The Replication Factor C Clamp Loader Requires Arginine Finger Sensors to Drive DNA Binding and Proliferating Cell Nuclear Antig

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 46, pp. 35531–35543, November 17, 2006 Printed in the U.S.A.

The Replication Factor C Clamp Loader Requires Arginine Finger Sensors to Drive DNA Binding and Proliferating Cell Nuclear Antigen Loading*ࡗ Received for publication, June 26, 2006, and in revised form, September 14, 2006 Published, JBC Papers in Press, September 15, 2006, DOI 10.1074/jbc.M606090200 Aaron Johnson‡, Nina Y. Yao‡, Gregory D. Bowman§, John Kuriyan¶ʈ, and Mike O’Donnell‡** From the **Laboratory of DNA Replication, Howard Hughes Medical Institute and ‡Rockefeller University, New York, New York 10021, the ¶Howard Hughes Medical Institute, Department of Molecular and Cell Biology and Department of Chemistry, University of California, Berkeley, California 94720, the ʈPhysical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, and the §Department of Biophysics, Johns Hopkins University, Baltimore, Maryland 21218

,Replication factor C (RFC) is an AAA؉ heteropentamer that The DNA polymerase binds to the processivity clamp protein couples the energy of ATP binding and hydrolysis to the loading which completely encircles DNA. The clamp slides on DNA Downloaded from of the DNA polymerase processivity clamp, proliferating cell and is pulled along by the polymerase during chain extension, nuclear antigen (PCNA), onto DNA. RFC consists of five sub- continuously holding polymerase to DNA and constraining it units in a spiral arrangement (RFC-A, -B, -C, -D, and -E, corre- to act in a highly processive manner. sponding to subunits RFC1, RFC4, RFC3, RFC2, and RFC5, The eukaryotic ring-shaped processivity factor, proliferating -respectively). The RFC subunits are AAA؉ family proteins and cell nuclear antigen (PCNA),2 is a trimer of three identical sub http://www.jbc.org/ the complex contains four ATP sites (sites A, B, C, and D) units arranged head-to-tail to generate a ring with a large cen- located at subunit interfaces. In each ATP site, an arginine res- tral cavity for encircling DNA (3, 4). PCNA confers processivity idue from one subunit is located near the ␥-phosphate of ATP on DNA polymerase ␦ (5–7) and also interacts with other fac- bound in the adjacent subunit. These arginines act as “arginine tors involved in DNA metabolism such as DNA polymerase ⑀, fingers” that can potentially perform two functions: sensing that flap endonuclease-1, DNA ligase, mismatch repair proteins, ATP is bound and catalyzing ATP hydrolysis. In this study, the and many others (8, 9). An interface between two PCNA mono- at Rockefeller University Library on July 30, 2015 arginine fingers in RFC were mutated to examine the steps in the mers must be disrupted and opened to place DNA into the PCNA loading mechanism that occur after RFC binds ATP. This center of the ring. The ring must then be re-closed for the clamp report finds that the ATP sites of RFC function in distinct steps to remain bound to the DNA and function with the polymerase. during loading of PCNA onto DNA. ATP binding to RFC powers The eukaryotic clamp loader complex, replication factor C recruitment and opening of PCNA and activates a ␥-phosphate (RFC), uses the energy of ATP binding and hydrolysis to recruit sensor in ATP site C that promotes DNA association. ATP the processivity clamp to DNA, break one clamp interface, and hydrolysis in site D is uniquely stimulated by PCNA, and we topologically link the clamp to primed template DNA (10–15). propose that this event is coupled to PCNA closure around Studies in the Escherichia coli system have shown that the DNA, which starts an ordered hydrolysis around the ring. PCNA clamp loader and DNA polymerase compete for the clamp, and closure severs contact to RFC subunits D and E (RFC2 and therefore the clamp loader must eject from the clamp for the RFC5), and the ␥-phosphate sensor of ATP site C is switched off, DNA polymerase to use it (16). Likewise, RFC and Pol ␦ also resulting in low affinity of RFC for DNA and ejection of RFC compete for binding to PCNA (17, 18). Therefore, after PCNA from the site of PCNA loading. is linked to DNA, RFC ejects from the clamp to allow the polymerase access to the clamp (19) (summarized in Fig. 1A). A crystal structure of Saccharomyces cerevisiae RFC-ATP␥S- All cellular organisms utilize a multiprotein ATP-driven PCNA reveals RFC to consist of a circular collar from which the DNA replicase, which functions with other proteins to dupli- five ATP-binding modules are suspended (Fig. 1B) (20). This cate chromosomal DNA prior to cell division (1). DNA repli- subunit arrangement is similar to the E. coli ␥ complex clamp cases are composed of a DNA polymerase, a ring-shaped pro- loader (21), which loads the ring-shaped ␤ clamp onto DNA cessivity clamp, and a clamp loader ATPase (reviewed in Ref. 2). (22). The current study refers to the five RFC subunits based on their sequential order in the pentamer: RFC-A for RFC1, RFC-B * This work was supported by National Institutes of Health Grant GM38839 for RFC4, RFC-C for RFC3, RFC-D for RFC2, and RFC-E for (to M. O. D.), an institutional training grant (to A. J.), the Ruth L. Kirschstein RFC5 (see Fig. 1C). This alphabetical nomenclature facilitates National Research Service Award through the National Institute of General the comparison of subunits in analogous positions in RFC and ␥ Medical Sciences (to G. D. B.), and by National Institutes of Health Grant GM45547 (to J. K.). The costs of publication of this article were defrayed in complex. 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. 2 The abbreviations used are: PCNA, proliferating cell nuclear antigen; RFC, ࡗ This article was selected as a Paper of the Week. replication factor C; ATP␥S, adenosine 5Ј-O-(thiotriphosphate); ssDNA, sin- 1 To whom correspondence should be addressed: 1230 York Ave., Box 228, gle-stranded DNA; SRC, Ser-Arg-Cys; SSB, single-stranded DNA-binding New York, NY 10021-6399. Tel.: 212-327-7255; Fax: 212-327-7253; E-mail: protein; BSA, bovine serum albumin; DTT, dithiothreitol; CHAPS, 3-[(3-chol- [email protected]. amidopropyl)dimethylammonio]-1-propanesulfonic acid.

NOVEMBER 17, 2006•VOLUME 281•NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 35531 The ATP Cycle of RFC

closed ring on DNA. This final stage of the clamp loading mechanism requires hydrolysis of ATP and is currently viewed as a single step, although multiple ATP molecules are hydrolyzed, and thus this stage of the reaction is likely composed of multiple steps. The five RFC subunits each contain three domains (domains I–III), and the primary sequence of these domains is homologous among the five subunits; only the RFC-A (RFC1) subunit has additional N- and C-terminal regions (34, 35). The RFC pentamer complex is mainly held together by a tightly Downloaded from packed “collar” region consisting of domain III of each RFC subunit (see Fig. 1, A and B). The N-terminal ATPase modules, composed of domains I and II, are arranged in a http://www.jbc.org/ right-handed spiral with an overall pitch that closely matches double- stranded DNA (20). A cavity exists in the center of this protein spiral

that is sufficiently large to accom- at Rockefeller University Library on July 30, 2015 modate double-stranded DNA. Fur- thermore, conserved polar and positively charged residues line this cavity and are proposed to interact with DNA (20, 31). There is a gap between two subunits, RFC-A and RFC-E (illustrated in Fig. 1, A and C), which is presumed to provide an exit path for single-stranded DNA from the central cavity (20). Muta- FIGURE 1. ATP-coupled mechanism of RFC. A, schematic model of the ATP binding- and hydrolysis-coupled tion of the conserved residues that steps of PCNA loading. After PCNA is loaded, RFC must disengage from PCNA to allow the polymerase access line the central cavity in ␥ complex to the clamp. B, crystal structure of RFC-ATP␥S-PCNA (20). RFC is partially engaged with the closed PCNA ring. The C-terminal collar of RFC (top) is composed of domain III of each RFC subunit and domains I and II form the causes a decrease in DNA binding, AAAϩ modules that bind ATP and PCNA. C, right: schematic of the arrangement of ATP sites in the AAAϩ which supports the hypothesis that modules of the RFC heteropentamer. Each ATP site is at a subunit interface. The neighboring subunit contains DNA binds in the central cavity of an arginine finger in a conserved SRC motif that interacts with the ␥-phosphate of the ATP bound to the adjacent subunit. Left: schematic depicting ATP site C from the crystal structure of RFC-PCNA (left). ATP (black) these clamp loaders (32). A similar bound to RFC-C (RFC3) interacts with the RFC-C P-loop (yellow). The SRC arginine finger (blue) of RFC-D is study in RFC shows that residues ␥ proximal to the -phosphate of the ATP. within the central cavity are required for DNA binding (36). The structural similarity between prokaryotic and eukaryotic A recent electron microscopic reconstruction of Pyrococcus clamps and clamp loaders suggests that the clamp loading furiosus RFC reveals that PCNA adopts a spiral conformation mechanisms may be similar, in general terms, and biochemical when held open in a complex of RFC-ATP␥S-PCNA-DNA (37). studies in both systems support this suggestion (23–30). The Consistent with this observation, molecular dynamics simula- clamp loader binds ATP to promote clamp binding and open- tions of yeast PCNA indicate that PCNA alone may readily ing. The clamp loader-ATP-open clamp complex then associ- adopt a right-handed spiral when open (38). The spiral confor- ates with primed template DNA. The association of clamp load- mation of open PCNA may allow a more intimate contact er-ATP-clamp complex with DNA does not appear to require between the clamp and the five-subunit spiral of RFC. ATP hydrolysis, and it is thought that DNA is positioned inside The ATPase modules of the five RFC subunits are similar in the central channel of the clamp during this step (20, 31–33). structure to the AAAϩ family of ATPases (39). This family DNA binding triggers ATP hydrolysis in the clamp loader, includes a wide variety of factors that couple ATP binding and which then closes the clamp and ejects from DNA, leaving the hydrolysis to remodel protein substrates (40). A common fea-

35532 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281•NUMBER 46•NOVEMBER 17, 2006 The ATP Cycle of RFC

ture of oligomeric AAAϩ complexes is the location of ATP in this site leads to closure of PCNA around DNA. The crystal sites at the interface of two subunits. Interactions between structure of RFC-PCNA shows that closed PCNA no longer AAAϩ modules of RFC subunits create four interfacial ATP interacts with RFC-D and RFC-E, and thus ring closure is pre- sites that are competent for hydrolysis (20, 41). The four ATP sumed to lead to loss of RFC affinity for PCNA and dissociation sites of RFC have previously been referred to numerically, with of RFC from the PCNA-DNA complex. ATP site 1 at the RFC-D/E interface. In this study we adopt an alphabetical nomenclature for the ATP sites to correspond with EXPERIMENTAL PROCEDURES the RFC subunit nomenclature. Thus, the four ATPase sites are Materials—Unlabeled ADP and ATP were purchased from referred to as ATP sites A–D (Fig. 1C), with site A at the RFC- Sigma, unlabeled ATP␥S and eosin-5-maleimide were from A/B interface and site D at the RFC-D/E interface. RFC-E Roche Diagnostics, and radioactive nucleoside triphosphates (RFC5) also binds nucleotide (20), but the nucleotide-binding were from PerkinElmer Life Sciences, Inc. The following pro- pocket of RFC-E is not competent for hydrolysis nor is it needed teins were purified as described: PCNA and PCNA (Cys81- for RFC activity (25). Only) (50) and E. coli SSB (51). PCNA and RFC concentrations Each of the four interfacial ATP sites in RFC consist of one were determined by absorbance at 280 nm in 6 M guanidinium subunit that binds the ATP, plus side chains from the adjacent hydrochloride using extinction coefficients calculated from the ⑀ ϭ Ϫ1 Ϫ1 subunit (Fig. 1C). One of the most prominent residues emanat- Trp and Tyr content (PCNA, 280 5120 M cm ; RFC, ⑀ ϭ Ϫ1 Ϫ1 Downloaded from ing from the adjacent subunit is a highly conserved arginine 280 158,880 M cm ). All other protein concentrations that is part of a Ser-Arg-Cys (SRC) motif found in RFC Box VII were determined by Bradford assay reagent (Bio-Rad) using (34), a region of sequence similarity found in all clamp loaders. BSA as a standard. PCNAHK is a PCNA construct with a His-tag This arginine side chain is referred to as an “arginine finger” by and a N-terminal 6-residue kinase recognition site (52). analogy to an arginine residue inserted at the active site of the PCNAHK was purified in one step by nickel chelate chromatog- Ras protein by the GTPase-activating proteins (42). The RFC-B raphy. PCNAHK was labeled to a specific activity of 100 dpm/ http://www.jbc.org/ arginine finger functions in ATP site A, and the arginine finger fmol with [␥-32P]ATP using the recombinant catalytic subunit of RFC-C functions in ATP site B and so on (see Fig. 1C). In the of cAMP-dependent protein kinase produced in E. coli (a gift E. coli ␥ complex clamp loader these arginine fingers have been from Dr. Susan Taylor, University of California at San Diego). shown to have two functions: they sense bound ATP and also The following oligonucleotides were synthesized and gel-puri- Ј play a catalytic role in ATP hydrolysis (43, 44). The sensor func- fied by Integrated DNA Technologies: 30-mer, 5 -GCA ATA at Rockefeller University Library on July 30, 2015 tions of the arginine fingers in the ␥ complex clamp loader are ACT GGC CGT CGT TTG AAG ATT TCG-3Ј; 66-mer, thought to be coupled to conformational changes that promote 5Ј-CCA TTC TGT AAC GCC AGG GTT TTC GCA GTC AAC binding to the clamp and DNA (44). The arginine fingers are ATT CGA AAT CTT CAA ACG ACG GCC AGT TAT TGC- needed for ATP hydrolysis (43) and are presumed to act by 3Ј. A 102-mer 3Ј-biotinylated oligonucleotide was synthesized stabilizing the growing negative charge as the ␥-phosphate is and gel-purified by the W. M. Keck Facility at Yale University: cleaved from ATP (21, 42, 45). In both RFC and ␥ complex, 5Ј-CCA TTC TGT AAC GCC AGG GTT TTC GCA GTC AAC ATP␥S binding promotes ring opening and DNA binding, but ATT CGA AAT CTT CAA ACG ACG GCC AGT TAT TGC the clamp does not re-close (12, 27, 33, 46, 47); DNA binding is TCT TCT TGA GTT TGA TAG CCA AAA CGA CCA TTA stabilized instead by direct contacts between the clamp loader TAG-3Ј (Biotin). To form synthetic primed template, the and DNA (27, 32, 33, 36, 48). 30-mer primer oligonucleotide was mixed with either the Previous studies of prokaryotic and eukaryotic clamp loaders 66-mer or 102-mer template strand in a 1:1.2 ratio of primer to suggest that individual ATP sites play specific roles in clamp template in 50 ␮lof5mM Tris-HCl, 150 mM NaCl, 15 mM loading (29, 44, 49). Mutational analysis in yeast RFC has shown sodium citrate (final pH 8.5), then incubated in a 95 °C water that ATP binding to sites C and D of RFC is essential for DNA bath, which was allowed to cool to room temperature over a binding (29), but it is not clear how ATP binding is coupled to 30-min interval. For reactions using primed M13mp18 ssDNA, DNA binding. In E. coli ␥ complex, the arginine finger in ATP the M13mp18 ssDNA was purified (53) and primed with a site D functions allosterically in promoting ␤ clamp association, 30-mer DNA oligonucleotide as described (54). The Bio-Gel and the arginine fingers in sites B and/or C are involved in A-15m resin was purchased from Bio-Rad. Nitrocellulose promoting DNA binding (44). These findings support the sug- membrane circles were purchased from Schleicher and Schuell, gestion that there is a division of activities among ATP sites in and polyethyleneimine-cellulose TLC plates were purchased clamp loaders. Perhaps specific ATP sites control other impor- from EM Science. Streptavidin-coated Dynabead M-280 mag- tant clamp loader actions such as clamp closing and clamp netic beads were purchased from Dynal Biotech. loader ejection from the clamp and DNA. Buffers—Buffer A is 30 mM Hepes-NaOH (pH 7.5), 0.5 mM This report examines different RFC complexes that have one EDTA, 2 mM DTT, and 10% glycerol (v/v). Buffer B is 20 mM or more arginine finger residues mutated to alanine. We show Tris-HCl (pH 7.5), 0.5 mM EDTA, 2 mM DTT, and 10% glycerol that none of the arginine fingers of RFC are needed for the steps (v/v). Membrane Wash Buffer is: 20 mM Tris-HCl (pH 7.5), 10

of PCNA interaction and ring opening. However, our results mM MgCl2, and 150 mM NaCl. Reaction buffer is 20 mM Tris- demonstrate that certain ATP sites of RFC play distinct roles HCl (pH 7.5), 0.1 mM EDTA, 5 mM DTT, 4% glycerol (v/v), and downstream of PCNA opening. The arginine finger in ATP site 40 ␮g/ml BSA. Gel filtration buffer is 20 mM Tris-HCl (pH 7.5), ␮ C is needed for RFC to bind DNA. ATP hydrolysis in site D is 0.5 mM EDTA, 2 mM DTT, 10 mM MgCl2, 100 g/ml BSA, and specifically triggered by PCNA, and we propose that hydrolysis 4% glycerol (v/v). Clamp loading buffer is 30 mM Hepes-NaOH

NOVEMBER 17, 2006•VOLUME 281•NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 35533 The ATP Cycle of RFC

(pH 7.5), 7 mM MgCl2, 130 mM NaCl, 1 mM DTT, 1 mM CHAPS. was applied to a 5-ml Bio-Gel A-15m column (Bio-Rad) equil- ATPase buffer is 20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 5 mM ibrated in gel filtration buffer containing 100 mM NaCl at 23 °C. DTT, and 10% glycerol (v/v). Thirty-five fractions of 180 ␮l each were collected, and 100 ␮l Construction and Purification of RFC(SAC) Mutants—Indi- was analyzed by liquid scintillation. 32P-PCNAHK-DNA com- vidual RFC-B (RFC4), RFC-C (RFC3), RFC-D (RFC2), and plex elutes in the early fractions (fractions 9–15), while the free RFC-E (RFC5) genes were mutated using the QuikChange 32P-PCNAHK elutes later (fractions 18–31). The amount of method (Stratagene). The following mutations were made: PCNA in each fraction was determined from its known specific RFC-B (RFC4) Arg157 to Ala, RFC-C (RFC3) Arg160 to Ala, activity. RFC-D (RFC2) Arg183 to Ala, RFC-E (RFC5) Arg184 to Ala. All Magnetic Bead PCNA Clamp Loading Assay—The 30-mer/ S. cerevisiae five-subunit RFC complexes used in this study 102-mer biotinylated DNA was conjugated to Dynabeads were expressed in E. coli using a two-plasmid system described M-280 Streptavidin (Dynal Biotech), and the DNA-bead conju- previously (55). In all RFC constructs, the first 283 residues of gate was incubated with 5 mg/ml BSA in 10 mM Tris-HCl (pH the RFC-A (RFC1) subunit were deleted and RFC-A contained 7.5), 1 mM EDTA, 150 mM NaCl at room temperature for 30 min an N-terminal His-kinase tag (52). All RFC complexes were with agitation and washed three times in the same buffer until purified as follows. Plasmids were transformed into E. coli no BSA remained in the supernatant. Typical yield was ϳ100 strain BL21(DE3) and grown in 12 liters of Luria broth medium pmol of DNA/mg of Dynabeads, as analyzed by comparing containing 100 ␮g/ml ampicillin and 50 ␮g/ml kanamycin at DNA ethidium bromide staining intensities in a polyacrylamide Downloaded from 37 °C. Upon reaching an OD of 0.6, cells were brought to 15 °C, gel using known amounts of primed DNA as a standard. Clamp and isopropyl ␤-D-thiogalactopyranoside was added to 0.5 mM, loading reactions were performed at 16 °C in loading buffer. followed by a 12-h incubation with shaking at 15 °C. Cells were Mixtures containing final concentrations of 0.5 mM ATP, 170 32 HK harvested by centrifugation, resuspended in 100 ml of Buffer A nM DNA, 510 nM E. coli SSB (tetramer), 700 nM P-PCNA containing 150 mM NaCl, and lysed using a French press at (trimer), but lacking RFC, were agitated by pipette and incu- http://www.jbc.org/ 22,000 p.s.i. 2 mM PMSF was added to the cell lysate, and the bated for 1 min at 16 °C, then clamp loading was initiated upon lysate was clarified by centrifugation. The supernatant was adding RFC to a final concentration of 100 nM. Reactions were applied to a 100 ml of SP-Sepharose (Amersham Biosciences) quenched with a final concentration of 21 mM EDTA (pH 7.5) at column equilibrated in Buffer A containing 150 mM NaCl. Pro- the indicated time points. Quenched reactions were placed on

tein was eluted in a 750-ml gradient from 150–600 mM NaCl. ice for 1 min, then the DNA beads were isolated with a magnetic at Rockefeller University Library on July 30, 2015 The five-subunit RFC complex eluted at ϳ510 mM NaCl, sepa- concentrator (1 min) at 4 °C. Beads were washed twice in clamp rating from RFC subcomplexes that eluted in earlier fractions. loading buffer containing 300 mM NaCl at 4 °C. Protein was Fractions from the SP-Sepharose column containing five-sub- stripped from the beads using 1% SDS and counted by liquid unit RFC were pooled, diluted in Buffer B to a conductivity scintillation. equal to 100 mM NaCl, and loaded onto a 15-ml Fast Flow PCNA Ring Opening Assay—The following residues in the Q-Sepharose column (Amersham Biosciences) equilibrated in S. cerevisiae PCNA gene were mutated by the QuikChange pro- 22 30 62 Buffer B containing 90 mM NaCl. Protein was eluted using an cedure (Stratagene): Cys to Ser, Cys to Ser, Cys to Ser. One 81 80-ml gradient of Buffer B from 90–500 mM NaCl. Fractions buried native cysteine residue (Cys ) at each trimer interface 81 containing RFC eluted at ϳ250 mM NaCl and were pooled, remained in this PCNA construct, referred to as PCNA (Cys - aliquoted, and stored at Ϫ80 °C. The protein yield was ϳ50 mg Only). PCNA ring opening was tested by treatment with eosin- from 12 liters of E. coli culture. 5-maleimide, a thiol-reactive dye. Ring opening reactions con- ATP␥S Binding Assays—Nitrocellulose membrane circles tained 20 mM Tris-HCl (pH 7.1), 200 mM NaCl, 0.5 mM EDTA, ␮ ␥ ␮ 81 ␮ (25 mm) were washed with 0.5 M NaOH, rinsed immediately 10 mM MgCl2, 100 M ATP S, 3 M PCNA (Cys -Only), 3 M with water, and equilibrated in membrane wash buffer before RFC, and 30 ␮M eosin-5-maleimide (final concentrations in 15 35 use. Reactions contained 1 ␮M RFC and [ S]ATP␥S (0–25 ␮M) ␮l). Reactions were incubated without eosin-5-maleimide for 1 ϩ in Buffer B 160 mM NaCl and 10 mM MgCl2 in a total volume min on ice, initiated with eosin-5-maleimide, and quenched of 15 ␮l. After 1-min incubation on ice, 10 ␮l of the reaction was after 30 s with 5 ␮lof1M DTT. Control assays without RFC filtered through a pretreated nitrocellulose membrane on a contained 1.1 mg/ml BSA. Quenched reactions were analyzed glass microanalysis filter assembly (Fisher) at a rate of 60 in the dark on a 10% SDS-polyacrylamide gel, and labeled ␮l/min. The membrane was immediately washed with 150 ␮lof PCNA was visualized in a UV scan (305 nm) on a Fluor-S ice-cold membrane wash buffer at ϳ1 ml/min. Membrane cir- MultiImager (Bio-Rad). cles were dried, and radioactivity was measured by liquid scin- Fluorescent DNA Binding Assays—RFC binding to DNA was

tillation counting. An apparent Kd was determined using the measured using a fluorescently labeled primed template DNA. simple binding model, RFC ϩ ATP␥S 7 RFC-ATP␥S, to fit the A synthetic 30-mer oligonucleotide, modified at the 3Ј-hy- data using KaleidaGraph (Synergy Software). droxyl with a C6-spacer primary amino group, was reacted with Clamp Loading Assays Using Primed M13mp18 ssDNA— a Rhodamine Red-X-NHS-ester (Integrated DNA Technolo- 32 HK Clamp loading was measured using P-PCNA (29.4 nM as gies) to form a Rhodamine Red-X-conjugated 30-mer. “Rh- trimer), which was incubated for 10 min at 30 °C with RFC P/T” primed template DNA was prepared by mixing 1.0 nmol of BCDE(SAC) or wild-type RFC (16.7 nM), in 60 ␮l of Reaction Rhodamine Red-X-conjugated 30-mer (Integrated DNA Tech- Buffer containing primed M13mp18 ssDNA (17 nM), SSB (4.2 nologies) with 1.2 nmol of unlabeled 66-mer in 100 ␮lof5mM ␮ M as tetramer), 0.5 mM ATP, and 10 mM MgCl2. The reaction Tris-HCl, 150 mM NaCl, 15 mM sodium citrate (final pH 8.5)

35534 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281•NUMBER 46•NOVEMBER 17, 2006 The ATP Cycle of RFC

and annealed at 37 °C for 1 h. RFC complexes were titrated from C(SAC), RFC D(SAC), and RFC E(SAC). RFC-A does not con- 0–420 nM into reactions of 60 ␮l final volume containing 20 tain an SRC motif. Therefore, no point mutants of RFC-A were

mM Tris-HCl (pH 7.5), 175 mM NaCl, 10 mM MgCl2,5mM DTT, used in this study. 0.5 mM EDTA, 100 ␮M ATP␥S or ADP, and 35 nM Rh-P/T We also constructed a quadruple arginine finger mutant, DNA. When present, PCNA trimer was at a concentration of RFC BCDE(SAC) complex, which lacks arginine fingers 810 nM. The fluorophore was excited at 570 nm, and a 580–680 entirely. Residual activities inherent to RFC BCDE(SAC) may nm scan was taken. Relative intensity of the peak at 588 nm was be presumed to be independent of the arginine fingers. All of

plotted versus RFC concentration. Apparent Kd measurements the single RFC(SAC) mutants should therefore be able to pro- were determined using a simple model (RFC ϩ DNA 7 RFC- gress through the PCNA loading mechanism at least as far as DNA) to fit the data using the KaleidaGraph program (Synergy the RFC BCDE(SAC) mutant. The RFC BCDE(SAC) complex Software). Attempts to measure primer-template association and the single RFC(SAC) complexes were expressed and puri- by a change in fluorescence anisotropy were complicated by a fied similarly to wild-type RFC and have comparable appear- low signal of RFC that bound nonspecifically to regions other ance in an SDS-polyacrylamide gel (Fig. 2A), suggesting that the than the primer-template junction. Measurement of fluores- RFC(SAC) mutants fold properly. cence intensity change of the probe at the primer-template Previous studies of the arginine fingers in the E. coli ␥ com- junction avoids the influence of this nonspecific binding activ- plex clamp loader concluded that these residues do not contrib- ity. Mutation of DNA-interacting residues of RFC-B, -C, and -D ute to ATP binding (43). To test whether this is true for the RFC Downloaded from was performed as described previously (36). arginine fingers, we examined the ability of the RFC Steady-state ATP Hydrolysis of RFC(SAC) Mutants—ATPase BCDE(SAC) mutant to bind nucleoside triphosphate. Using a ␣ 32 ␮ assays contained 500 nM RFC, 1 mM [ - P]ATP, 2 M PCNA nitrocellulose filter binding assay we determined the Kd value of trimer (when present), and 1 ␮M synthetic 30/66 DNA (when wild-type RFC for ATP␥Stobeϳ3.3 ␮M. The RFC BCDE(SAC)

␮ ␥ ␮ http://www.jbc.org/ present) in a final volume of 30 l of ATPase buffer containing quadruple mutant displayed a Kd for ATP Sof5.5 M, compa- 150 mM NaCl. The synthetic primer/template DNA is linear rable with wild-type RFC (Fig. 2B). This result indicates that the and allows PCNA to slide off the ends after it is loaded. Hence, arginine fingers of RFC do not significantly contribute to ATP PCNA is continuously recycled during these assays. Reactions binding. were brought to 30 °C and initiated upon addition of RFC. Ali- The RFC BCDE(SAC) Complex Cannot Load PCNA—To test ␮ quots of 5 l were removed at 1 min intervals from 0–3 min (for whether ATP binding to RFC can promote PCNA loading in at Rockefeller University Library on July 30, 2015 “ϩDNA” and “ϩPCNAϩDNA” samples) or at 5 min intervals the absence of the arginine fingers, we measured the ability of from 0–20 min (for “alone” and “ϩPCNA” samples) and the RFC BCDE(SAC) mutant to recruit PCNA to a primer- quenched with an equal volume of 0.5 M EDTA (pH 7.5). 1 ␮lof template DNA. A circular single-stranded M13mp18 phage each quenched aliquot was spotted on a polyethyleneimine cel- DNA, uniquely primed with a DNA oligonucleotide and coated lulose TLC sheet (EM Science) and developed in 0.6 M potas- with E. coli SSB, was used as a substrate for PCNA loading. To sium phosphate buffer (pH 3.4). The TLC sheet was dried, and follow clamp loading on DNA we used a PCNA construct con- [␣-32P]ATP and [␣-32P]ADP were quantitated using a Phos- taining a six-residue N-terminal kinase recognition sequence phorImager (GE Healthcare). Three independent experiments that enables it to be labeled with [32P]phosphate. The large were performed and the standard deviation is indicated in the DNA template, and 32P-PCNA attached to it, elutes in the early figure chart. A similar procedure was used when PCNA was fractions (fractions 9–15) of a Bio-Gel A-15m gel filtration col- titrated into a fixed concentration of either wild-type RFC or umn, while the comparatively small 32P-PCNA that is not RFC E(SAC), except that each reaction was analyzed at a fixed 2 attached to DNA elutes in later fractions (fractions 18–31) (Fig. min time point. 2C). Incubation of 32P-PCNA with the primed DNA, ATP, and wild-type RFC generates a peak of 32P-PCNA that co-elutes RESULTS with DNA in the early fractions (Fig. 2C). However, the RFC Arginine Finger Mutants of RFC Bind ATP Similarly to Wild BCDE(SAC) mutant does not load PCNA onto DNA. A small Type—The N-terminal region of RFC-A, containing the first amount of 32P-PCNA in the DNA-containing fractions was 283 residues, is not required for cell viability, displays nonspe- observed for the RFC BCDE(SAC) sample. This profile is cific DNA binding activity, and deletion of this region results in observed when 32P-PCNA is applied to the BioGel column in a complex with higher specific activity than RFC containing the absence of clamp loader and DNA (data not shown) and full-length RFC-A (56, 57). All RFC complexes used in this therefore is likely due to a small amount of aggregated 32P- study carry a deletion of this region. The RFC complex contain- PCNA in the protein preparation. Therefore, one or more argi- ing the truncated RFC-A with no additional point mutations to nine fingers are needed to load PCNA onto DNA. any subunit is referred to herein as “wild-type” RFC. The Arginine Fingers of ATP Sites C and D Are Required for Arginine to alanine mutations were constructed in each of Efficient PCNA Loading—We next addressed which arginine the four S. cerevisiae RFC subunits with SRC motifs. Five-sub- fingers are required for RFC to load PCNA by testing single unit complexes of RFC-A/B/C/D/E containing the mutation in RFC(SAC) mutants for PCNA loading activity. Since wild-type either RFC-B, RFC-C, RFC-D, or RFC-E were expressed in RFC can load PCNA onto DNA in a very short time frame, we E. coli using a two-plasmid system and purified as described designed a PCNA loading assay that allows rate measurements previously for wild-type RFC (55). These single mutant five- over a 25-s time course (see scheme in Fig. 3). In this assay, a subunit complexes are referred to as: RFC B(SAC), RFC synthetic primed template is first conjugated to a magnetic

NOVEMBER 17, 2006•VOLUME 281•NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 35535 The ATP Cycle of RFC Downloaded from

FIGURE 3. RFC D(SAC) and RFC E(SAC) are severely deficient in PCNA loading. The scheme at the top illustrates the magnetic bead-conjugated primed DNA assay to measure the kinetics of 32P-PCNA loading. E. coli SSB blocks the end of the DNA to prevent 32P-PCNA sliding off the linear DNA. The time- 32

course of P-PCNA loading by the indicated RFC complexes was performed http://www.jbc.org/ as described under “Experimental Procedures.”

ing of E. coli SSB, which blocks PCNA from sliding off (51). 32P-Labeled PCNA was added to the magnetic bead-coupled primed DNA, and clamp loading was initiated upon addition of

RFC. Reactions were rapidly quenched at timed intervals upon at Rockefeller University Library on July 30, 2015 adding EDTA and then placed at 4 °C. PCNA remains stably associated with the SSB-coated DNA with a half-life of ϳ50 min at 4 °C (data not shown). The magnetic bead-conjugated DNA template enabled rapid separation of PCNA-DNA complexes from free PCNA within 1 min. Western analysis showed that RFC does not remain bound to the PCNA-DNA complex (data not shown). The single (SAC) mutants displayed a spectrum of PCNA loading rates (Fig. 3). The RFC B(SAC) mutant was comparable with wild-type RFC. The RFC C(SAC) mutant was moderately deficient, displaying ϳ35% the rate of clamp loading by wild- type RFC. In contrast, the RFC D(SAC) and RFC E(SAC) mutants were less than 5% the rate of wild-type RFC, suggesting that ATP sites C (location of the RFC-D(SAC) mutation) and ATP site D (location of the RFC-E(SAC) mutation) play important roles in driving PCNA loading. The Arginine Finger Mutants of RFC Can Bind and Open PCNA—Which steps in clamp loading are the arginine finger FIGURE 2. The RFC arginine fingers are required for PCNA loading, but not mutants of RFC deficient in? Do any of the steps occur prior to for ATP binding. A, analysis of 6 ␮g each of wild-type and mutant RFC com- ATP hydrolysis? We used the non-hydrolyzable ATP analogue, plexes in a 10% SDS-PAGE stained with Coomassie Brilliant Blue (G250). ␥ Migration positions of the five RFC subunits are indicated to the left. B, wild- ATP S, to test the single RFC(SAC) mutants for a role prior to type RFC (black diamonds) and RFC BCDE(SAC) (red circles) were incubated ATP hydrolysis. PCNA ring opening and DNA binding do not with the indicated amounts of [35S]ATP␥S followed by analysis of bound nucleotide on nitrocellulose filters as described under “Experimental Proce- require ATP hydrolysis by RFC, and therefore we developed dures.” C, wild-type RFC (black diamonds) and RFC BCDE(SAC) (red circles) assays to examine these two steps. were tested for ability to load 32P-PCNA onto singly primed circular M13mp18 First, we developed a PCNA opening assay based on a similar ssDNA coated with E. coli SSB (see scheme at top). DNA-bound 32P-PCNA migrates early in the elution profile (fractions 9–15) while unbound 32P-PCNA assay for ring opening that we used previously for the E. coli migrates later (fractions 18–31). system (27, 58). We prepared a construct of PCNA in which the three surface-exposed cysteine residues were mutated to serine, bead. To prevent PCNA sliding off the free end of the linear leaving only one cysteine, Cys81, that is buried at each trimer DNA, the template was designed with sufficient single- interface (Fig. 4A). This “PCNA (Cys81-Only)” clamp is resist- stranded DNA on both sides of the primer to facilitate the bind- ant to labeling by the thiol-reactive dye, eosin-5-maleimide

35536 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281•NUMBER 46•NOVEMBER 17, 2006 The ATP Cycle of RFC

ciency of the RFC D(SAC) and RFC E(SAC) mutants (e.g. see Fig. 3) must be blocked at a step in the PCNA loading mechanism that occurs after PCNA opening. A ␥-Phosphate Sensor in ATP Site C of RFC Regulates DNA Binding—We next examined the RFC mutants for ability to bind DNA. ATP binding is known to promote RFC association with DNA, even in the absence of PCNA (28). To analyze DNA binding by RFC, we used a synthetic DNA with a fluorescent rhodamine derivative attached to the 3Ј terminus of the primer strand (see scheme in Fig. 5). We refer to this template as Rh- P/T. A change in fluorescence intensity indicates association of RFC at the primer-template junction and we have shown previously that RFC binds this DNA substrate with an affinity comparable with that of unmodified DNA (28, 36). In Fig. 5A, we measured the affinity for DNA of wild-type and arginine finger mutant RFC complexes by titrating RFC into a fixed concentration of Rh-P/T DNA in the presence of ATP␥S. Downloaded from Wild-type RFC produces an increase in fluorescence intensity in this assay. Fig. 5B shows that ADP does not suffice to power the conformational change in wild-type RFC needed for DNA binding. The RFC C(SAC) and RFC B(SAC) mutants had affin- ities for the primed DNA that were equal or 2-fold reduced, http://www.jbc.org/ respectively, compared with wild-type RFC; the RFC E(SAC) mutant displayed a 6-fold reduced affinity for the primed DNA. However, RFC D(SAC) lost essentially all detectable DNA bind- Ͼ ␮ ing in this assay (Kd 5 M). Therefore, the arginine finger of

RFC-D is likely needed to recognize ATP in site C to promote at Rockefeller University Library on July 30, 2015 the conformational change in RFC required for DNA binding. The RFC BCDE(SAC) complex did not display significant DNA binding activity, presumably due to the D(SAC) mutation FIGURE 4. PCNA ring opening assays. A, space-filling representation of the PCNA clamp structure. The three PCNA monomers are colored blue, yellow, (Fig. 5C). Surprisingly, when the RFC BCDE(SAC) mutant was and cyan. Cysteine 81 (sulfur atom in orange) of PCNA is buried at each trimer titrated into a reaction with the Rh-P/T DNA in the presence of interface of the ring (left diagram). In the diagram to the right, one monomer saturating PCNA, a significant amount of DNA binding activity is removed to expose Cys81 at the trimer interface. B, scheme of eosin-5- maleimide labeling assays using PCNA (Cys81 only). The top panel is a control was observed (Fig. 5C). ADP could not substitute for ATP␥Sto reaction comparing eosin-labeling reactions containing no RFC, wild-type promote this PCNA-dependent DNA binding (Fig. 5C), which RFC, or RFC lacking domains I and II of RFC-E (RFC E(⌬I,II)). The middle panel compares single RFC(SAC) mutants and wild-type RFC in the PCNA opening may reflect the need for RFC to bind ATP to interact with assay. The bottom panel compares PCNA ring opening reactions with no RFC, PCNA. This result demonstrates that PCNA partially rescues wild-type RFC, or RFC BCDE(SAC). Proteins were separated by 10% SDS-PAGE the DNA binding deficiency of the RFC D(SAC) mutant. and visualized by UV scanning at 305 nm on a Fluor-S Multi-Imager (Bio-Rad). Removing DNA Interaction Residues in RFC-B, -C, or -D Causes a Deficiency in DNA Binding—The result that RFC (Fig. 4B). RFC and ATP␥S stimulate labeling of Cys81 in PCNA D(SAC) is deficient in association with primed template DNA (Cys81-Only), which is likely due to PCNA ring opening, thus indicates that ATP site C is important in modulating RFC-DNA increasing exposure of the buried Cys81 for reaction with the interaction. Three conserved positively charged residues of fluorescent maleimide. As a control we tested an RFC complex RFC-D are predicted to interact with the phosphate backbone that lacks domains I and II of RFC-E (RFC5), which binds but of double-stranded DNA that is bound in the central chamber does not open or unload PCNA from DNA (36). This mutant of RFC (Fig. 6A) (20, 31, 32). Previously, we demonstrated that RFC did not stimulate labeling of the PCNA (Cys81-only) in the removal of these residues from RFC-D, in combination with presence of ATP␥S (Fig. 4B, top). We were unable to perform a removal of the corresponding residues in RFC-B and RFC-C, control reaction in the absence of ATP␥S, because in the abolished DNA binding by RFC (36). We used the DNA binding absence of nucleotide, RFC and PCNA form an insoluble assay described above to test an RFC complex in which only the aggregate.3 three RFC-D residues were mutated. The result demonstrates Next, we tested various RFC(SAC) mutants for ability to that the RFC D(R101A,R107A,R175A) mutant does not pro- open PCNA in the presence of ATP␥S. The single RFC(SAC) duce a change in fluorescence intensity like wild-type RFC (Fig. mutants were all active for PCNA opening (Fig. 4B, middle). In 6B), suggesting that removal of these residues is sufficient to fact, even the RFC BCDE(SAC) mutant was capable of opening break the strong interaction between RFC and the primed tem- PCNA (Fig. 4B, bottom). Therefore the clamp loading defi- plate. Interestingly, RFC complexes with the corresponding three mutations in either RFC-B or RFC-C were similarly defi- 3 G. D. Bowman and J. Kuriyan, unpublished observation. cient in DNA binding (Fig. 6B). This disruption of the interac-

NOVEMBER 17, 2006•VOLUME 281•NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 35537 The ATP Cycle of RFC

loading after formation of the RFC- PCNA-DNA complex, resulting in closure of the clamp and ejection of RFC. Next we examine the effect of individual arginine finger mutations on the steady-state rate of ATP hydrolysis of RFC using a synthetic primed/template in the presence of PCNA. The short linear primed DNA allows PCNA to slide off the DNA after it is loaded, and thus RFC can perform repeated clamp loading events without running out of PCNA and DNA substrate. Wild- type RFC produced a steady-state rate of ϳ123 ATP hydrolyzed/RFC/ min (Fig. 7A). The RFC BCDE(SAC) Downloaded from mutant gave no ATP hydrolysis within the time frame of the assay. Next, we examined the rate of ATP hydrolysis of the single RFC(SAC) mutants. If all four ATP sites con- http://www.jbc.org/ tribute equally to the ATPase cycle of RFC, each single RFC(SAC) mutant may be expected to hydrolyze ATP at ϳ75% the rate of wild-

type RFC. However, the results at Rockefeller University Library on July 30, 2015 show that two of the RFC(SAC) mutants, RFC D(SAC) and RFC E(SAC), hydrolyze ATP at less than 20% the rate of wild-type RFC. We have shown earlier in this report that the RFC D(SAC) mutant is deficient in DNA binding. If the RFC D(SAC) mutant does not bind DNA, ATP hydrolysis will be com- promised since DNA stimulates ATP hydrolysis by RFC (Fig. 7B). However, the ATPase reaction is performed in the presence of sufficient PCNA to enable the RFC FIGURE 5. The RFC-D arginine finger is required for DNA binding by RFC. The fluorescent primed template contains a 3Ј-terminal rhodamine moiety attached to the primer strand. Addition of RFC in the presence of D(SAC) mutant to bind to DNA. ATP␥S results in an increase in fluorescence intensity (see scheme at top). A, wild-type RFC and RFC(SAC) Therefore the RFC D(SAC) and mutants were titrated into a reaction containing the fluorescent primed template and the relative fluorescence RFC E(SAC) mutants are likely intensity change (I/I0) was measured. The Kd values for RFC interaction with the primed template are indicated to the right. B, wild-type RFC was titrated into reactions containing the fluorescent primed template in either bound to PCNA and DNA, but the the presence of ATP␥S(black diamonds) or ADP (red open circles). C, RFC BCDE(SAC) was titrated into reactions ATP hydrolysis cycle is blocked, ␥ containing the fluorescent primed template and ATP S in the presence (closed circles) or absence (closed preventing clamp loading. One pos- diamonds) of a saturating concentration of PCNA (810 nM). RFC BCDE(SAC) was also titrated into the reaction in the presence of ADP and PCNA (crosses). sible explanation for this is that ATP bound at the mutant site must tion between any one RFC subunit and the phosphate backbone hydrolyze before the remaining ATP sites can fire. The RFC of DNA appears to be sufficient to prevent RFC-DNA C(SAC) and RFC B(SAC) mutants each hydrolyze ATP at about interaction. half the rate of wild-type RFC. Therefore, inability to hydrolyze The Arginine Fingers of RFC-D and RFC-E Are Most Impor- ATP in sites A or B also hinders hydrolysis in other sites, but the tant for the ATPase Cycle of RFC—The experiments described block is less severe than when sites C or D are mutated. These above show that the arginine fingers of RFC are not needed for assays were performed in the presence of PCNA and DNA. ATP binding or PCNA opening but are needed for DNA bind- Addition of either PCNA or DNA alone to RFC also stimulates ing (i.e. the RFC-D arginine finger). The arginine fingers are the rate of ATP hydrolysis by RFC but to a lesser extent than also needed for ATP hydrolysis which drives the steps of PCNA when they are both present with RFC simultaneously.

35538 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281•NUMBER 46•NOVEMBER 17, 2006 The ATP Cycle of RFC Downloaded from http://www.jbc.org/ at Rockefeller University Library on July 30, 2015

FIGURE 7. DNA stimulates ATP hydrolysis in ATP site C of RFC. All ATPase assays were performed as described under “Experimental Procedures.” Reac- tions were performed in triplicate and error bars indicate one standard deviation of the data set. A, steady-state ATPase activity of single RFC(SAC) and FIGURE 6. Residues in the central chamber of RFC are required for DNA RFC BCDE(SAC) mutants in the presence of PCNA and the synthetic primed binding. Three residues from RFC-B, RFC-C, and RFC-D that are proposed to template. B, ATPase activity of RFC(SAC) triple mutants (i.e. three mutant sub- interact with DNA were mutated to alanine (A, from RFC-PCNA crystal struc- units in each complex) in the absence (Ϫ) or presence (ϩ) of DNA. ture with DNA modeled through the central cavity of RFC and PCNA (20)). B, DNA binding assays were performed with a primer-template DNA labeled at the 3Ј end of the primer (see scheme at top). An increase in the relative fluo- PCNA Triggers ATP Hydrolysis in ATP Site D—PCNA syner- rescence (I/I0) indicates association of RFC with the DNA. RFC complexes with mutations in DNA-interacting residues of either RFC-B (R84A,R90A,K149A), gistically stimulates ATP hydrolysis by wild-type RFC in the RFC-C (R88A,R94A,K152A), or RFC-D (R101A,R107A,R175A) were assayed presence of DNA, presumably reflecting repeated cycles of along with wild-type RFC. PCNA loading. In contrast, the RFC E(SAC) mutant displayed more steady-state ATP hydrolysis activity in the presence of DNA Triggers ATP Hydrolysis in Site C—At 1 ␮M DNA, DNA alone than in the presence of PCNA and DNA (Fig. 7A which is saturating in the ATPase assay, ATP hydrolysis by and data not shown). This PCNA effect is examined more wild-type RFC and all four single RFC(SAC) mutants reaches a closely in Fig. 8A by titrating PCNA into reactions containing maximum, but the RFC D(SAC) mutant was deficient in DNA- RFC and primed template DNA. The steady-state rate of ATP stimulated ATP hydrolysis in the presence or absence of PCNA hydrolysis by wild-type RFC in the presence of primer-template (Fig. 7A and data not shown). This result suggests that DNA DNA is stimulated by PCNA, while PCNA has the opposite may trigger ATP hydrolysis in the unmutated ATP site C of effect on the ATPase activity of the RFC E(SAC) mutant. PCNA wild-type RFC. To test this possibility we compared the rate of inhibition of ATP hydrolysis by the RFC E(SAC) mutant DNA-stimulated ATP hydrolysis of the triple RFC(SAC) implies that when ATP in site D cannot be hydrolyzed, PCNA mutants. Triple RFC(SAC) mutants contain only one ATP site blocks the ATPase cycle. that is competent for hydrolysis. The RFC BCE(SAC) mutant, In Fig. 8B we tested the RFC E(SAC) mutant and the other which only contains a competent ATP site C, is most strongly single RFC(SAC) mutants for ATPase activity in the absence stimulated by DNA and in fact displays a rate of ϳ23 ATP and presence of PCNA, without DNA. The RFC E(SAC) mutant hydrolyzed/RFC/min, about 80% the rate of wild-type RFC (Fig. is not significantly stimulated by PCNA. This result suggests 7B). This result indicates that DNA triggers ATP hydrolysis in that association of PCNA with RFC-ATP normally stimulates site C and suggests that it may drive hydrolysis in additional the hydrolysis of ATP bound to site D. To test this proposal, we ATP sites. examined the triple RFC(SAC) mutants for PCNA-stimulated

NOVEMBER 17, 2006•VOLUME 281•NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 35539 The ATP Cycle of RFC Downloaded from http://www.jbc.org/ at Rockefeller University Library on July 30, 2015

FIGURE 8. PCNA triggers ATP hydrolysis in ATP site D of RFC. Reactions were performed as in Fig. 7 unless indicated. Error bars indicate one standard deviation of the data set. A, PCNA was titrated into a reaction containing primed DNA and either wild-type RFC or RFC E(SAC); ADP production was analyzed after 2-min incubation. B, ATPase activity of RFC(SAC) single site mutants was analyzed in either the absence (Ϫ) or presence (ϩ) of PCNA. C, ATPase activity of RFC(SAC) triple site mutants was analyzed in either the absence (Ϫ) or presence (ϩ) of PCNA.

ATPase activity (Fig. 8C). The results show that PCNA has the in clamp loading. The results reveal individual functions of dif- greatest stimulatory effect on ATP hydrolysis by the RFC ferent ATP sites during the clamp loading cycle. BCD(SAC) triple mutant, which only has a competent ATP site PCNA Is Open Prior to ATP Hydrolysis—In the absence of D (Fig. 8C). PCNA stimulates the basal rate of the triple RFC ATP, neither E. coli ␥ complex nor eukaryotic RFC bind to their BCD(SAC) mutant 10-fold, which is significantly more than the respective clamp or to DNA (28, 48, 59, 60). ATP binding pow- 3–4 fold stimulation by PCNA on wild-type RFC ATP ers a conformational change that enables the clamp loader to hydrolysis. bind the clamp and DNA (12, 27, 33, 46, 47). Use of ATP␥S DISCUSSION promotes ring opening and ring loading onto DNA, but the ring stays open and the clamp loader also remains with the open The spiral heteropentameric RFC clamp loader from S. cer- clamp on DNA. This was demonstrated in the E. coli system evisiae has four bipartite ATP sites, each located at subunit- ␤ subunit interfaces (see Fig. 1). At each site one subunit contrib- using a labeled maleimide and a clamp containing a cysteine utes a catalytic arginine finger that is contained within a highly residue at a buried position within the clamp interface (27). In ␥ conserved SRC motif in RFC box VII. The other subunit con- the presence of ATP S, clamp opening is signaled by an tains the P-loop and other ATP binding elements. In this report, increase in reactivity of the buried cysteine residue at the inter- conserved arginine fingers are mutated individually and in face. Several lines of evidence exist for RFC holding PCNA open combination and the resulting ATP site mutant RFC complexes while it is bound to DNA with ATP␥S: 1) an electron micro- are examined for ability to progress through the different steps graphic reconstruction of a P. furiosus RFC-ATP␥S-PCNA-

35540 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281•NUMBER 46•NOVEMBER 17, 2006 The ATP Cycle of RFC Downloaded from http://www.jbc.org/ at Rockefeller University Library on July 30, 2015

FIGURE 9. Model of ATP site function by RFC during clamp loading. Schematic models depicting the findings of this study in the context of the PCNA loading cycle. A, ATP binding powers binding and opening of PCNA by RFC. The arginine fingers of RFC are not involved in the formation of this RFC-ATP-(open)PCNA complex. B, ␥-phosphate recognition by the ATP site C arginine finger coordinates RFC-(open)PCNA binding to DNA through the central chamber. C, ATP hydrolysis in site D is coupled to closure of PCNA by breaking the RFC-D/E contact to the clamp. D, DNA triggers ATP hydrolysis first in site C, which proceeds to site B and then site A. RFC dissociates from the site of loading, aided by the lowered affinity for DNA due to the lack of triphosphate in site C.

DNA complex shows an open PCNA ring in a right-handed The current study of arginine finger mutants of RFC demon- helix (37), 2) A FRET study of yeast RFC indicates that the strates that the arginine finger of RFC-D is required for RFC to clamp remains open upon DNA binding (33), similar to the EM bind DNA. This ATP site corresponds to site C of E. coli ␥ study of the P. furiosus clamp loader, and 3) study of RFC complex (at the interface of two ␥ subunits), the one site that mutants indicates that the ring is open in the RFC-ATP␥S- remains closed in ␥ complex when it is cocrystallized with PCNA-DNA complex and that RFC, not PCNA provides the ATP␥S (61). Furthermore, the ␥ complex structure with two grip that holds RFC-ATP␥S-PCNA to DNA (36). bound ATP␥S molecules is in essentially the same conforma- The PCNA ring is closed in the RFC-ATP␥S-PCNA struc- tion as inactive ␥ complex with no bound nucleotide. There- ture in which the four arginine fingers of RFC were mutated to fore, binding of ATP to site C is presumed to power the confor- glutamine to inhibit nucleotide hydrolysis (20). The reason for mational change in ␥ complex required to bind both ␤ and this result is not understood at present. However, the studies of DNA. The ATP site C arginine finger mutation of RFC is the ¡ the current report show that the RFC with four SRC SAC only one that blocks a step powered by ATP binding, and thus mutations (i.e. RFC BCDE(SAC)) is still active in binding and one may infer that ATP site C of RFC may carry a similar impor- opening PCNA. The RFC BCDE(SAC) mutant is also capable of tance to the ATP-dependent conformation of RFC as ATP site assembling PCNA onto DNA with ATP␥S, forming the RFC- CinE. coli ␥ complex. However, structures of the fully ATP ATP␥S-PCNA-DNA complex (Fig. 5C),4 which is consistent bound state of ␥ complex and of the unliganded state of RFC with the ability of RFC BCDE(SAC) to open PCNA (Fig. 4B, will be needed to confirm whether this prediction is indeed the bottom). case. The ATP Cycle of RFC—The scheme in Fig. 9 summarizes a 4 A. Johnson, N. Y. Yao, G. D. Bowman, J. Kuriyan, and M. O’Donnell, unpub- model of the ATP hydrolysis cycle of RFC inferred from the lished results. findings of this report. ATP binding powers all the steps that

NOVEMBER 17, 2006•VOLUME 281•NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 35541 The ATP Cycle of RFC lead to formation of the complex of RFC bound to an open, yields a complex that no longer interacts tightly with DNA spiral PCNA clamp (diagram A). To take the next step, binding (44). Thus, there are striking parallels between arginine fin- to DNA, the arginine finger of RFC-D is needed to sense the ger mutants of RFC and ␥ complex. The arginine finger of ␥-phosphate of ATP bound to site C. This ATP sensing step RFC in ATP site C is needed for DNA binding and thus has brings the RFC-PCNA complex into the proper conformation similar behavior to the arginine finger mutation in ATP sites to bind primed DNA (diagram B). This conformational change BandCin␥ complex. If RFC and ␥ complex act in a similar could be either subtle, for example by bringing the DNA bind- fashion, then one may predict that the arginine finger in ATP ing residues of RFC-D into proper alignment for interaction site C of ␥ complex, and not the arginine in ATP site B, is with the phosphate backbone, or could be a more global change, responsible for the ATP binding-induced DNA affinity of ␥ such as altering the spiral pitch of the AAAϩ domains of the complex. Mutation of the ␥ complex arginine finger in ATP five subunits of RFC, thus bringing all the subunits into register site D (between the D and E positions), like the same muta- for DNA interaction. The results presented in Fig. 6B indicate tion in RFC, prevents ATPase activity in response to the that removal of DNA-interacting residues from either RFC-B, clamp (43). Interestingly, the RFC-D and RFC-E subunits of -C, or -D eliminates RFC-DNA contact, suggesting that local RFC are needed to open the PCNA clamp (36). However, in a changes in RFC-D may be sufficient to modulate DNA binding significant departure between the activities of the eukaryotic of the RFC complex. RFC and ␥ complex, the ␦ wrench (A subunit) of ␥ complex Downloaded from RFC-PCNA binds to primed DNA through the slot between opens the ␤ clamp, while RFC-A lacks this activity (36). It is RFC subunits A and E, and through the open gap in PCNA. interesting to note that the proposed order of ATP hydroly- Once the DNA and PCNA substrates are properly oriented, the sis in ␥ complex (43) is also opposite to that proposed here ATP hydrolysis phase may begin. We propose in Fig. 9 that ATP for RFC. site D may fire first, allowing PCNA to close (diagram C), fol- The RFC of Archaeoglobus fulgidus is similar to E. coli ␥ com- http://www.jbc.org/ lowed by hydrolysis at sites C, B, and A. We have shown previ- plex and eukaryotic RFC in the ability to load a clamp onto ously that RFC-E, possibly along with RFC-D, opens the PCNA DNA prior to ATP hydrolysis, and in all cases the clamp loader clamp (36). We show here that hydrolysis in ATP site D is spe- remains on DNA with the clamp. One may presume that the cifically stimulated by PCNA, and we propose that hydrolysis clamp remains open from the work in the E. coli system and leads to PCNA closing around DNA, thereby severing the con- from the electron micrographic reconstruction of the RFC- tact to RFC-D/E (diagram C). ADP formation in the ATP bind- at Rockefeller University Library on July 30, 2015 ATP␥S-PCNA-DNA complex from the archaeon P. furiosus ing pocket of RFC-D (ATP site D) may then act by a local allo- (37). Archael RFC complexes consist of two different subunits: steric mechanism to position the RFC-D arginine finger for RFC-s (s for small) and RFC-l (l for large), in a 4:1 ratio, respec- catalysis of ATP in site C. This wave of cause-and-effect may tively. Studies on A. fulgidus RFC suggest that four ATP mole- lead to hydrolysis proceeding around the pentamer to ATP site cules bind to archaeal RFC and are hydrolyzed during a full B and then ATP site A, which is located across the subunit gap clamp loading cycle (49). A. fulgidus RFC with arginine finger from ATP site D. The fact that RFC activity is least affected by mutations in the four RFC-s subunits still binds ATP but is no mutation of ATP site A, compared with the other ATP sites, is consistent with hydrolysis of this site occurring at the end of the longer functional in formation of the RFC-PCNA-DNA ternary hydrolysis cycle. complex (62), suggesting that a step prior to hydrolysis is dis- Hydrolysis of ATP in site C is sensed by the arginine finger of rupted by this mutation. This step correlates with the finding RFC-D, thus lowering the affinity of RFC for DNA. A decrease here that the arginine finger of RFC-D is needed to sense ATP in affinity of RFC for DNA is also promoted by the absence of binding in site C for RFC to bind DNA. nucleotide in ATP sites C and D as indicated by a study of Clamp loader subunit sequences and clamp loader structures P-loop mutants of RFC that no longer bind ATP (29). In the are similar in all three domains of life: bacteria, archaea, and model of Fig. 9, ATP hydrolysis continues to site B and then the eukaryotes (20, 21, 34, 37, 63–66). ATP sites C and D in RFC, ATP site of RFC-A is last to fire. RFC-A forms a tight connec- which are critical for efficient PCNA loading, also are present in tion to PCNA (20), and hydrolysis in site A may loosen this bacterial and archaeal clamp loaders. These two ATP sites are contact (diagram D). Hence, the ATP hydrolysis cycle of Fig. 9 also present in the alternate RFC clamp loaders in which the A first causes release of two RFC subunits (D and E) from PCNA, subunit (RFC1) exchanges with another protein (e.g. Rad24 then loosens the interaction of RFC with DNA (site C) followed (Rad17 in humans), Ctf18, or Elg1). These “alternate” RFC com- by severing the remaining contact between RFC-A and PCNA, plexes act in other cellular processes such as DNA damage thus ejecting the clamp loader. repair and sister chromatid cohesion (15). For example, the Comparison with Other Clamp Loaders—Unlike RFC, E. coli Rad24 subunit substitutes for RFC-A to form Rad24-RFC ␥ complex contains only three ATP sites. These sites corre- which loads the 9-1-1 heterotrimer clamp onto DNA (67, 68). spond to ATP sites B, C, and D of RFC. In ␥ complex, the The Ctf18-RFC acts in sister chromatid cohesion and is effi- arginine finger of ATP site D is contained in ␦Ј (E subunit), cient in unloading PCNA, leading to the suggestion that it may and mutation of this arginine yields a clamp loader complex serve in this capacity when replication forks deal with cohesins that has lower affinity for the ␤ clamp (44). Arginine fingers (69–71). Since ATP sites C and D are present in all alternate of the other ATP sites cannot be mutated individually since clamp loaders, these two sites may be expected to perform sim- the ␥ subunits are encoded by the same gene. However, ilar functions to those shown here for the replicative RFC mutation of the arginine fingers in both ATP sites B and C complex.

35542 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281•NUMBER 46•NOVEMBER 17, 2006 The ATP Cycle of RFC

REFERENCES 38. Kazmirski, S. L., Zhao, Y., Bowman, G. D., O’Donnell, M., and Kuriyan, J. 1. Kornberg, A., and Baker, T. (1992) DNA replication, 2nd Ed., W.H. Free- (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 13801–13806 man, New York 39. Neuwald, A. F., Aravind, L., Spouge, J. L., and Koonin, E. V. (1999) Ge- 2. Johnson, A., and O’Donnell, M. (2005) Annu. Rev. Biochem. 74, 283–315 nome. Res. 9, 27–43 3. Krishna, T. S., Kong, X. P., Gary, S., Burgers, P. M., and Kuriyan, J. (1994) 40. Erzberger, J. P., and Berger, J. M. (2006) Annu. Rev. Biophys. Biomol. Struct. Cell 79, 1233–1243 35, 93–114 4. Gulbis, J. M., Kelman, Z., Hurwitz, J., O’Donnell, M., and Kuriyan, J. (1996) 41. Yao, N., Coryell, L., Zhang, D., Georgescu, R. E., Finkelstein, J., Coman, Cell 87, 297–306 M. M., Hingorani, M. M., and O’Donnell, M. (2003) J. Biol. Chem. 278, 5. Tan, C. K., Castillo, C., So, A. G., and Downey, K. M. (1986) J. Biol. Chem. 50744–50753 261, 12310–12316 42. Ahmadian, M. R., Stege, P., Scheffzek, K., and Wittinghofer, A. (1997) Nat. 6. Prelich, G., Tan, C. K., Kostura, M., Mathews, M. B., So, A. G., Downey, Struct. Biol. 4, 686–689 K. M., and Stillman, B. (1987) Nature 326, 517–520 43. Johnson, A., and O’Donnell, M. (2003) J. Biol. Chem. 278, 14406–14413 7. Prelich, G., Kostura, M., Marshak, D. R., Mathews, M. B., and Stillman, B. 44. Snyder, A. K., Williams, C. R., Johnson, A., O’Donnell, M., and Bloom, L. B. (1987) Nature 326, 471–475 (2004) J. Biol. Chem. 279, 4386–4393 8. Warbrick, E. (2000) Bioessays 22, 997–1006 45. Ogura, T., Whiteheart, S. W., and Wilkinson, A. J. (2004) J. Struct. Biol. 9. Maga, G., and Hubscher, U. (2003) J. Cell Sci. 116, 3051–3060 146, 106–112 10. Tsurimoto, T., and Stillman, B. (1989) EMBO J. 8, 3883–3889 46. Lee, S. H., and Hurwitz, J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 11. Tsurimoto, T., and Stillman, B. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5672–5676

1023–1027 47. Burgers, P. M. (1991) J. Biol. Chem. 266, 22698–22706 Downloaded from 12. Tsurimoto, T., and Stillman, B. (1991) J. Biol. Chem. 266, 1950–1960 48. Ason, B., Handayani, R., Williams, C. R., Bertram, J. G., Hingorani, M. M., 13. Lee, S. H., Kwong, A. D., Pan, Z. Q., and Hurwitz, J. (1991) J. Biol. Chem. O’Donnell, M., Goodman, M. F., and Bloom, L. B. (2003) J. Biol. Chem. 266, 594–602 278, 10033–10040 14. Fien, K., and Stillman, B. (1992) Mol. Cell. Biol. 12, 155–163 49. Seybert, A., and Wigley, D. B. (2004) EMBO J. 23, 1360–1371 15. Majka, J., and Burgers, P. M. (2004) Prog. Nucleic Acids Res. Mol. Biol. 78, 50. Ayyagari, R., Impellizzeri, K. J., Yoder, B. L., Gary, S. L., and Burgers, P. M.

227–260 (1995) Mol. Cell. Biol. 15, 4420–4429 http://www.jbc.org/ 16. Naktinis, V., Turner, J., and O’Donnell, M. (1996) Cell 84, 137–145 51. Yao, N., Hurwitz, J., and O’Donnell, M. (2000) J. Biol. Chem. 275, 17. Mossi, R., Jonsson, Z. O., Allen, B. L., Hardin, S. H., and Hubscher, U. 1421–1432 (1997) J. Biol. Chem. 272, 1769–1776 52. Kelman, Z., Yao, N., and O’Donnell, M. (1995) Gene 166, 177–178 18. Oku, T., Ikeda, S., Sasaki, H., Fukuda, K., Morioka, H., Ohtsuka, E., Yo- 53. Turner, J., and O’Donnell, M. (1995) Methods Enzymol. 262, 442–449 shikawa, H., and Tsurimoto, T. (1998) Genes Cells 3, 357–369 54. Studwell, P. S., and O’Donnell, M. (1990) J. Biol. Chem. 265, 1171–1178 19. Podust, V. N., Tiwari, N., Stephan, S., and Fanning, E. (1998) J. Biol. Chem.

55. Finkelstein, J., Antony, E., Hingorani, M. M., and O’Donnell, M. (2003) at Rockefeller University Library on July 30, 2015 273, 31992–31999 Anal. Biochem. 319, 78–87 20. Bowman, G. D., O’Donnell, M., and Kuriyan, J. (2004) Nature 429, 56. Uhlmann, F., Cai, J., Gibbs, E., O’Donnell, M., and Hurwitz, J. (1997) J. Biol. 724–730 Chem. 272, 10058–10064 21. Jeruzalmi, D., O’Donnell, M., and Kuriyan, J. (2001) Cell 106, 429–441 57. Gomes, X. V., Gary, S. L., and Burgers, P. M. (2000) J. Biol. Chem. 275, 22. Stukenberg, P. T., Studwell-Vaughan, P. S., and O’Donnell, M. (1991) 14541–14549 J. Biol. Chem. 266, 11328–11334 58. Turner, J., Hingorani, M. M., Kelman, Z., and O’Donnell, M. (1999) EMBO 23. O’Donnell, M., Jeruzalmi, D., and Kuriyan, J. (2001) Curr. Biol. 11, J. 18, 771–783 R935–R946 59. Naktinis, V., Onrust, R., Fang, L., and O’Donnell, M. (1995) J. Biol. Chem. 24. Ellison, V., and Stillman, B. (1998) J. Biol. Chem. 273, 5979–5987 270, 13358–13365 25. Cai, J., Yao, N., Gibbs, E., Finkelstein, J., Phillips, B., O’Donnell, M., and 60. Gerik, K. J., Gary, S. L., and Burgers, P. M. (1997) J. Biol. Chem. 272, Hurwitz, J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 11607–11612 1256–1262 26. Podust, V. N., Tiwari, N., Ott, R., and Fanning, E. (1998) J. Biol. Chem. 273, 61. Kazmirski, S. L., Podobnik, M., Weitze, T. F., O’Donnell, M., and Kuriyan, 12935–12942 J. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 16750–16755 27. Hingorani, M. M., and O’Donnell, M. (1998) J. Biol. Chem. 273, 24550–24563 62. Seybert, A., Singleton, M. R., Cook, N., Hall, D. R., and Wigley, D. B. (2006) 28. Gomes, X. V., and Burgers, P. M. (2001) J. Biol. Chem. 276, 34768–34775 EMBO J. 25, 2209–2218 29. Schmidt, S. L., Gomes, X. V., and Burgers, P. M. (2001) J. Biol. Chem. 276, 63. O’Donnell, M., Onrust, R., Dean, F. B., Chen, M., and Hurwitz, J. (1993) 34784–34791 Nucleic Acids Res. 21, 1–3 30. Hingorani, M. M., and Coman, M. M. (2002) J. Biol. Chem. 277, 64. Chen, M., Pan, Z. Q., and Hurwitz, J. (1992) Proc. Natl. Acad. Sci. U. S. A. 47213–47224 89, 2516–2520 31. Bowman, G. D., Goedken, E. R., Kazmirski, S. L., O’Donnell, M., and 65. Chen, M., Pan, Z. Q., and Hurwitz, J. (1992) Proc. Natl. Acad. Sci. U. S. A. Kuriyan, J. (2005) FEBS Lett. 579, 863–867 89, 5211–5215 32. Goedken, E. R., Kazmirski, S. L., Bowman, G. D., O’Donnell, M., and 66. Guenther, B., Onrust, R., Sali, A., O’Donnell, M., and Kuriyan, J. (1997) Kuriyan, J. (2005) Nat Struct Mol Biol 12, 183–190 Cell 91, 335–345 33. Zhuang, Z., Yoder, B. L., Burgers, P. M., and Benkovic, S. J. (2006) Proc. 67. Bermudez, V. P., Lindsey-Boltz, L. A., Cesare, A. J., Maniwa, Y., Griffith, Natl. Acad. Sci. U. S. A. J. D., Hurwitz, J., and Sancar, A. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 34. Cullmann, G., Fien, K., Kobayashi, R., and Stillman, B. (1995) Mol. Cell. 1633–1638 Biol. 15, 4661–4671 68. Majka, J., and Burgers, P. M. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 35. Uhlmann, F., Gibbs, E., Cai, J., O’Donnell, M., and Hurwitz, J. (1997) J. Biol. 2249–2254 Chem. 272, 10065–10071 69. Mayer, M. L., Gygi, S. P., Aebersold, R., and Hieter, P. (2001) Mol. Cell 7, 36. Yao, N. Y., Johnson, A., Bowman, G., Kuriyan, J., and O’Donnell, M. (2006) 959–970 J. Biol. Chem. 281, 17528–17539 70. Bermudez, V. P., Maniwa, Y., Tappin, I., Ozato, K., Yokomori, K., and 37. Miyata, T., Suzuki, H., Oyama, T., Mayanagi, K., Ishino, Y., and Morikawa, Hurwitz, J. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 10237–10242 K. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 13795–13800 71. Bylund, G. O., and Burgers, P. M. (2005) Mol. Cell. Biol. 25, 5445–5455

NOVEMBER 17, 2006•VOLUME 281•NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 35543 DNA: Replication, Repair, and Recombination: The Replication Factor C Clamp Loader Requires Arginine Finger Sensors to Drive DNA Binding and Proliferating Cell Nuclear Antigen Loading

Aaron Johnson, Nina Y. Yao, Gregory D. Bowman, John Kuriyan and Mike O'Donnell Downloaded from J. Biol. Chem. 2006, 281:35531-35543. doi: 10.1074/jbc.M606090200 originally published online September 15, 2006 http://www.jbc.org/

Access the most updated version of this article at doi: 10.1074/jbc.M606090200

Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites.

at Rockefeller University Library on July 30, 2015 Alerts: • When this article is cited • When a correction for this article is posted

Click here to choose from all of JBC's e-mail alerts

This article cites 69 references, 44 of which can be accessed free at http://www.jbc.org/content/281/46/35531.full.html#ref-list-1