows the balance between clamp loading and unloading to flow in the direction of unloading ³²P-clamp from DNA (3, 28-30). S. cerevisiae RFC may also be expected to unload PCNA. Fig. 1A illustrates an unloading reaction using a version of yeast RFC that we used throughout this study. This particular RFC contains an Rfc1 subunit that lacks the N-terminal 282 amino acids, which we refer to as RFC_1N. The N-terminal region of Rfc1 binds DNA nonspecifically, and removal of this region has no observable replication phenotype and results in a more active RFC clamp loader relative to the wild type (10, 11, 31, 32). The experiment in Fig. 1 compares the unloading activity of RFC_1N with that of wild-type RFC. The result shows that RFC_1N removed PCNA clamps from circular duplex DNA (Fig. 1A), but wild-type RFC, containing full-length Rfc1, was much less efficient in this unloading reaction (Fig. 1B). This result is consistent with the inability to observe unloading by wild-type RFC in a recent study (33). Presumably, wild-type RFC is a poor clamp unloader compared with its activity as a clamp loader, and the reaction lies far in the direction of clamps being assembled onto DNA, even at very low concentrations of free PCNA. The previous study did not detect unloading by RFC_1N, but this discrepancy may be explained by the higher amounts of protein used here.

Rfc1 Is Not Required for PCNA Opening—The touch points between PCNA and RFC are located within the N-terminal ATPase domains of each subunit contained within the AAA+ region of homology (8). The AAA+ domains are the first two (i.e. N-terminal) of the three domains that compose the small RFC subunits, Rfc2-5. The third domain (i.e. C-terminal) mediates the major pentameric contacts that form a "collar" that holds the subunits together. Rfc1 contains these three domains as well, but also contains N- and C-terminal extensions relative to the small subunits. If Rfc1 acts in a fashion analogous to the wrench in the complex, the Rfc1 AAA+ region, which contacts the PCNA clamp, should be required for clamp opening. Hence, we expressed an RFC mutant in which the N and C termini of Rfc1 were pared down to only the "collar domain," which mediates oligomerization with the other subunits. We refer to this RFC mutant as RFC(Rfc1 AAA+); it behaved similarly to wild-type RFC and RFC_1N during purification. Although the RFC(Rfc1 AAA+) mutant was inactive in loading PCNA onto DNA (data not shown), analysis of ³²P-PCNA removal from DNA showed that this RFC mutant retained clamp unloading activity and thus was still capable of opening PCNA (Fig. 2A). The amount of PCNA released from DNA by RFC(Rfc1 AAA+) was similar to that released by RFC_1N (compare Figs. 1A and 2A). This result suggests that interaction between PCNA and the AAA+ domain of Rfc1, which includes the PCNA-binding region, is not required for PCNA clamp opening.

Rfc5 Is Required for PCNA Opening—We also expressed an RFC mutant missing the AAA+ region of Rfc5, but containing the C-terminal collar domain of Rfc5, as well as full-length Rfc2-4 and RFC_1N. This RFC mutant is referred to here as RFC(Rfc5 AAA+). In contrast to RFC(Rfc1 AAA+), the RFC(Rfc5 AAA+) mutant could not unload PCNA (Fig. 2B), implying that contact between Rfc5 and PCNA is needed to open the PCNA clamp.

To further investigate the role of Rfc5 in PCNA opening, we studied subcomplexes of RFC. Because Rfc1-PCNA interaction is not necessary for clamp opening, one may predict that an Rfc2·3·4·5 subcomplex, lacking Rfc1 entirely, will still open and unload PCNA from DNA. Fig. 3A demonstrates that the Rfc2·3·4·5 subcomplex was able to unload PCNA from DNA. This result is consistent with an observation in the human system indicating that an RFC subassembly analogous to Rfc2·3·4 can unload PCNA (30).

View larger version (23K): [in this window] [in a new window]   FIGURE 3. RFC subcomplexes containing Rfc5 can open PCNA to unload the clamp. Clamp unloading reactions were performed using singly primed M13mp18 single strand DNA coated with E. coli single-stranded DNA-binding protein and Rfc2·3·4·5 (A), Rfc2·5 (B), or Rfc3·4 (C). Reactions were performed as described under "Experimental Procedures." The red circles are controls in which the 32P-PCNA·DNA complex was treated with buffer only. RFC subcomplexes were titrated in PCNA unloading assays, and the amount of 32P-PCNA unloaded from DNA was quantitated (D).

View larger version (30K): [in this window] [in a new window]   FIGURE 4. Rfc5 and Rfc2 bind PCNA. A, the crystal structure of the RFC·PCNA·ATPS complex (left) shows that Rfc2 and Rfc5 do not contact the closed PCNA clamp. A schematic of the crystal structure is shown (right). B, surface plasmon resonance experiments use PCNA that is passed over the top of immobilized Rfc5(domain 3) (left panel) or Rfc2 (right panel). Points at which PCNA and buffer lacking PCNA were injected are shown. C, the scheme suggests how Rfc5 and Rfc2 may contact the PCNA clamp after the clamp opens out of plane into a helix, forming an intermediate in which all five RFC subunits interact with the open clamp.

Next, we examined the efficiency of these complexes in PCNA unloading relative to RFC_1N (Fig. 3D). The results demonstrate that Rfc2·3·4·5 was 100-fold less efficient than RFC_1N. The lower efficiency of Rfc2·3·4·5 compared with RFC(Rfc1 AAA+) suggests that disruption of the circular collar affects the efficiency of the clamp unloading activity. The unloading activity of Rfc2·5 was comparable with that of Rfc2·3·4·5. These results suggest that Rfc2·5 is primarily responsible for the PCNA clamp-opening function of RFC.

Rfc2 and Rfc5 Bind PCNA—The above results demonstrate that Rfc2·5 unloads PCNA and therefore suggest that one or both of these subunits interact with PCNA. The RFC·PCNA·ATPS structure shows no contact between Rfc2 and Rfc5 with the closed PCNA ring (8). However, molecular simulations indicate that the PCNA ring opens out of plane to form a right-handed spiral (34). The bottom diameter of RFC matches the dimensions of the PCNA ring, and thus, it seems likely that all of the RFC subunits contact PCNA in the open spiral form. Indeed, an electron microscopic reconstruction study of an archaeal RFC·PCNA·ATPS complex indicated that the open PCNA ring interacts extensively with RFC (35). Hence, we examined Rfc2 and Rfc5 for direct contact with PCNA.

Full-length Rfc2 can be purified individually as recombinant protein expressed in E. coli (25). However, we have not been able to obtain soluble Rfc5 when expressed without other subunits. We have also tried expressing Rfc5 as a fusion with either galactose- or maltose-binding protein at the N or C terminus, but the resulting fusions were completely insoluble. However, we were successful in expressing and purifying the N-terminal AAA+ domain of Rfc5.

Interaction of Rfc2 and Rfc5(domain 1) with PCNA was studied by surface plasmon resonance (Fig. 4). Either Rfc2 or Rfc5(domain 1) was immobilized on the sensor chip, and a solution of PCNA was passed over the top. The results show a clear interaction of both Rfc2 and Rfc5(domain 1) with PCNA. Similar analyses in which PCNA was immobilized, and Rfc2 or Rfc5(domain 1) was passed over it also yielded an interaction between them (data not shown). The K_d values calculated from the data of Fig. 4 are 480 nM for Rfc2·PCNA and 50 µM for Rfc5(domain 1)·PCNA. These values are 10- and 1000-fold weaker, respectively, than the K_d value reported for RFC·PCNA determined by surface plasmon resonance in an earlier study (10). Hence, the full strength of interaction between RFC and PCNA likely requires all or most of the subunits.

View larger version (49K): [in this window] [in a new window]   FIGURE 5. DNA binds in the central chamber of RFC. A, the model of RFC·DNA shows only the Rfc2-4 subunits and the conserved positively charged residues located in the central cavity of RFC. The residues listed to the right are among the conserved residues and were each substituted with alanine. B, the RFC mutant (red squares) was analyzed for DNA binding using a rhodamine-labeled primer·template DNA as described under "Experimental Procedures." DNA binding by RFC1N is shown for comparison (blue circles).

To test these residues for DNA binding in the central chamber of RFC, we mutated three conserved positively charged residues in each of the Rfc2-4 subunits and purified the resulting RFC mutant (referred to here as the RFC DNA-binding mutant). These residues are near the DNA modeled into the center of RFC (Fig. 5A). Next, we developed a fluorescent DNA binding assay using two short synthetic oligonucleotides that hybridize to form a primed template with a rhodamine moiety on the 3'-nucleotide of the primer to report RFC binding. Titration of RFC into the fluorescent primed template in the presence of ATPS (no PCNA) resulted in a change in intensity with an observed K_d of 29.7 nM (Fig. 5B). This value compares favorably with the previously observed K_d of 15 nM for yeast RFC binding to a primed site with ATPS (no PCNA) determined using a Biacore system (10).

Analysis of the RFC DNA-binding mutant in the fluorescent DNA binding assay demonstrated vastly reduced DNA binding (Fig. 5B). Assuming that full binding produces a similar maximal intensity change as RFC, the K_d value for interaction of the RFC mutant with DNA is 1 µM. It remains possible that one or more of the amino acid replacements may have altered the proper folding of the RFC DNA-binding mutant. However, the ability of the RFC DNA-binding mutant to bind and unload PCNA from DNA indicates that the RFC mutant is properly folded (described below).

ATP Hydrolysis Is Not Required to Open PCNA for Clamp Unloading— In Fig. 6, we examined the ability of the RFC DNA-binding mutant to function with PCNA in clamp loading and unloading assays. As expected, the RFC mutant was unable to load PCNA onto DNA (Fig. 6A). However, the RFC mutant inhibited PCNA loading when mixed with wild-type RFC (Fig. 6A). This observation indicates that the RFC DNA-binding mutant is still able to bind PCNA, sequestering it away from active RFC.

Next, we examined the RFC DNA-binding mutant for ability to unload ³²P-PCNA from DNA (Fig. 6B, right panel). Surprisingly, the RFC DNA-binding mutant could still unload clamps from DNA (Fig. 6B). It is interesting to note that the PCNA unloading activity of the RFC DNA-binding mutant was observed even when ATPS was substituted for ATP. With RFC, ATPS promoted ring opening and PCNA loading, but RFC stayed with PCNA on DNA; therefore, clamp unloading was not observed (Fig. 6B, left panel). This is presumably due to the tight interaction of the RFC·PCNA·ATPS complex with DNA. However, the possibility remained that PCNA unloading requires ATP hydrolysis and thus is not supported by ATPS. The fact that ATPS induced the RFC DNA-binding mutant to unload PCNA from DNA may be explained by its ability to open PCNA yet inability to bind DNA, thereby leading to dissociation of the RFC·open PCNA·ATPS complex from DNA. These observations indicate that the PCNA-opening step does not require ATP hydrolysis. Thus, it may be presumed that RFC uses ATPS to open PCNA and to bind DNA, but that hydrolysis is needed to break the grip of RFC on DNA, allowing it to eject from the PCNA·DNA complex.

View larger version (32K): [in this window] [in a new window]   FIGURE 6. ATP binding is sufficient for clamp opening by RFC. A, clamp loading assays. The RFC DNA-binding mutant was tested for ability to load PCNA onto DNA (blue squares). Analysis of PCNA loading by RFC1N is shown for comparison (red circles). The presence of the RFC DNA-binding mutant inhibited clamp loading by RFC1N (green triangles). Reactions contained 100 nM RFC1N and/or RFC DNA-binding mutant. B, clamp unloading assays. Right panel, the RFC DNA-binding mutant could unload PCNA in the presence of either ATP (blue squares) or ATPS (red circles). A control reaction carried out in the absence of ATP is also shown (green triangles). Left panel, RFC1N utilized ATP, but not ATPS, for PCNA unloading. Unloading reactions contained 100 nM RFC.

Does Rad·RFC open the clamp when it binds PCNA? To test this possibility, ³²P-PCNA was loaded onto a singly nicked plasmid DNA and purified from free ³²P-PCNA, RFC, and ATP by gel filtration. The ³²P-PCNA·DNA complex was treated with Rad·RFC or the equivalent volume of buffer, and then the reaction was analyzed by gel filtration a second time to determine whether ³²P-PCNA was unloaded from DNA. Rad·RFC removed most of the ³²P-PCNA from DNA (Fig. 7B). To evaluate the efficiency of PCNA unloading by Rad·RFC, the experiment was repeated using different concentrations of Rad·RFC. The results show that Rad·RFC was quite efficient and was half-maximal at 50 nM. The results with Rad·RFC are also consistent with the other experiments in this study demonstrating that Rfc1 is not required for clamp opening.

View larger version (18K): [in this window] [in a new window]   FIGURE 7. Rad·RFC is a PCNA clamp unloader. A, Rad·RFC (100 nM) was tested for ability to unload PCNA from DNA (green triangles). The buffer control is shown for comparison (red circles). B, Rad·RFC was titrated in separate clamp unloading reactions. The amount of PCNA·DNA complex remaining is plotted versus concentration of Rad·RFC used in each reaction.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The RFC·PCNA structure shows that Rfc2 and Rfc5 do not contact PCNA (8). Molecular simulations of PCNA opening indicate that the clamp protomers open out of plane to form a right-handed helix, which complements the helical arrangement of RFC and may allow an open PCNA clamp to bind all five RFC subunits (34). A recent electron microscopic reconstruction of archaeal RFC bound to PCNA provides experimental support for this conclusion (35). In addition, a recent fluorescence resonance energy transfer study of yeast RFC-PCNA interaction indicates that PCNA is held open by RFC in response to bound nucleotide (40). Surface plasmon resonance measurements in this study demonstrated that Rfc2 and Rfc5 both interact with PCNA. We presume that Rfc5 binds to the hydrophobic pocket between the PCNA domains of the third protomer and that, like Rfc4, Rfc2 binds to an interface between PCNA protomers.

Eukaryotic Versus Prokaryotic Clamp Opening—The Rfc1 subunit of the clamp loader is in the position analogous within the heteropentamer to the wrench of the E. coli complex. However, the E. coli wrench can open the E. coli clamp and unload it from DNA by itself (20). plugs into a hydrophobic pocket of and interacts with the interface, distorting it to a form that is incompatible with ring closure (21). In contrast, the studies described herein indicate that the spatially analogous Rfc1 subunit is not needed for clamp opening. The evidence is as follows. 1) An RFC mutant containing an Rfc1 subunit deleted in the PCNA-interacting AAA+ region retains function in PCNA clamp unloading. 2) An Rfc2·3·4·5 complex (no Rfc1) is capable of unloading PCNA from DNA. 3) The RFC·PCNA·ATPS structure shows extensive interaction of Rfc1 with PCNA, yet the PCNA ring remains shut. 4) Finally, the Rad·RFC DNA damage checkpoint response clamp loader, which lacks Rfc1, opens PCNA and unloads it from DNA nearly as efficiently as RFC_1N. The clamp-interacting helix of Rfc1, unlike that of , does not interact with or disrupt the PCNA interface as observed for the wrench.

This study demonstrates that the eukaryotic RFC clamp loader requires subunits other than Rfc1 for PCNA clamp opening. In particular, Rfc5 is required to open PCNA. Of the three hydrophobic pockets in PCNA, the only one that is not filled in the crystal structure is the pocket positioned below Rfc5. As the clamp is closed in the RFC·PCNA·ATPS structure, interaction of Rfc5 (and Rfc2) with PCNA may be needed to open PCNA. Furthermore, the interface of PCNA that opens for clamp loading onto DNA is presumed to be the interface positioned below the Rfc1 and Rfc5 subunits because DNA must pass between these two subunits to gain access to the central chamber of the clamp loader. Hence, if Rfc5 destabilizes the PCNA ring, it seems reasonable to suggest that the PCNA clamp is opened from the opposite side of the clamp interface that the E. coli wrench acts upon (Fig. 8A).

The wrench can open and unload it from DNA, but cannot load onto DNA. Clamp unloading is an important cellular function, as clamps are used in stoichiometric fashion on the lagging strand (6, 22). One clamp is required for each Okazaki fragment, and as each Okazaki fragment is completed, the clamp is left on DNA and must be recycled. The cellular excess of relative to the other subunits and the efficiency of in unloading from DNA have led to the suggestion that the excess recycles clamps from DNA as they are left behind by the moving replisome (41).

This study demonstrates that the Rfc2·3·4·5 and Rfc2·5 subcomplexes are capable of unloading PCNA clamps from DNA in vitro. Could these same subcomplexes exist in vivo to aid in the recycling of PCNA during replication? Measurements of the intracellular levels of the RFC subunits have been made in a global analysis of protein expression in yeast by Ghaemmaghami et al. (42). The copy numbers of Rfc5 (5040) and Rfc2 (4610) are about twice as abundant as those of Rfc1 (2360), Rfc3 (3140), and Rfc4 (2160). These intracellular copy numbers predict an excess of Rfc2·5 complex in the cell, leaving 2000 copies of free Rfc2·5 complex to perform activities such as PCNA unloading. Assuming a 1-µm diameter of the yeast cell nucleus, 2000 copies of Rfc2·5 complex correspond to a concentration of 1.9 µM. This value is within the range of the concentration of Rfc2·5 required in this study to observe PCNA unloading activity.

View larger version (30K): [in this window] [in a new window]   FIGURE 8. Scheme of RFC action and comparison with the E. coli clamp-opening mechanism. A, Rfc5, required to open PCNA, is positioned above the clamp protomer to the left of the open interface (left). In contrast, the wrench subunit of the E. coli complex is positioned above the clamp protomer to the right of the interface (right). B, the model shows when the Rfc2 and Rfc5 subunits contact PCNA. The first diagram shows that all five subunits bind an open PCNA. Primed DNA enters the central cavity of RFC and is positioned through PCNA (second diagram). The PCNA clamp-RFC connection loosens as the Rfc5 and Rfc2 subunits detach from PCNA (third diagram). ATP hydrolysis completes detachment of all RFC subunits from PCNA and DNA (fourth diagram).

Interestingly, the RFC DNA-binding mutant can use either ATP or ATPS to open the clamp and unload PCNA from DNA. This nonhydrolyzable ATP analog typically leads to an RFC·PCNA complex that remains tightly stuck to DNA (17, 18). In light of the ability of the RFC DNA-binding mutant to unload PCNA with ATPS, we expect that the tight attachment of the wild-type RFC·PCNA·ATPS complex to DNA is mediated mainly through RFC-DNA contacts rather than PCNA-DNA interaction. It is possible that the PCNA ring is held in the open state in the ATPS·RFC·PCNA·DNA complex because the affinity for DNA lies in RFC and not necessarily a topologically closed PCNA ring as an anchor. Indeed, a previous study demonstrated that the E. coli clamp is open while attached to the complex and DNA in the presence of ATPS (16). In addition, a recent electron microscopic image reconstruction of the archaeal RFC·PCNA complex showed that the PCNA ring is held open in the presence of ATPS (35). A recent fluorescence resonance energy transfer study of yeast RFC-PCNA complex also demonstrates that PCNA is held in an open lockwasher configuration (40).

Rad24·RFC Damage Checkpoint Clamp Loader Unloads PCNA— The 911 heterotrimeric clamp is assembled onto DNA by the Rad·RFC clamp loader, in which Rfc1 is replaced by Rad24 protein (Rad17 in humans) (12). Although Rad·RFC is not known to load PCNA onto DNA, it has an intrinsic DNA-dependent ATPase activity that is stimulated by PCNA (37, 43). Hence, Rad·RFC must bind the PCNA clamp, although its function with PCNA is not known. We have shown here that Rad·RFC unloads PCNA from DNA. A yeast proteomic study of the intracellular copy number of proteins places the Rad24 protein at a level of 752 molecules/cell (42). At this level and assuming full formation with the other RFC subunits, Rad·RFC would exist in the nucleus at a concentration of 710 nM. This level is 7-fold above the concentration needed to saturate the clamp unloading assays in this work. Hence, there may be sufficient amounts of Rad·RFC in the cell to participate in the recycling of PCNA clamps from DNA.

Other alternative clamp loaders that contain Rfc2-5 could also play a role in PCNA unloading. Ctf18·RFC is an alternative clamp loader involved in chromosome cohesion, and it is capable of loading PCNA onto DNA (44). A recent study demonstrated that only 3-10 nM Ctf18·RFC is needed to unload PCNA (33). Bylund and Burgers (33) did not observe PCNA unloading by either Rad·RFC or RFC_1N. The different results observed herein can be explained by the different conditions used in this study relative to those used in the earlier study. For example, the earlier study used much lower concentrations of clamp loader complexes (10 nM), which, compared with this study, are at or below the threshold levels needed to observe PCNA unloading activity.

It seems possible that PCNA clamp unloading could be accomplished by any of several different complexes. Perhaps different clamp loaders assort to different locales in the cell. For example, it has been suggested that Ctf18·RFC may unload PCNA when replication forks meet the cohesion apparatus (33). The Rad·RFC clamp loader is responsible for loading 911 clamps onto DNA in the damage checkpoint response. Rad·RFC may also clear pre-existing PCNA clamps from DNA, perhaps to make room for 911 clamps. An important role of the DNA damage checkpoint is to halt ongoing DNA synthesis. Hence, the inability of Rad·RFC to load PCNA and the ability to unload PCNA may contribute to replication shutdown during DNA damage.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grants GM38839 (to M. O. D.) and GM45547 (to J. K.) and by a Ruth L. Kirschstein National Research Service award from NIGMS, National Institutes of Health (to G. D. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: Dept. of Biophysics, The Johns Hopkins University, Baltimore, MD 21218.

² To whom correspondence should be addressed: Lab. of DNA Replication, Howard Hughes Medical Inst., The Rockefeller University, 1230 York Ave., New York, NY 10021. Tel.: 212-327-7255; Fax: 212-327-7253; E-mail: odonnel{at}mail.rockefeller.edu.

³ The abbreviations used are: PCNA, proliferating cell nuclear antigen; RFC, replication factor C; ATPS, adenosine 5'-O-(thiotriphosphate); DTT, dithiothreitol.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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