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Dynamics of Loading the β Sliding Clamp of DNA Polymerase III onto DNA

Dynamics of Loading the β Sliding Clamp of DNA Polymerase III onto DNA THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 48, Issue of November 29, pp. 30699–30708, 1996 © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Dynamics of Loading the b Sliding Clamp of DNA Polymerase III onto DNA* (Received for publication, July 22, 1996, and in revised form, September 13, 1996) ¶ ¶ i Linda B. Bloom‡§, Jennifer Turner , Zvi Kelman , Joseph M. Beechem , Mike O’Donnell , and Myron F. Goodman‡ From the ‡Department of Biological Sciences, Hedco Molecular Biology Laboratories, University of Southern California, Los Angeles, California 90089-1340, the Microbiology Department, the Hearst Research Foundation, and the Howard Hughes Medical Institute, Cornell University Medical College, New York, New York 10021, and the Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232 DNA during chain elongation (9). X-ray diffraction analysis A “minimal” DNA primer-template system, consisting of an 80-mer template and 30-mer primer, supports reinforces the biochemical data, showing that the b subunit is processive DNA synthesis by DNA polymerase III core in a doughnut-shaped dimer with an inner diameter sufficient to the presence of the b sliding clamp, g complex clamp form a ring around duplex DNA, enabling it to act as a sliding loader, and single-stranded binding protein from Esch- clamp for the polymerase (12). Therefore, proper assembly of erichia coli. This primer-template system was used to the b sliding clamp by the g complex is an absolute prerequisite measure the loading of the b sliding clamp by the g for accurate and efficient chromosome duplication. It has re- complex in an ATP-dependent reaction. Bound protein- cently been shown that the t subunit acts as a bridge between DNA complexes were detected by monitoring fluores- two core polymerases (13, 14); the t subunit also brings one g cence depolarization of DNA. Steady state and time-re- complex clamp loader into the holoenzyme structure (15, 16). solved anisotropies were measured, and stopped-flow This asymmetric dimer of two polymerases within one holoen- pre-steady state fluorescence measurements allowed zyme is therefore capable of simultaneous replication of both visualization of the loading reactions in real time. The strands of a duplex chromosome as hypothesized (17). E. coli rate of loading b onto DNA was 12 s , demonstrating SSB enhances processive DNA synthesis by suppressing DNA that clamp assembly is rapid on the time scale required secondary structure that would otherwise act to stall the pol III for lagging strand Okazaki fragment synthesis. The as- holoenzyme complex (1). sociation rate appears to be limited by an intramolecu- The b clamp slides freely off the ends of linear DNA mole- lar step occurring prior to the clamp-loading reaction, cules (9); therefore, previous studies of the g complex clamp- possibly the opening of the toroidal b dimer. loading action have been limited to use of circular DNAs, such as large bacteriophage ssDNA circular genomes, as primed templates. Application of biophysical techniques to the study of The replicative DNA polymerase in Escherichia coli is the pol how the g complex loads b clamps onto DNA has been hindered III holoenzyme composed of 10 subunits (reviewed in Refs. 1 by the use of these large DNA templates. In this report we have and 2). The pol III core contains three subunits (3); the a developed a short linear template with ssDNA overhangs of subunit contains the DNA polymerase activity (4), the 39 to 59 sufficient length to slow the dissociation of b from the DNA and proofreading exonuclease resides in e (5, 6), and the third have used this template to study the interaction of b and g subunit, u, lacks a well defined function and is nonessential for complex with the DNA template via the technique of fluores- cell growth (3, 7). The core polymerase is extremely inefficient cence depolarization. because it dissociates rapidly from primer-template DNA, caus- DNA in solution undergoes rotational diffusion that varies ing DNA synthesis to be predominantly nonprocessive. Approx- with molecular weight. Rotational motion of DNA is slowed imately 10–20 nt are synthesized per template binding event down when proteins are bound. The p/t DNA was labeled with by pol III core in vitro (8). The activity of the accessory proteins a fluorescent dye molecule, X-rhodamine. The rotational mo- g complex and b tethers the core to the p/t DNA, resulting in a tion of the dye, detected by time-resolved and steady state processivity of greater than 5000 nt/template binding event fluorescence depolarization, was used to measure rotation of (4, 9). the DNA in the presence and absence of combinations of b and The g complex contains five subunits: g, d, d9, x, and c (10, g complex. Pre-steady state stopped-flow measurements were 11). The key function of the g complex is to load the b subunit made allowing us to visualize the loading of the b sliding clamp onto DNA. Biochemical data suggest that b encircles DNA and onto DNA in real time. interacts directly with the pol III core, tethering the core to the EXPERIMENTAL PROCEDURES * This work was supported by National Institutes of Health (NIH) Enzymes—E. coli DNA pol III proteins were purified as described: a, Grants GM21422, GM42554, GM38839, National Science Foundation e, and g (18); b (12); d and d9 (19); x and c (20); and u (7). Subassemblies Grant MCB-9303921, and NIH Postdoctoral Fellowship GM15304 (to of g complex and core were constituted as described (7, 11). Enzyme L. B. B.). The costs of publication of this article were defrayed in part by reaction buffer contained 20 mM TriszHCl, pH 7.5, 40 mg/ml BSA, 5 mM the payment of page charges. This article must therefore be hereby dithiothreitol, and 8 mM MgCl . For fluorescence experiments, 50 mM marked “advertisement” in accordance with 18 U.S.C. Section 1734 NaCl was included in the reaction buffer to reduce nonspecific protein- solely to indicate this fact. DNA interactions. § Present address: Dept. of Chemistry and Biochemistry, Arizona Oligonucleotides—Oligonucleotides were synthesized using standard State University, Tempe, AZ 85287-1604. b-cyanoethyl phosphoramidite chemistry. The abbreviations used are: pol III, E. coli DNA polymerase III; The 80-mer template used for primer extension reaction is 59-TGA p/t, primer-template; nt, nucleotide; SSB, E. coli single-stranded bind- ing protein; ssDNA, single-stranded DNA; AMPPNP, adenyl-59-yl GGG TGG CGG TTC TGA GGG TGG CGG TTC TAA GGG TGG CGG imidodiphosphate. TAC TAA ACC TCC TGA GTA CGG TGA TAC ACC TAT TCC GG 39. This paper is available on line at http://www-jbc.stanford.edu/jbc/ 30699 This is an Open Access article under the CC BY license. 30700 pol III Accessory Proteins b and g Complex The primer-template used for fluorescence measurements is as slit width. The emission polarizer rotated from vertical to horizontal follows. every 30 s during data acquisition. Data were acquired over a 30-min period and averaged. The instrument G factor was determined by 59 CT GGT AAT ATC CAG rotating the excitation beam to the horizontal position and acquiring 39 TCT TCT TGA GTT TGA TAG CCG GAA C GA CCA TTA TAG GTC signal-averaged data for approximately 15 30-s periods for both hori- zontal and vertical polarized emissions. Two 160-ml samples were pre- AAC AAT ATT ACC GCC A 3 9 pared containing 25 nM p/t (X-rhodamine label on 59-primer end) and 2 TTG TTA TAA TGG CGG TCG GTA ACG TTG TCC TTT TTG CGA GT 5 9 mM ATP in reaction buffer with NaCl and less than 1% glycerol. One SEQUENCE 1 sample contained 630 nM b as dimer and 200 nM g complex, and the second did not. A59-amino group on a 6-carbon atom linker arm (Glen Research) was General Procedure for Stopped-flow Anisotropy Measurements of Pro- added to oligonucleotides that were to be labeled with rhodamine. tein-DNA Association Reactions—Stopped-flow anisotropy measure- X-rhodamine (Molecular Probes, catalog number X-491) was attached ments were made using instrumentation as described previously (24). to the amino linker as described previously (21). A 30-nt primer and a Samples were excited at 580 nm (4-nm band pass), and the emission complementary 80-nt template were labeled with X-rhodamine. The signal passed through a 620-nm cut on filter. In general, the three labeled primer was annealed to an unlabeled template, and the double- syringes on the stopped-flow apparatus were loaded, and reactions were stranded p/t was purified from the single-stranded oligonucleotides by performed as follows. The first syringe was loaded with protein in nondenaturing polyacrylamide gel electrophoresis. The labeled tem- reaction buffer, the second was loaded with reaction buffer only, and the plate was annealed to an unlabeled primer in enzyme reaction buffer third was loaded with p/t DNA in reaction buffer. Association reactions using a ratio of 1 template to 1.5 primers. This p/t system was not were initiated by mixing 100 ml of the protein solution with 100 mlof gel-purified. DNA solution. Control preshot reactions were done by mixing 100 mlof The template used in reactions with core polymerase was synthe- DNA with 100 ml of reaction buffer only to measure the anisotropy of sized with a single phosphorothioate linkage to the last nucleotide on DNA alone. Data points were typically taken every 2 ms. Multiple runs the 39-terminus. A diasteriomeric mixture of products is formed where (10–25 runs) were averaged to increase signal to noise ratio. one phosphorothioate isomer is resistant to exonucleolytic cleavage and Association Kinetics at Different Concentrations of g Complex—One the second is not. Both T4 DNA polymerase and the e subunit of pol III syringe contained 100 nM p/t DNA (X-rhodamine label on 59-template show the same stereoselectivity in excision reactions (22). The mixture end), 800 nM b as dimer, and 1 mM ATP in reaction buffer with NaCl of phosphorothioate isomers was degraded on a preparative scale by and less than 1% glycerol. The second syringe contained 200, 400, or incubation with T4 DNA polymerase to leave only the nonhydrolyzable 800 nM g complex in reaction buffer with NaCl and less than 1% isomer, which was purified by polyacrylamide gel electrophoresis. glycerol. Control preshots contained 50 nM p/t DNA, 400 nM b, and 0.5 Primer Extension Processivity Assays—Primers were 59-end-labeled mM ATP. with P using T4 polynucleotide kinase (U.S. Biochemical Corp.) in Association Kinetics for Different Orders of the Addition of Subunits enzyme reaction buffer at 37 °C. Primer-templates were annealed in to DNA—Two reactions were performed. In the first, 100 nM p/t DNA enzyme reaction buffer using a ratio of 1 primer to 1.2 templates by heating to 80 °C in enzyme reaction buffer and cooling to room temper- (X-rhodamine label on 59-primer end), 800 nM b dimer, and 0.5 mM ATP ature. Four different primers were annealed to the 80-mer template, in reaction buffer with NaCl and less than 1% glycerol were loaded into and six separate primer extension reactions were performed for each p/t one syringe. The second syringe contained 600 nM g complex and 0.5 mM combination containing (A) core only, (B) core, b, and g complex, (C) core ATP in reaction buffer with NaCl and less than 1% glycerol. Control and SSB, (D) core, SSB, b, and g complex, (E) core, SSB, and b,or(F) preshots contained 50 nM p/t DNA, 400 nM b, and 0.25 mM ATP. In the core, SSB, and g complex (Fig. 1). Primer-templates were preincubated second reaction, one syringe contained 100 nM p/t DNA (X-rhodamine with SSB for 2 min at 37 °C prior to a second preincubation with b and label on 59-primer end) and 0.5 mM ATP in reaction buffer with NaCl. g complex for 5 min at 37 °C. In reactions where SSB, b,or g complex The second syringe contained 800 nM b dimer, 600 nM g complex, and were omitted, preincubations were performed with reaction buffer only. 0.5 mM ATP in reaction buffer with NaCl and less than 1% glycerol. Reactions were initiated by the addition of core polymerase and Control preshots contained 50 nM p/t DNA and 0.25 mM ATP. quenched after 10 s with 40 mlof20mM EDTA in 95% formamide. All Dissociation Kinetics—M13 trap DNA consisted of single-stranded reactions contained 2 nM p/t DNA, enzyme reaction buffer, 4% glycerol, wild type M13 DNA with two 30-nt oligonucleotides annealed to differ- 0.5 mM ATP, and 60 mM dATP, dCTP, dGTP, and dTTP in a 20-ml ent sites. In this experiment, p/t DNA was preincubated with b and g reaction volume. Reactions with accessory proteins contained 40 nM complex, and reactions were initiated by the addition of M13 trap DNA. SSB, 20 nM b dimer, 3.5 nM g complex, and 18 nM core. Reaction One syringe was loaded with 100 nM p/t DNA (X-rhodamine label on the products were separated on 10% denaturing polyacrylamide gels and 59-primer end), 800 nM b dimer, 400 nM g complex, and 2 mM ATP in visualized using a PhosphorImager (Molecular Dynamics). reaction buffer with NaCl and less than 1% glycerol. The second con- Steady State Anisotropy Measurements—Steady state anisotropy tained 500 nM M13 trap DNA in reaction buffer with NaCl. Control measurements were made using a QuantaMaster QM-1 fluorometer preshot reactions contained 50 nM p/t DNA, 400 nM b dimer, 200 nM g (Photon Technology International) with a single emission channel (L complex, and 1 mM ATP. format) and 400–800-nm dichroic sheet polarizers (Oriel). Samples were excited with vertically polarized light at 580 nm (8 nm band pass), RESULTS and both vertical and horizontal emission was monitored at 610 nm A series of protein-protein interactions govern the assembly (8-nm band pass). Since the emission polarizer was manually rotated, of the pol III holoenzyme complex, and protein-protein and three separate anisotropy measurements were made on each sample, and the values were averaged. Anisotropy values were calculated using protein-DNA interactions, coordinated with the use of ATP, are a G factor (polarization bias) (23) determined under identical experi- required to load pol III holoenzyme onto p/t DNA. We are mental conditions. investigating protein-DNA interactions accompanying the Titration experiments were performed starting with 188 mlof53nM loading of the b clamp by the g complex using a sensitive p/t DNA (X-rhodamine label on 59-template end) in reaction buffer with optical technique, fluorescence depolarization, to detect and NaCl and ATP. After measuring the anisotropy of the p/t, a 4-ml aliquot analyze binding of the proteins to DNA (21, 25). Fluorescence of 20 mM b dimer and an 8-ml aliquot of 5 mM g complex were added. Anisotropy was measured after each addition of protein. Final concen- depolarization measures the rotational diffusion of fluores- trations were 50 nM p/t, 400 nM b dimer, 200 nM g complex, and 0.5 mM cence-labeled DNA alone or when present in a DNA-protein ATP. The anisotropy of this solution was measured after it stood at complex. The rotational motion depends predominantly on the room temperature for 15 min. After the addition of a 2-ml aliquot of 45 molecular weight of the DNA-protein complex. Therefore, it is mM ATP, the anisotropy measurement was repeated. possible to distinguish free DNA from DNA bound by combina- Time-resolved Decays of Fluorescence—Time-resolved measurements tions of b and g complex by measuring steady state fluores- were performed in cuvettes with 3-mm path lengths using instrumen- tation described in Ref. 21. Samples were excited at 580 nm. The cence anisotropy. A pre-steady state anisotropy measurement emission signal passed through a Glan-Thompson polarizer, 620-nm cut allows protein-DNA complex formation to be visualized in real on filter, and a SPEX 0.22-m monochromator set at 640 nm with a 1-mm time. Minimal Primer-Template Requirements for Processive Syn- L. B. Bloom and M. F. Goodman, unpublished results. thesis by E. coli pol III Core Polymerase in the Presence of b and pol III Accessory Proteins b and g Complex 30701 FIG.1. Requirements for processive DNA synthesis by pol III on an 80-nucleotide template. Primer extension reactions by pol III core polymerase on an 80-mer template in the presence of different combinations of accessory proteins (b, g complex, and SSB) were analyzed by denaturing polyacrylamide gel electrophoresis. Four different 30 nucleotide primers were annealed to an 80-nucleotide template to create regions of single-stranded DNA on the 39-template end (39-template overhangs) of 10, 15, 20, and 25 nucleotides. Six separate extension reactions were performed at 37 °C on each primer-template combination. All reactions contained 2 nM p/t DNA, 0.5 mM ATP, and 60 mM dATP, dCTP, dGTP, and dTTP. Reactions P (lanes 1, 8, 15, and 22) contained primer-template only as a marker. Reactions A (lanes 2, 9, 16, and 23) contained 18 nM core only. Reactions B (lanes 3, 10, 17, and 24) contained 18 nM core, 20 nM b (dimer), and 3.5 nM g complex. Reactions C (lanes 4, 11, 18, and 25) contained 18 nM core and 40 nM SSB (as monomer). Reactions D (lanes 5, 12, 19, and 26) contained 18 nM core, 20 nM b (as dimer), 3.5 nM g complex, and 40 nM SSB (as monomer). Reactions E (lanes 6, 13, 20, and 27) contained 18 nM core, 20 nM b (as dimer), and 40 nM SSB as monomer. Reactions F (lanes 7, 14, 21, and 28) contained 18 nM core, 3.5 nM g complex, and 40 nM SSB (as monomer). g Complex—To observe maximum changes in rotational anisot- the absence of SSB, did not significantly enhance the proces- ropy signals during transitions between free DNA and bound sivity of the core polymerase, although a small fraction of DNA-protein complexes it is important to utilize the smallest primers were extended to the end of the template (Fig. 1, lane possible DNA capable of supporting processive pol III DNA 24). The addition of SSB alone to the core inhibited synthesis, synthesis. On large DNA templates such as fX and M13 DNA, as shown by faint gel bands (Fig. 1, lane 25). However, proces- the processivity of DNA synthesis by pol III core DNA polym- sive synthesis was observed by the core polymerase in the erase is increased substantially by the addition of b and g presence of b, g complex, and SSB (Fig. 1, lane 26). A dark gel complex. The g complex loads b on DNA, and b acts as a sliding band was present for primers extended to the end of the tem- clamp to tether the core polymerase to the DNA template, plate, while faint bands due to dissociation of the core prior to reviewed in Ref. 26. reaching the end of the template were also present. The addi- We found that a DNA template as small as 80 nt in length tion of either b and SSB in the absence of g complex (Fig. 1, lane supports processive synthesis by core polymerase in the pres- 27)or g complex and SSB in the absence of b (Fig. 1, lane 28) ence of b and g complex. Processive synthesis on this 80-mer did not result in processive synthesis by core polymerase. Re- template requires b, g complex, and single-stranded binding action products resembled those by synthesis of core in the protein, SSB (Fig. 1, lanes 23–28).A59- P-labeled 30-nt presence of SSB alone. primer was annealed to an 80-nt template so that 25-nt single- The efficiency of synthesis on the 80-nt template depended stranded regions were present on both the 59- and 39-ends of the both on the length of the primer and its position on the tem- template. The primers were extended by core polymerase and plate. On an 80-mer template, a 30-nt primer was extended different combinations of accessory proteins (b, g complex, and more efficiently by core polymerase in the presence of b, g SSB). After 2 min at 37 °C with SSB only, the p/t DNA was complex, and SSB than a 20-nt primer (data not shown). A preincubated at 37 °C for 5 min with different combinations of 15-nt primer was not extended at all in a 20-s reaction (data accessory proteins in separate reactions. Reactions were initi- not shown). ated by the addition of the core polymerase and quenched after To achieve processive synthesis, the 30-nt primer had to be 10 s. Reaction products were analyzed by polyacrylamide gel positioned on the 80-mer template so that a single-stranded electrophoresis. region of DNA greater than 15 nt long was present at the Gel bands represent sites where the polymerase either dis- 39-template end (Fig. 1, lanes 5, 12, 19, and 26). 59- P-labeled sociated or paused after incorporating one or more nt. Since primers, 30 nt in length, were annealed to an 80-nt template so DNA synthesis by the core polymerase alone has been demon- that the length of the single-stranded region of DNA on the strated to be distributive (8), dark gel bands most likely indi- 39-template end (39-template overhang) varied. Primer exten- cate a high probability of dissociation at a given site. Synthesis sion reactions on these p/t DNAs were performed with core by core polymerase alone was essentially distributive. Dark gel polymerase along with different combinations of accessory pro- bands were present for DNA products that were extended by 1, teins as above. In the presence of b, g complex, and SSB, 2, 3, 4, and 5 nt (Fig. 1, lane 23). Bands caused by the formation processive synthesis by the core polymerase was not observed of longer products were less intense. The core alone was unable when the 39-template overhang was 10 or 15 nt long (Fig. 1, to synthesize DNA to the end of the template. lanes 5 and 12). When the 39-template overhang was increased The addition of b and g complex to the core polymerase, in to 20 nt, a faint gel band representing the extension of primers 30702 pol III Accessory Proteins b and g Complex FIG.2. Increase in steady state anisotropy for X-rhodamine-labeled DNA when g complex loads b on DNA. A, the change in steady state anisotropy for a X-rhodamine-labeled primer-template when b and then g complex were added to the DNA. An 80-nucleotide template was labeled at the 59-end with X-rhodamine and annealed to a 30-nucleotide primer. The steady state anisotropy of the primer-template in reaction buffer containing ATP and MgCl was measured (DNA). The anisotropy of this DNA solution was measured again after adding a small aliquot of b (1 b) and then g complex (1 g complex). Concentrations after the addition of g complex were 50 nM primer-template, 400 nM b (dimer), 200 nM g complex, and 0.5 mM ATP. The anisotropy of this solution was measured again after the solution stood at room temperature for 15 min (15 min). An aliquot of ATP was then added, and the anisotropy was measured again (1 ATP). B, the change in steady state anisotropy for an X-rhodamine-labeled primer-template when g complex and then b were added to the DNA. The conditions for this titration were the same as for A except that the order of the addition of b and g complex was reversed. In this experiment, g complex was added first, and then b was added to establish that both b and g complex are required to give the large increase in steady state anisotropy (r ' 0.26) for the DNA. A sketch of the reaction scheme is drawn at the top of the figure. to the end of the template was observed for a reaction contain- proximity to b after b is loaded by g complex. ing core polymerase, b, g complex, and SSB (Fig. 1, lane 19). Loading of b on Primer-Template DNA by g Complex—Inter- More efficient primer extension to the end of the template by actions of b and g complex with p/t DNA under steady state core in the presence of b, g complex, and SSB was observed conditions were observed by monitoring changes in fluores- when the 39-template overhang was increased to 25 nt (Fig. 1, cence anisotropy of p/t DNA labeled with an extrinsic fluores- lane 26). This probably reflects a requirement for SSB to pre- cent probe, X-rhodamine. An 80-nt template was labeled at the vent b from “sliding off” the end of the template. 59-terminus with X-rhodamine. A 30-nt primer was annealed to Although an excess of polymerase over DNA was used in the center of the template so that 25-nt regions of single- primer extension reactions (core:p/t 5 9:1), only a fraction of stranded DNA were present on both the 59- and 39-ends of the primers were extended during our short 10-s reaction time. In template. This was the optimal p/t configuration supporting reactions with pol III core alone, inefficient primer extension processive DNA synthesis by the pol III core in the presence of may result from a weak association of core with DNA. In the b, g complex, and SSB (Fig. 1). A DNA synthesis reaction using presence of b, g complex, and SSB, inefficient primer extension a rhodamine-labeled template, carried out as shown in Fig. 1, may be caused by a slow association of the core polymerase demonstrated that the rhodamine label has no measurable with b to form a processive bzcore complex (or a slow displace- effect on the activity of the pol III proteins (data not shown). ment of SSB from the template). Note that it may also be The steady state anisotropy of the rhodamine-labeled p/t, in difficult to load b onto all of the p/t molecules because g com- the absence of pol III accessory proteins, was 0.166 (Fig. 2A). plex may actively remove b resulting in a steady state popula- The addition of b (molecular mass 81 kDa for a b dimer) to the tion of DNA containing b and free DNA. rhodamine-labeled p/t DNA (52 nM DNA, 420 nM b dimer) did In primer extension assays, the fraction of primers extended not affect the anisotropy of the DNA (i.e. in the absence of g increased with concentration of core polymerase in 10-s reac- complex, there was no measurable interaction between b and tions (data not shown) and also increased with reaction time p/t DNA). The addition of g complex to the solution of b and (data not shown), indicating that essentially all of the primers DNA (50 nM DNA, 400 nM b dimer, and 200 nM g complex) were extendible. Experiments are under way to measure the resulted in a large increase in the steady state anisotropy of the kinetics of association of the core polymerase with b to deter- labeled DNA, from 0.166 to 0.257 (Fig. 2A). The rotational mine if a rate-determining association step limits the rate of diffusion of the DNA decreased upon addition of b and g primer extension. In the holoenzyme complex, the core and g complex. complex form part of a larger complex so that association of the To determine if the increase in steady state anisotropy in the core with b is likely to be more efficient, since it is in close presence of b and g complex was caused by g complex loading pol III Accessory Proteins b and g Complex 30703 FIG.3. Association of g complex and bzg complex with DNA substrates as determined by an increase in steady state anisotropy. Three different DNA substrates, a single-stranded 30-mer (L), a single-stranded 80-mer (M), and a 30-mer/80-mer p/t (E) were labeled with rhodamine and were titrated with g complex. Each solution contained reaction buffer consisting of 20 mM TriszHCl, pH 7.5, 50 mM NaCl, 8 mM MgCl ,5mM dithiothreitol, 40 mg/ml BSA, and 1 mM ATP. Data were taken by hand-mixing reagents in a cuvette and measuring steady state anisotropy values immediately after the addition of g complex (A)or b (B). A, g complex was added to a solution of 50 nM DNA in reaction buffer. A new solution was made for each concentration of g complex. B, after each g complex addition in A, b was added to a concentration of 500 nM b (as dimer). b on the p/t or by g complex alone binding to p/t, the order of the oligonucleotide, single-stranded 80-nt oligonucleotide, and 30- addition of g complex and b to the p/t DNA was reversed (Fig. mer/80-mer p/t were measured in the presence of 1 mM ATP 2B). The solution of labeled p/t DNA in the absence of b and g (Fig. 3A). We previously used 0.5 mM ATP for steady state complex gave a steady state anisotropy of 0.165 as observed anisotropy measurements with 200 nM g complex (Fig. 2). The previously. The addition of g complex (201 kDa) to DNA (51 nM higher concentration of ATP was used to ensure that it was not DNA and 200 nM g complex) resulted in a small increase in completely consumed during the 2–3 min taken to measure anisotropy to 0.176. However, the subsequent addition of b to anisotropies using up to 800 nM g complex (Fig. 3). g complex the solution (50 nM DNA, 400 nM b dimer, 200 nM g complex) was found to interact with the single-stranded 80-nt DNA resulted in a much larger increase in anisotropy to 0.248. Thus, substrate, although this association is weak (apparent K 5 the much larger increase in anisotropy was caused by g com- 450 nM). Interaction of g complex with 30-mer/80-mer p/t DNA plex loading b on the p/t DNA and not by direct interaction of or with 30-mer ssDNA was barely detectable (Fig. 3A, lower g complex alone with DNA. two curves). However, when b was added to form bzg complex, In both titration experiments above, the anisotropy of the p/t the interaction with ssDNA and p/t DNA was strengthened DNA decreased when solutions of DNA, b, and g complex considerably (Fig. 3B). We want to emphasize that for the p/t remained for 15 min at room temperature (Fig. 2). The anisot- DNA, the increase in anisotropy with increasing concentration ropy regained its former level in both experiments when a fresh of g complex is most likely caused by loading of b onto DNA. aliquot of ATP was added (Fig. 2). These results demonstrate The replication assays (Fig. 1) show that a processive replica- that the interactions of b and g complex with DNA required tion complex can be formed on this p/t, demonstrating that g hydrolysis of ATP and that the ATP initially present was con- complex is fully capable of loading b on this p/t in a biologically sumed during the 15-min incubation. The g complex has been relevant manner. shown to be a DNA-dependent ATPase, and ATP is required for The association of g complex with either single-stranded 3 32 g complex to load b on DNA (27). Hydrolysis of a- P-labeled DNA or p/t DNA required ATP hydrolysis. In the presence of ATP was measured in solutions containing an unlabeled p/t, b, the nonhydrolyzable ATP analog, AMPPNP, g complex alone and g complex (data not shown). ATP was converted to ADP and a bzg complex did not associate with either single-stranded under these conditions. Loading of b by the g complex did not 80-mer or with 30-mer/80-mer p/t DNA (data not shown). Since take place when ATP was replaced in the reaction with either g complex was observed to bind ssDNA and p/t DNA, in the UTP or by the nonhydrolyzable ATP analog AMPPNP (data absence of b in an ATP-dependent reaction, a preliminary not shown). experiment was carried out to see if binding of individual Binding of g Complex to DNA—Association of g complex and subunits of g complex to ssDNA could also take place in the loading of b on DNA by g complex are not simple equilibrium presence of ATP. No measurable binding to either a single- protein-DNA binding interactions. Association of g complex stranded 30-mer or 80-mer was observed for g, d, d9,or xzc at with DNA is dependent on ATP hydrolysis, and loading of b concentrations of at least 600 nM protein and 50 nM DNA onto DNA depends on both the catalytic activity of g complex (data not shown). and the hydrolysis of ATP. Association constants for these Time-resolved Anisotropy Measurements—Time-resolved in- proteins to DNA were not measured under true equilibrium tensity and anisotropy measurements (ns time scale) were binding conditions; instead, association was measured as a made on p/t DNA containing a rhodamine-labeled primer-59- function of protein concentration to give apparent binding af- terminus. These measurements were made in the presence and finities under steady state conditions (Fig. 3). The binding of g absence of b and g complex. When b and g complex are present, complex to different DNA substrates, single-stranded 30-nt the system is dynamic, i.e. b and g complex continuously asso- ciate and dissociate from the DNA in an ATP-dependent reac- J. Turner and M. O’Donnell, unpublished results. tion on a ms time scale. The time-resolved measurements re- 30704 pol III Accessory Proteins b and g Complex FIG.5. Reaction time courses for loading b on p/t DNA: the addition of g complex to b and X-rhodamine-labeled DNA. Four separate time course reactions are shown. Each reaction contained 50 nM p/t (X-rhodamine label on 59-template end), 400 nM b as dimer, and 0.5 mM ATP as described under “Experimental Procedures.” Reactions were initiated by adding solutions differing in the concentration of g complex to a solution of b, DNA, and ATP. Final concentrations of g complex were 0 (showing a constant anisotropy), 100, 200, and 400 nM. The anisotropy increased upon the addition of g complex. The magni- tude of the anisotropy change increased with increasing concentration of g complex, while the observed rates (12 s ) of increase in anisotropy FIG.4. Time-resolved decays of total intensity and anisotropy remained constant. A sketch of the reaction scheme is drawn at the top. for rhodamine-labeled DNA in the presence and absence of b and g complex. A, time-resolved decay of total intensity for 25 nM p/t with an X-rhodamine label on the 59-primer terminus in the presence and absence of 200 nM g complex and 630 nM b dimer. Both reactions reflective of the rotational diffusion of the protein-DNA contained 20 mM TriszHCl, pH 7.5, 50 mM NaCl, 8 mM MgCl ,2mM ATP, complex. and less than 1% glycerol as described under “Experimental Proce- Pre-steady State Association of b and g Complex with p/t dures.” Data were collected over 30 min. The concentration of ATP was increased from 0.5 to 2 mM so that ATP would not be completely DNA Observed in Real Time—The kinetics of loading the b consumed during the time course of the measurement. The presence of sliding clamp onto p/t DNA by the g complex were observed in b and g complex does not affect the lifetime of the X-rhodamine probe; real time by measuring the steady state anisotropy of rhoda- the decay curves overlap. B, time-resolved decays of anisotropy for the mine-labeled p/t in stopped-flow reactions (Fig. 5). Note that same samples. The upper data curve shows the slower rate of anisotropy the protein-DNA association kinetics occurring on a pre-steady decay for the p/t in the presence of b and g complex. The lower data curve shows the more rapid rate of anisotropy decay for the p/t alone. state (ms) time scale are detected by changes in the steady state rotational anisotropy. Four curves are shown in Fig. 5. flect the steady state populations of bound and free DNA. The lower horizontal curve shows a constant value for the The decay in fluorescence intensity, i.e. the fluorescence life- steady state anisotropy of p/t DNA in the presence of b, which time, of the rhodamine-labeled p/t was the same for the naked does not interact with p/t under these conditions (Fig. 2). The DNA and when the b and g complex were present (Fig. 4A). The three upper curves illustrate loading of a constant amount of b fluorescence lifetimes for the p/t with 59-template label were (400 nM dimer) using three levels of g complex (100, 200, or 400 also the same in the presence and absence of b and g complex nM). The concentrations of b, g complex, p/t, and ATP for the (data not shown). Time-resolved decays in anisotropy showed reaction containing 200 nM g complex were the same as in the an increase in the rotational correlation time for the rhodamine steady state experiment in Fig. 2. probe in the presence of b and g complex, demonstrating that b The reaction was carried out using the following scheme (Fig. and/or g complex is associated with the p/t (Fig. 4B). 5, sketch of reaction). b (800 nM b ) was present in one syringe Steady state anisotropy values (Fig. 2) are a function of both along with p/t DNA (100 nM), ATP (1 mM), and buffer contain- the lifetime and rotational correlation time of a fluorophore (23, ing Mg . The g complex was present in a second syringe in the 24). Since the fluorescent lifetime of the rhodamine probe was same buffer with Mg but without ATP. The X-rhodamine the same in the presence and absence of b and g complex, the label was present at the 80-mer template-59-terminus. The changes in steady state anisotropy that we measured using contents of the two syringes were delivered to the reaction rhodamine-labeled DNA are the result of changes in rotational chamber, and anisotropy data points were collected at 2-ms motion of the probe only. Therefore, the signal in Fig. 2 is truly intervals (see “Experimental Procedures”). Multiple runs were pol III Accessory Proteins b and g Complex 30705 FIG.6. Reaction time courses for loading b on p/t DNA: different orders of the addition of g complex and b to X-rhodamine-labeled DNA. A, reaction time course for the addition of a solution of g complex and ATP to a solution of b, p/t DNA (X-rhodamine label on the 59-primer end), and ATP. Final concentrations were 50 nM p/t, 300 nM g complex, 400 nM b dimer, and 0.5 mM ATP as described under “Experimental Procedures.” The curve showing a constant anisotropy is a control reaction without g complex where reaction buffer only was added to the solution of b, p/t DNA, and ATP. B, reaction time course where a solution of b, g complex, and ATP was added to a solution of p/t DNA and ATP. Final reaction conditions in both panels are the same. The curve with constant anisotropy shows the addition of reaction buffer to DNA and ATP. When the reaction was initiated by the addition of b, g complex, and ATP to p/t, the observed increase in anisotropy was more rapid (much of the reaction is missed in the dead time of the instrument) than when the reaction was initiated by the addition of g complex and ATP to b, p/t DNA, and ATP. A sketch of the reaction scheme is drawn at the top. summed to increase the signal:noise ratio, and the data shown loading it on DNA (9, 16). A third possibility is that g complex were run-averaged over 5 points, while the raw data were fit by initially binds the p/t at multiple sites. A nonproductively a double exponential (Fig. 5, solid curves). bound g complex may have to dissociate or may have to be The data showed an increase in anisotropy for the labeled p/t displaced before productive loading of b onto the p/t can occur with an observed rate of 12 6 2s , resulting from association to achieve steady state loading and dissociation. A fourth, of b and g complex with p/t DNA. In this experiment, b was much more interesting, possibility might be a dramatic confor- either equal to or in excess of g complex, and both were in mational change in the bzg complex that affects the rotational excess of p/t DNA. The amplitudes of the reaction time courses mobility of the protein-DNA complex. increased with an increase in g complex and reflect an increase Stopped-flow anisotropy measurements were made using dif- in the total amount of protein bound to DNA. Although more ferent orders of the addition of b and g complex to 30-mer/80- protein bound to p/t DNA as the concentration of g complex was mer p/t DNA (Fig. 6). Depending on which interactions or steps increased, the observed reaction rate remained constant. Each are rate-limiting, the order of the addition of these components protein-bound DNA curve exhibited a small, but significant, to one another should affect the observed reaction rate. In these decrease between 250 and 350 ms prior to reaching steady state reactions, the 59-primer terminus, rather than the template, (t . 500 ms). was labeled with X-rhodamine. Since the rate of association of There are several possible explanations for this small de- b and g complex with DNA was not affected by the concentra- crease in anisotropy. First, the dip could reflect an initial tion of g complex, but the magnitude of the anisotropy increase sliding off of b from the relatively short 30-mer/80-mer p/t DNA was a function of the g complex concentration (Fig. 5), a con- in the absence of SSB, relaxing to a steady state characterized centration of 300 nM g complex was used in these experiments by repeated sliding off and reloading of b onto p/t DNA. Note to give a large anisotropy signal change during the time course that SSB which was present in the polymerization reactions to of these reactions. The concentrations of b, DNA, and ATP were keep the b dimer from sliding off the p/t DNA was not present the same as in Fig. 5. during the anisotropy measurements because the presence of In Fig. 6A, b (800 nM dimer) was preincubated with p/t DNA SSB bound to the DNA would limit the increase in anisotropy (100 nM) and ATP in one syringe, and g complex (600 nM) and that would be observed when b and g complex bind. A second ATP were in the second syringe. In Fig. 6B, b (800 nM), g explanation is that the decreased anisotropy reflects dissocia- complex (600 nM), and ATP were present together in one sy- tion of some fraction of g complex from the protein-DNA com- ringe, and p/t DNA (100 nM) and ATP were in the second plex. g complex has been shown to dissociate from b after syringe. There was a much more rapid increase in anisotropy 30706 pol III Accessory Proteins b and g Complex when the solution of b and g complex were added to p/t DNA (Fig. 6B) than when g complex was added to b and DNA (Fig. 6A). An estimate of the loading rate of b following preincuba- tion with g complex is 70 s , which is at least 6 times faster than the rate of loading b in the absence of preincubation with g complex (Figs. 5 and 6A). Inclusion of ATP along with the g complex prior to encountering b and DNA resulted in the same observed rate of increase in anisotropy, 12 s , as in reactions where ATP was added to g complex at the same time as b and DNA (Fig. 5). Thus, the rate-determining step appears to be different for reactions where b, g complex, and ATP were pre- incubated prior to the addition of the p/t in comparison with reactions where either g complex alone or g complex and ATP were added to a solution of b and p/t DNA. The decrease in anisotropy observed between 250 and 350 ms appears to be more pronounced in the reaction in Fig. 6B than in Fig. 6A. Both data sets were fit to the sum of an exponential increase and an exponential decrease in anisotropy. Since the rate of the increase in Fig. 6B is more rapid, more of the decrease in anisotropy is observed. In a similar experiment, a solution of b and g complex in the absence of ATP was added to a solution of p/t and ATP (data not shown). The observed rate of increase in anisotropy for this reaction was 11 s , indicating that this combination is not sufficient to bypass the rate-limiting step. These results are consistent with previous data showing that a stable multipro- tein complex consisting of b and g complex forms in an ATP- FIG.7. Reaction time course for the dissociation of b or b and dependent reaction in the absence of DNA (28). g complex from X-rhodamine-labeled DNA. The bottom trace shows Dissociation of b or bzg Complex from p/t DNA in Real a reaction initiated by the addition of p/t DNA (X-rhodamine label on Time—In steady state reactions (Fig. 2), ATP was rapidly hy- 59-primer end), b, g complex, and ATP to a solution of M13 trap DNA as drolyzed, most likely by repeated loading of b, which rapidly described under “Experimental Procedures.” Final concentrations were 50 nM p/t, 400 nM b dimer, 200 nM g complex, and 1 mM ATP. The dissociated from the short synthetic p/t DNA. Under our reac- concentrations of b, g complex, and p/t are the same as in Figs. 2 and 5. tion conditions, two possible dissociation pathways may exist: a The concentration of ATP was increased from 0.5 to 1 mM to ensure that two-step dissociation process where g complex dissociates from ATP was not completely consumed during the preincubation of b, g b and DNA after loading b and then b diffuses off the p/t, or a complex, and p/t. The observed rate of dissociation of proteins from one-step process where a bzg complex dissociates from the DNA was 6.4 s . The top trace shows constant anisotropy for a control reaction where a solution of p/t DNA, b, g complex, and ATP is added to DNA. The rate of dissociation of b or bzg complex from the reaction buffer rather than to trap DNA. A sketch of the reaction 30-mer/80-mer p/t DNA (depending on which pathway occurs) scheme is drawn at the top. was measured by preincubating b , g complex, ATP, and DNA in one syringe, while in the other syringe was placed an excess loading reaction carried out by the five-protein g complex can of single-stranded M13 DNA with two 30-nt primers. When the be detected, with high sensitivity, by measuring changes in the contents of the two syringes were mixed together in the steady state rotational anisotropy of fluorescence-tagged DNA. stopped-flow reaction chamber, any b and g complex that had A binding analysis, using stopped-flow techniques, has allowed either dissociated from the p/t DNA or had never been bound to us to visualize the clamp-loading reaction in real time. the p/t became trapped by the M13 DNA so that it could not Loading the b clamp onto DNA by the g complex is a prereq- reload onto the 30-mer/80-mer p/t. uisite to achieving processive leading and lagging strand DNA The rotational anisotropy remained essentially unchanged synthesis in E. coli. To study the reaction steps that involve on a 1-s time scale in the absence of trap DNA (Fig. 7, top trace). loading of the b sliding clamp onto p/t DNA, we have designed A reduction in the steady state rotational anisotropy reflects an a simple model system that supports processive synthesis by increase in the rotational motion of the p/t DNA as b (or bzg the pol III core polymerase in the presence of b, g complex, and complex) dissociated from the p/t, presumably by sliding off SSB. We find that processive synthesis is supported by a 30-nt over the end (Fig. 7, bottom trace). Dissociation was rapid, with primer annealed to the central region of an 80-nt template, so a first order off-rate constant of 6.4 s . The data can be that 25-nt single-stranded regions of DNA exist on both the 59- represented by a model in which b rapidly slides off the p/t and 39-ends of the template. This 30-mer/80-mer p/t DNA sys- DNA after being loaded. A cycle of repeated loading of b that tem is convenient for studying the binding of accessory proteins requires ATP hydrolysis by g complex, followed by rapid loss of (this report) and will also make possible study of the effects of b, would lead to consumption of ATP with time. A rapid disso- processivity on the fidelity of DNA synthesis. ciation of b would explain the requirement for SSB in primer Extensions of ssDNA of at least 20 nt were necessary on both extension assays to trap b on a linear p/t for enough time for the ends of the p/t DNA, in order to support processive synthesis by core to associate with b (Fig. 1). pol III core polymerase in the presence of b and g complex (Fig. DISCUSSION 1). Processive synthesis was observed only when SSB was Here we have shown that a simple DNA oligonucleotide included in reactions with core, b, and g complex. These single- primer-template that supports processive DNA synthesis can stranded regions are most likely needed to bind SSB efficiently. be used as a model system for analyzing interactions between SSB inhibited synthesis in reactions by core alone except when pol III accessory proteins and core pol III with DNA. In the the single-stranded region of DNA on the 59-template end was present study, we have demonstrated that the b sliding clamp- only 10 nt long, suggesting that SSB cannot bind to this short pol III Accessory Proteins b and g Complex 30707 FIG.8. Model showing g complex loading of b clamp onto DNA for lag- ging strand DNA synthesis. g complex binds b clamp in an ATP-dependent reac- tion. A rate-limiting step of about 12 s may possibly reflect opening of the toroi- dal b dimer clamp prior to loading onto p/t DNA. The last step shows dissociation of the complex from p/t DNA, which initiates the next clamp-loading cycle. region of ssDNA. Binding of SSB to single-stranded regions of was independent of the concentration of g complex. Instead, the the template on both sides of the primer may help stabilize b on slow step is more likely to be intramolecular in nature, because short p/t DNA long enough for a complex between b and core to it does not depend on the concentration of g complex. Perhaps assemble. a conformational change within b or g complex may be limiting The 30-mer/80-mer p/t DNA was labeled either at the 59- the rate of loading b. Two prominent possibilities for this step primer terminus or the 59-template terminus with a fluorescent are a conformational change in g complex or a conformational probe, X-rhodamine, and the fluorescence anisotropy of this change in b, such as “opening” of the ring prior to placement on probe was then used to detect interactions with the pol III DNA. accessory proteins, b and g complex. Association of b and g The pol III holoenzyme contains two DNA polymerase cores complex with the labeled p/t DNA decreased the rate of rota- (for simultaneous replication of both strands of a duplex chro- tional motion of the DNA and thus increased the steady state mosome) and only one g complex, all connected together by a anisotropy of the probe. When b and g complex were added to dimer of the t subunit (15). The g complex acts catalytically labeled p/t DNA, the anisotropy increased, demonstrating load- during lagging strand replication to load b clamps onto RNA ing of b by g complex. In contrast to the requirement for SSB to primers as they are produced by the helicase/primase (29). The obtain processive synthesis on the short p/t DNA (Fig. 1), SSB lagging polymerase, upon finishing a fragment, rapidly disen- was not required for b loading, further supporting the idea that gages from its b clamp, leaving it behind on DNA, and cycles SSB can provide a block keeping b from sliding off the ends of back to the newly assembled b clamp on the upstream primer the primer. Consistent with the idea that SSB serves as a block to extend the next lagging strand fragment (Fig. 8). Since the to stabilize b on linear DNA, a rapid rate of dissociation of b replication fork moves at a rate of approximately 1 kilobase/s was observed in the absence of SSB (Fig. 7). In these experi- and lagging strand fragments average 1–2 kilobase in length, a ments, a block is not necessary for loading per se, but rather to new fragment is produced every 1 or 2 s. Thus, the g complex keep b on the template so that core can bind to it and harness must be capable of clamping b onto primers within 1 s. The its ability to serve as a processivity factor. speed of clamp assembly observed in this report (12 s , t1 5 We investigated binding of g complex to individual ssDNA 58 ms) is well within the speed required for g complex action on and p/t DNA components. In the absence of b clamp, g complex the lagging strand. Perhaps even more relevant is that the rate bound extremely weakly to 30-mer ssDNA and 30-mer/80-mer of clamp assembly when b and g complex were preincubated p/t DNA (Fig. 3A). A somewhat higher affinity binding was together was about 70 s (t1 ' 10 ms), which is much faster observed with 80-mer ssDNA (Fig. 3A). Binding to each of the than required for recycling b clamp onto the lagging strand for forms of DNA was significantly enhanced in the presence of b rapid synthesis of Okazaki fragments. It is reasonable to sup- clamp (Fig. 3B). In preliminary experiments with the individ- pose that b clamp and g complex are, in fact, “preincubated” in ual subunits comprising g complex, we were unable to detect vivo, prior to the occurrence of the clamp-loading reaction. In binding of g, d, d9,or xzc subunits to the single-stranded 30-mer vivo, g complex loads b onto a template primed by primase with or 80-mer (data not shown). RNA. Although the rate of loading b onto a template primed Stopped-flow fluorescence anisotropy was used to measure with DNA was rapid enough to be consistent with loading rates the association of b and g complex with p/t DNA, on a ms time required during replication, the efficiency of loading b on a scale, in real time. Three different molecules, b, g complex, and template primed with RNA might be even greater. DNA, come together in these loading reactions, and g complex It has been shown previously that the g complex undergoes a hydrolyzes ATP to load b on DNA. Three types of loading structural alteration in the presence of ATP (28). It is believed reactions were performed in which the order of the addition of that this conformational change is necessary for g complex to these components to one another was varied. These experi- bind b, because ATP is required to isolate a b-g complex inter- ments demonstrated that preincubating g complex, ATP, and b action in vitro. However, preincubation of g complex with ATP caused b to load onto DNA at a rate in excess of 70 s (Fig. 6B), did not result in an increase in the rate of b loading, suggesting which appeared to bypass the rate-limiting step in the overall that the ATP-dependent conformational change in g complex loading reaction, which occurred at a rate of 12 s (Figs. 5 may not be rate-limiting during the process of b loading. There- and 6A). fore, the rate-limiting step is most likely an intramolecular The rate-limiting step is not likely to be the association of g event that occurs upon the interaction of b with g complex complex with b, because in reactions where the concentration (which has itself already changed its conformation upon inter- of g complex was varied (Fig. 5), the observed association rate action with ATP). The most attractive possibility then is that 30708 pol III Accessory Proteins b and g Complex 11785–11791 the rate-limiting step is opening of the toroidal b dimer. To 8. Fay, P. J., Johanson, K. O., McHenry, C. S., and Bambara, R. A. (1981) J. Biol. determine if this explanation is correct, we will investigate b Chem. 256, 976–983 mutant proteins in this assay that have different oligomeric 9. Stukenberg, P. T., Studwell-Vaughan, P. S., and O’Donnell, M. (1991) J. Biol. Chem. 266, 11328–11334 stability, the prediction being that a tighter dimer interface 10. Maki, S., and Kornberg, A. (1988) J. Biol. Chem. 263, 6555–6560 would decrease the rate of loading, while a weaker interface 11. Onrust, R., Finkelstein, J., Naktinis, V., Turner, J., Fang, L., and O’Donnell, might result in a faster rate. M. (1995) J. Biol. Chem. 270, 13348–13357 12. Kong, X.-P., Onrust, R., O’Donnell, M., and Kuriyan, J. (1992) Cell 69, In summary, we have shown that a simple DNA oligonucleo- 425–437 tide primer-template that supports processive DNA synthesis 13. McHenry, C. S. (1991) J. Biol. Chem. 266, 19127–19130 can be used as a model system for analyzing interactions be- 14. Studwell-Vaughan, P. S., and O’Donnell, M. (1991) J. Biol. Chem. 266, 19833–19841 tween pol III accessory proteins and core pol III with DNA. We 15. Onrust, R., Finkelstein, J., Turner, J., Naktinis, V., and O’Donnell, M. (1995) have demonstrated that the b sliding clamp-loading reaction J. Biol. Chem. 270, 13366–13377 16. Stukenberg, P. T., and O’Donnell, M. (1995) J. Biol. Chem. 270, 13384–13391 carried out by the five-protein g complex can be detected, with 17. Sinha, N. K., Morris, C. F., and Alberts, B. M. (1980) J. Biol. Chem. 263, high sensitivity, by measuring changes in steady state rota- 6561–6569 tional anisotropy of fluorescence-tagged DNA. A binding anal- 18. Studwell, P. S., and O’Donnell, M. (1990) J. Biol. Chem. 265, 1171–1178 ysis, using stopped-flow techniques, has allowed us to visualize 19. Dong, Z., Onrust, R., Skangalis, M., and O’Donnell, M. (1993) J. Biol. Chem. 268, 11758–11765 the loading reactions in real time. The future use of a series of 20. Xiao, H., Crombie, R., Dong, Z., Onrust, R., and O’Donnell, M. (1993) J. Biol. site-directed b mutants should help in defining the rate-limit- Chem. 268, 11773–11778 21. Perez-Howard, G. M., Weil, P. A., and Beechem, J. M. (1995) Biochemistry 34, ing step during clamp loading, and it should also be possible to 8005–8017 determine the properties of b that cause it to interact with two 22. Gupta, A., DeBrosse, C., and Benkovic, S. J. (1982) J. Biol. Chem. 257, dissimilar E. coli DNA polymerases, pol III core and polymer- 7689–7692 ase II (30). 23. Lakowicz, J. R. (1983) Principles of Fluorescence Spectroscopy, pp. 145–147, Plenum Press, New York REFERENCES 24. Otto, M. R., Lillo, M. P., and Beechem, J. M. (1994) Biophys. J. 67, 2511–2521 25. LeTilly, V., and Royer, C. A. (1993) Biochemistry 32, 7753–7758 1. Kornberg, A., and Baker, T. A. (1992) DNA Replication, 2nd Ed., pp. 169–170, 26. Kelman, Z., and O’Donnell, M. (1994) Curr. Opin. Genet. & Dev. 4, 185–195 W. H. Freeman and Co., New York 27. Onrust, R., Stukenberg, P. T., and O’Donnell, M. (1991) J. Biol. Chem. 266, 2. Kelman, Z., and O’Donnell, M. (1995) Annu. Rev. Biochem. 64, 171–200 21681–21686 3. McHenry, C. S., and Crow, W. (1979) J. Biol. Chem. 254, 1748–1753 28. Naktinis, V., Onrust, R., Fang, F., and O’Donnell, M. (1995) J. Biol. Chem. 270, 4. Maki, H., and Kornberg, A. (1985) J. Biol. 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Dynamics of Loading the β Sliding Clamp of DNA Polymerase III onto DNA

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 48, Issue of November 29, pp. 30699–30708, 1996 © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Dynamics of Loading the b Sliding Clamp of DNA Polymerase III onto DNA* (Received for publication, July 22, 1996, and in revised form, September 13, 1996) ¶ ¶ i Linda B. Bloom‡§, Jennifer Turner , Zvi Kelman , Joseph M. Beechem , Mike O’Donnell , and Myron F. Goodman‡ From the ‡Department of Biological Sciences, Hedco Molecular Biology Laboratories, University of Southern California, Los Angeles, California 90089-1340, the Microbiology Department, the Hearst Research Foundation, and the Howard Hughes Medical Institute, Cornell University Medical College, New York, New York 10021, and the Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232 DNA during chain elongation (9). X-ray diffraction analysis A “minimal” DNA primer-template system, consisting of an 80-mer template and 30-mer primer, supports reinforces the biochemical data, showing that the b subunit is processive DNA synthesis by DNA polymerase III core in a doughnut-shaped dimer with an inner diameter sufficient to the presence of the b sliding clamp, g complex clamp form a ring around duplex DNA, enabling it to act as a sliding loader, and single-stranded binding protein from Esch- clamp for the polymerase (12). Therefore, proper assembly of erichia coli. This primer-template system was used to the b sliding clamp by the g complex is an absolute prerequisite measure the loading of the b sliding clamp by the g for accurate and efficient chromosome duplication. It has re- complex in an ATP-dependent reaction. Bound protein- cently been shown that the t subunit acts as a bridge between DNA complexes were detected by monitoring fluores- two core polymerases (13, 14); the t subunit also brings one g cence depolarization of DNA. Steady state and time-re- complex clamp loader into the holoenzyme structure (15, 16). solved anisotropies were measured, and stopped-flow This asymmetric dimer of two polymerases within one holoen- pre-steady state fluorescence measurements allowed zyme is therefore capable of simultaneous replication of both visualization of the loading reactions in real time. The strands of a duplex chromosome as hypothesized (17). E. coli rate of loading b onto DNA was 12 s , demonstrating SSB enhances processive DNA synthesis by suppressing DNA that clamp assembly is rapid on the time scale required secondary structure that would otherwise act to stall the pol III for lagging strand Okazaki fragment synthesis. The as- holoenzyme complex (1). sociation rate appears to be limited by an intramolecu- The b clamp slides freely off the ends of linear DNA mole- lar step occurring prior to the clamp-loading reaction, cules (9); therefore, previous studies of the g complex clamp- possibly the opening of the toroidal b dimer. loading action have been limited to use of circular DNAs, such as large bacteriophage ssDNA circular genomes, as primed templates. Application of biophysical techniques to the study of The replicative DNA polymerase in Escherichia coli is the pol how the g complex loads b clamps onto DNA has been hindered III holoenzyme composed of 10 subunits (reviewed in Refs. 1 by the use of these large DNA templates. In this report we have and 2). The pol III core contains three subunits (3); the a developed a short linear template with ssDNA overhangs of subunit contains the DNA polymerase activity (4), the 39 to 59 sufficient length to slow the dissociation of b from the DNA and proofreading exonuclease resides in e (5, 6), and the third have used this template to study the interaction of b and g subunit, u, lacks a well defined function and is nonessential for complex with the DNA template via the technique of fluores- cell growth (3, 7). The core polymerase is extremely inefficient cence depolarization. because it dissociates rapidly from primer-template DNA, caus- DNA in solution undergoes rotational diffusion that varies ing DNA synthesis to be predominantly nonprocessive. Approx- with molecular weight. Rotational motion of DNA is slowed imately 10–20 nt are synthesized per template binding event down when proteins are bound. The p/t DNA was labeled with by pol III core in vitro (8). The activity of the accessory proteins a fluorescent dye molecule, X-rhodamine. The rotational mo- g complex and b tethers the core to the p/t DNA, resulting in a tion of the dye, detected by time-resolved and steady state processivity of greater than 5000 nt/template binding event fluorescence depolarization, was used to measure rotation of (4, 9). the DNA in the presence and absence of combinations of b and The g complex contains five subunits: g, d, d9, x, and c (10, g complex. Pre-steady state stopped-flow measurements were 11). The key function of the g complex is to load the b subunit made allowing us to visualize the loading of the b sliding clamp onto DNA. Biochemical data suggest that b encircles DNA and onto DNA in real time. interacts directly with the pol III core, tethering the core to the EXPERIMENTAL PROCEDURES * This work was supported by National Institutes of Health (NIH) Enzymes—E. coli DNA pol III proteins were purified as described: a, Grants GM21422, GM42554, GM38839, National Science Foundation e, and g (18); b (12); d and d9 (19); x and c (20); and u (7). Subassemblies Grant MCB-9303921, and NIH Postdoctoral Fellowship GM15304 (to of g complex and core were constituted as described (7, 11). Enzyme L. B. B.). The costs of publication of this article were defrayed in part by reaction buffer contained 20 mM TriszHCl, pH 7.5, 40 mg/ml BSA, 5 mM the payment of page charges. This article must therefore be hereby dithiothreitol, and 8 mM MgCl . For fluorescence experiments, 50 mM marked “advertisement” in accordance with 18 U.S.C. Section 1734 NaCl was included in the reaction buffer to reduce nonspecific protein- solely to indicate this fact. DNA interactions. § Present address: Dept. of Chemistry and Biochemistry, Arizona Oligonucleotides—Oligonucleotides were synthesized using standard State University, Tempe, AZ 85287-1604. b-cyanoethyl phosphoramidite chemistry. The abbreviations used are: pol III, E. coli DNA polymerase III; The 80-mer template used for primer extension reaction is 59-TGA p/t, primer-template; nt, nucleotide; SSB, E. coli single-stranded bind- ing protein; ssDNA, single-stranded DNA; AMPPNP, adenyl-59-yl GGG TGG CGG TTC TGA GGG TGG CGG TTC TAA GGG TGG CGG imidodiphosphate. TAC TAA ACC TCC TGA GTA CGG TGA TAC ACC TAT TCC GG 39. This paper is available on line at http://www-jbc.stanford.edu/jbc/ 30699 This is an Open Access article under the CC BY license. 30700 pol III Accessory Proteins b and g Complex The primer-template used for fluorescence measurements is as slit width. The emission polarizer rotated from vertical to horizontal follows. every 30 s during data acquisition. Data were acquired over a 30-min period and averaged. The instrument G factor was determined by 59 CT GGT AAT ATC CAG rotating the excitation beam to the horizontal position and acquiring 39 TCT TCT TGA GTT TGA TAG CCG GAA C GA CCA TTA TAG GTC signal-averaged data for approximately 15 30-s periods for both hori- zontal and vertical polarized emissions. Two 160-ml samples were pre- AAC AAT ATT ACC GCC A 3 9 pared containing 25 nM p/t (X-rhodamine label on 59-primer end) and 2 TTG TTA TAA TGG CGG TCG GTA ACG TTG TCC TTT TTG CGA GT 5 9 mM ATP in reaction buffer with NaCl and less than 1% glycerol. One SEQUENCE 1 sample contained 630 nM b as dimer and 200 nM g complex, and the second did not. A59-amino group on a 6-carbon atom linker arm (Glen Research) was General Procedure for Stopped-flow Anisotropy Measurements of Pro- added to oligonucleotides that were to be labeled with rhodamine. tein-DNA Association Reactions—Stopped-flow anisotropy measure- X-rhodamine (Molecular Probes, catalog number X-491) was attached ments were made using instrumentation as described previously (24). to the amino linker as described previously (21). A 30-nt primer and a Samples were excited at 580 nm (4-nm band pass), and the emission complementary 80-nt template were labeled with X-rhodamine. The signal passed through a 620-nm cut on filter. In general, the three labeled primer was annealed to an unlabeled template, and the double- syringes on the stopped-flow apparatus were loaded, and reactions were stranded p/t was purified from the single-stranded oligonucleotides by performed as follows. The first syringe was loaded with protein in nondenaturing polyacrylamide gel electrophoresis. The labeled tem- reaction buffer, the second was loaded with reaction buffer only, and the plate was annealed to an unlabeled primer in enzyme reaction buffer third was loaded with p/t DNA in reaction buffer. Association reactions using a ratio of 1 template to 1.5 primers. This p/t system was not were initiated by mixing 100 ml of the protein solution with 100 mlof gel-purified. DNA solution. Control preshot reactions were done by mixing 100 mlof The template used in reactions with core polymerase was synthe- DNA with 100 ml of reaction buffer only to measure the anisotropy of sized with a single phosphorothioate linkage to the last nucleotide on DNA alone. Data points were typically taken every 2 ms. Multiple runs the 39-terminus. A diasteriomeric mixture of products is formed where (10–25 runs) were averaged to increase signal to noise ratio. one phosphorothioate isomer is resistant to exonucleolytic cleavage and Association Kinetics at Different Concentrations of g Complex—One the second is not. Both T4 DNA polymerase and the e subunit of pol III syringe contained 100 nM p/t DNA (X-rhodamine label on 59-template show the same stereoselectivity in excision reactions (22). The mixture end), 800 nM b as dimer, and 1 mM ATP in reaction buffer with NaCl of phosphorothioate isomers was degraded on a preparative scale by and less than 1% glycerol. The second syringe contained 200, 400, or incubation with T4 DNA polymerase to leave only the nonhydrolyzable 800 nM g complex in reaction buffer with NaCl and less than 1% isomer, which was purified by polyacrylamide gel electrophoresis. glycerol. Control preshots contained 50 nM p/t DNA, 400 nM b, and 0.5 Primer Extension Processivity Assays—Primers were 59-end-labeled mM ATP. with P using T4 polynucleotide kinase (U.S. Biochemical Corp.) in Association Kinetics for Different Orders of the Addition of Subunits enzyme reaction buffer at 37 °C. Primer-templates were annealed in to DNA—Two reactions were performed. In the first, 100 nM p/t DNA enzyme reaction buffer using a ratio of 1 primer to 1.2 templates by heating to 80 °C in enzyme reaction buffer and cooling to room temper- (X-rhodamine label on 59-primer end), 800 nM b dimer, and 0.5 mM ATP ature. Four different primers were annealed to the 80-mer template, in reaction buffer with NaCl and less than 1% glycerol were loaded into and six separate primer extension reactions were performed for each p/t one syringe. The second syringe contained 600 nM g complex and 0.5 mM combination containing (A) core only, (B) core, b, and g complex, (C) core ATP in reaction buffer with NaCl and less than 1% glycerol. Control and SSB, (D) core, SSB, b, and g complex, (E) core, SSB, and b,or(F) preshots contained 50 nM p/t DNA, 400 nM b, and 0.25 mM ATP. In the core, SSB, and g complex (Fig. 1). Primer-templates were preincubated second reaction, one syringe contained 100 nM p/t DNA (X-rhodamine with SSB for 2 min at 37 °C prior to a second preincubation with b and label on 59-primer end) and 0.5 mM ATP in reaction buffer with NaCl. g complex for 5 min at 37 °C. In reactions where SSB, b,or g complex The second syringe contained 800 nM b dimer, 600 nM g complex, and were omitted, preincubations were performed with reaction buffer only. 0.5 mM ATP in reaction buffer with NaCl and less than 1% glycerol. Reactions were initiated by the addition of core polymerase and Control preshots contained 50 nM p/t DNA and 0.25 mM ATP. quenched after 10 s with 40 mlof20mM EDTA in 95% formamide. All Dissociation Kinetics—M13 trap DNA consisted of single-stranded reactions contained 2 nM p/t DNA, enzyme reaction buffer, 4% glycerol, wild type M13 DNA with two 30-nt oligonucleotides annealed to differ- 0.5 mM ATP, and 60 mM dATP, dCTP, dGTP, and dTTP in a 20-ml ent sites. In this experiment, p/t DNA was preincubated with b and g reaction volume. Reactions with accessory proteins contained 40 nM complex, and reactions were initiated by the addition of M13 trap DNA. SSB, 20 nM b dimer, 3.5 nM g complex, and 18 nM core. Reaction One syringe was loaded with 100 nM p/t DNA (X-rhodamine label on the products were separated on 10% denaturing polyacrylamide gels and 59-primer end), 800 nM b dimer, 400 nM g complex, and 2 mM ATP in visualized using a PhosphorImager (Molecular Dynamics). reaction buffer with NaCl and less than 1% glycerol. The second con- Steady State Anisotropy Measurements—Steady state anisotropy tained 500 nM M13 trap DNA in reaction buffer with NaCl. Control measurements were made using a QuantaMaster QM-1 fluorometer preshot reactions contained 50 nM p/t DNA, 400 nM b dimer, 200 nM g (Photon Technology International) with a single emission channel (L complex, and 1 mM ATP. format) and 400–800-nm dichroic sheet polarizers (Oriel). Samples were excited with vertically polarized light at 580 nm (8 nm band pass), RESULTS and both vertical and horizontal emission was monitored at 610 nm A series of protein-protein interactions govern the assembly (8-nm band pass). Since the emission polarizer was manually rotated, of the pol III holoenzyme complex, and protein-protein and three separate anisotropy measurements were made on each sample, and the values were averaged. Anisotropy values were calculated using protein-DNA interactions, coordinated with the use of ATP, are a G factor (polarization bias) (23) determined under identical experi- required to load pol III holoenzyme onto p/t DNA. We are mental conditions. investigating protein-DNA interactions accompanying the Titration experiments were performed starting with 188 mlof53nM loading of the b clamp by the g complex using a sensitive p/t DNA (X-rhodamine label on 59-template end) in reaction buffer with optical technique, fluorescence depolarization, to detect and NaCl and ATP. After measuring the anisotropy of the p/t, a 4-ml aliquot analyze binding of the proteins to DNA (21, 25). Fluorescence of 20 mM b dimer and an 8-ml aliquot of 5 mM g complex were added. Anisotropy was measured after each addition of protein. Final concen- depolarization measures the rotational diffusion of fluores- trations were 50 nM p/t, 400 nM b dimer, 200 nM g complex, and 0.5 mM cence-labeled DNA alone or when present in a DNA-protein ATP. The anisotropy of this solution was measured after it stood at complex. The rotational motion depends predominantly on the room temperature for 15 min. After the addition of a 2-ml aliquot of 45 molecular weight of the DNA-protein complex. Therefore, it is mM ATP, the anisotropy measurement was repeated. possible to distinguish free DNA from DNA bound by combina- Time-resolved Decays of Fluorescence—Time-resolved measurements tions of b and g complex by measuring steady state fluores- were performed in cuvettes with 3-mm path lengths using instrumen- tation described in Ref. 21. Samples were excited at 580 nm. The cence anisotropy. A pre-steady state anisotropy measurement emission signal passed through a Glan-Thompson polarizer, 620-nm cut allows protein-DNA complex formation to be visualized in real on filter, and a SPEX 0.22-m monochromator set at 640 nm with a 1-mm time. Minimal Primer-Template Requirements for Processive Syn- L. B. Bloom and M. F. Goodman, unpublished results. thesis by E. coli pol III Core Polymerase in the Presence of b and pol III Accessory Proteins b and g Complex 30701 FIG.1. Requirements for processive DNA synthesis by pol III on an 80-nucleotide template. Primer extension reactions by pol III core polymerase on an 80-mer template in the presence of different combinations of accessory proteins (b, g complex, and SSB) were analyzed by denaturing polyacrylamide gel electrophoresis. Four different 30 nucleotide primers were annealed to an 80-nucleotide template to create regions of single-stranded DNA on the 39-template end (39-template overhangs) of 10, 15, 20, and 25 nucleotides. Six separate extension reactions were performed at 37 °C on each primer-template combination. All reactions contained 2 nM p/t DNA, 0.5 mM ATP, and 60 mM dATP, dCTP, dGTP, and dTTP. Reactions P (lanes 1, 8, 15, and 22) contained primer-template only as a marker. Reactions A (lanes 2, 9, 16, and 23) contained 18 nM core only. Reactions B (lanes 3, 10, 17, and 24) contained 18 nM core, 20 nM b (dimer), and 3.5 nM g complex. Reactions C (lanes 4, 11, 18, and 25) contained 18 nM core and 40 nM SSB (as monomer). Reactions D (lanes 5, 12, 19, and 26) contained 18 nM core, 20 nM b (as dimer), 3.5 nM g complex, and 40 nM SSB (as monomer). Reactions E (lanes 6, 13, 20, and 27) contained 18 nM core, 20 nM b (as dimer), and 40 nM SSB as monomer. Reactions F (lanes 7, 14, 21, and 28) contained 18 nM core, 3.5 nM g complex, and 40 nM SSB (as monomer). g Complex—To observe maximum changes in rotational anisot- the absence of SSB, did not significantly enhance the proces- ropy signals during transitions between free DNA and bound sivity of the core polymerase, although a small fraction of DNA-protein complexes it is important to utilize the smallest primers were extended to the end of the template (Fig. 1, lane possible DNA capable of supporting processive pol III DNA 24). The addition of SSB alone to the core inhibited synthesis, synthesis. On large DNA templates such as fX and M13 DNA, as shown by faint gel bands (Fig. 1, lane 25). However, proces- the processivity of DNA synthesis by pol III core DNA polym- sive synthesis was observed by the core polymerase in the erase is increased substantially by the addition of b and g presence of b, g complex, and SSB (Fig. 1, lane 26). A dark gel complex. The g complex loads b on DNA, and b acts as a sliding band was present for primers extended to the end of the tem- clamp to tether the core polymerase to the DNA template, plate, while faint bands due to dissociation of the core prior to reviewed in Ref. 26. reaching the end of the template were also present. The addi- We found that a DNA template as small as 80 nt in length tion of either b and SSB in the absence of g complex (Fig. 1, lane supports processive synthesis by core polymerase in the pres- 27)or g complex and SSB in the absence of b (Fig. 1, lane 28) ence of b and g complex. Processive synthesis on this 80-mer did not result in processive synthesis by core polymerase. Re- template requires b, g complex, and single-stranded binding action products resembled those by synthesis of core in the protein, SSB (Fig. 1, lanes 23–28).A59- P-labeled 30-nt presence of SSB alone. primer was annealed to an 80-nt template so that 25-nt single- The efficiency of synthesis on the 80-nt template depended stranded regions were present on both the 59- and 39-ends of the both on the length of the primer and its position on the tem- template. The primers were extended by core polymerase and plate. On an 80-mer template, a 30-nt primer was extended different combinations of accessory proteins (b, g complex, and more efficiently by core polymerase in the presence of b, g SSB). After 2 min at 37 °C with SSB only, the p/t DNA was complex, and SSB than a 20-nt primer (data not shown). A preincubated at 37 °C for 5 min with different combinations of 15-nt primer was not extended at all in a 20-s reaction (data accessory proteins in separate reactions. Reactions were initi- not shown). ated by the addition of the core polymerase and quenched after To achieve processive synthesis, the 30-nt primer had to be 10 s. Reaction products were analyzed by polyacrylamide gel positioned on the 80-mer template so that a single-stranded electrophoresis. region of DNA greater than 15 nt long was present at the Gel bands represent sites where the polymerase either dis- 39-template end (Fig. 1, lanes 5, 12, 19, and 26). 59- P-labeled sociated or paused after incorporating one or more nt. Since primers, 30 nt in length, were annealed to an 80-nt template so DNA synthesis by the core polymerase alone has been demon- that the length of the single-stranded region of DNA on the strated to be distributive (8), dark gel bands most likely indi- 39-template end (39-template overhang) varied. Primer exten- cate a high probability of dissociation at a given site. Synthesis sion reactions on these p/t DNAs were performed with core by core polymerase alone was essentially distributive. Dark gel polymerase along with different combinations of accessory pro- bands were present for DNA products that were extended by 1, teins as above. In the presence of b, g complex, and SSB, 2, 3, 4, and 5 nt (Fig. 1, lane 23). Bands caused by the formation processive synthesis by the core polymerase was not observed of longer products were less intense. The core alone was unable when the 39-template overhang was 10 or 15 nt long (Fig. 1, to synthesize DNA to the end of the template. lanes 5 and 12). When the 39-template overhang was increased The addition of b and g complex to the core polymerase, in to 20 nt, a faint gel band representing the extension of primers 30702 pol III Accessory Proteins b and g Complex FIG.2. Increase in steady state anisotropy for X-rhodamine-labeled DNA when g complex loads b on DNA. A, the change in steady state anisotropy for a X-rhodamine-labeled primer-template when b and then g complex were added to the DNA. An 80-nucleotide template was labeled at the 59-end with X-rhodamine and annealed to a 30-nucleotide primer. The steady state anisotropy of the primer-template in reaction buffer containing ATP and MgCl was measured (DNA). The anisotropy of this DNA solution was measured again after adding a small aliquot of b (1 b) and then g complex (1 g complex). Concentrations after the addition of g complex were 50 nM primer-template, 400 nM b (dimer), 200 nM g complex, and 0.5 mM ATP. The anisotropy of this solution was measured again after the solution stood at room temperature for 15 min (15 min). An aliquot of ATP was then added, and the anisotropy was measured again (1 ATP). B, the change in steady state anisotropy for an X-rhodamine-labeled primer-template when g complex and then b were added to the DNA. The conditions for this titration were the same as for A except that the order of the addition of b and g complex was reversed. In this experiment, g complex was added first, and then b was added to establish that both b and g complex are required to give the large increase in steady state anisotropy (r ' 0.26) for the DNA. A sketch of the reaction scheme is drawn at the top of the figure. to the end of the template was observed for a reaction contain- proximity to b after b is loaded by g complex. ing core polymerase, b, g complex, and SSB (Fig. 1, lane 19). Loading of b on Primer-Template DNA by g Complex—Inter- More efficient primer extension to the end of the template by actions of b and g complex with p/t DNA under steady state core in the presence of b, g complex, and SSB was observed conditions were observed by monitoring changes in fluores- when the 39-template overhang was increased to 25 nt (Fig. 1, cence anisotropy of p/t DNA labeled with an extrinsic fluores- lane 26). This probably reflects a requirement for SSB to pre- cent probe, X-rhodamine. An 80-nt template was labeled at the vent b from “sliding off” the end of the template. 59-terminus with X-rhodamine. A 30-nt primer was annealed to Although an excess of polymerase over DNA was used in the center of the template so that 25-nt regions of single- primer extension reactions (core:p/t 5 9:1), only a fraction of stranded DNA were present on both the 59- and 39-ends of the primers were extended during our short 10-s reaction time. In template. This was the optimal p/t configuration supporting reactions with pol III core alone, inefficient primer extension processive DNA synthesis by the pol III core in the presence of may result from a weak association of core with DNA. In the b, g complex, and SSB (Fig. 1). A DNA synthesis reaction using presence of b, g complex, and SSB, inefficient primer extension a rhodamine-labeled template, carried out as shown in Fig. 1, may be caused by a slow association of the core polymerase demonstrated that the rhodamine label has no measurable with b to form a processive bzcore complex (or a slow displace- effect on the activity of the pol III proteins (data not shown). ment of SSB from the template). Note that it may also be The steady state anisotropy of the rhodamine-labeled p/t, in difficult to load b onto all of the p/t molecules because g com- the absence of pol III accessory proteins, was 0.166 (Fig. 2A). plex may actively remove b resulting in a steady state popula- The addition of b (molecular mass 81 kDa for a b dimer) to the tion of DNA containing b and free DNA. rhodamine-labeled p/t DNA (52 nM DNA, 420 nM b dimer) did In primer extension assays, the fraction of primers extended not affect the anisotropy of the DNA (i.e. in the absence of g increased with concentration of core polymerase in 10-s reac- complex, there was no measurable interaction between b and tions (data not shown) and also increased with reaction time p/t DNA). The addition of g complex to the solution of b and (data not shown), indicating that essentially all of the primers DNA (50 nM DNA, 400 nM b dimer, and 200 nM g complex) were extendible. Experiments are under way to measure the resulted in a large increase in the steady state anisotropy of the kinetics of association of the core polymerase with b to deter- labeled DNA, from 0.166 to 0.257 (Fig. 2A). The rotational mine if a rate-determining association step limits the rate of diffusion of the DNA decreased upon addition of b and g primer extension. In the holoenzyme complex, the core and g complex. complex form part of a larger complex so that association of the To determine if the increase in steady state anisotropy in the core with b is likely to be more efficient, since it is in close presence of b and g complex was caused by g complex loading pol III Accessory Proteins b and g Complex 30703 FIG.3. Association of g complex and bzg complex with DNA substrates as determined by an increase in steady state anisotropy. Three different DNA substrates, a single-stranded 30-mer (L), a single-stranded 80-mer (M), and a 30-mer/80-mer p/t (E) were labeled with rhodamine and were titrated with g complex. Each solution contained reaction buffer consisting of 20 mM TriszHCl, pH 7.5, 50 mM NaCl, 8 mM MgCl ,5mM dithiothreitol, 40 mg/ml BSA, and 1 mM ATP. Data were taken by hand-mixing reagents in a cuvette and measuring steady state anisotropy values immediately after the addition of g complex (A)or b (B). A, g complex was added to a solution of 50 nM DNA in reaction buffer. A new solution was made for each concentration of g complex. B, after each g complex addition in A, b was added to a concentration of 500 nM b (as dimer). b on the p/t or by g complex alone binding to p/t, the order of the oligonucleotide, single-stranded 80-nt oligonucleotide, and 30- addition of g complex and b to the p/t DNA was reversed (Fig. mer/80-mer p/t were measured in the presence of 1 mM ATP 2B). The solution of labeled p/t DNA in the absence of b and g (Fig. 3A). We previously used 0.5 mM ATP for steady state complex gave a steady state anisotropy of 0.165 as observed anisotropy measurements with 200 nM g complex (Fig. 2). The previously. The addition of g complex (201 kDa) to DNA (51 nM higher concentration of ATP was used to ensure that it was not DNA and 200 nM g complex) resulted in a small increase in completely consumed during the 2–3 min taken to measure anisotropy to 0.176. However, the subsequent addition of b to anisotropies using up to 800 nM g complex (Fig. 3). g complex the solution (50 nM DNA, 400 nM b dimer, 200 nM g complex) was found to interact with the single-stranded 80-nt DNA resulted in a much larger increase in anisotropy to 0.248. Thus, substrate, although this association is weak (apparent K 5 the much larger increase in anisotropy was caused by g com- 450 nM). Interaction of g complex with 30-mer/80-mer p/t DNA plex loading b on the p/t DNA and not by direct interaction of or with 30-mer ssDNA was barely detectable (Fig. 3A, lower g complex alone with DNA. two curves). However, when b was added to form bzg complex, In both titration experiments above, the anisotropy of the p/t the interaction with ssDNA and p/t DNA was strengthened DNA decreased when solutions of DNA, b, and g complex considerably (Fig. 3B). We want to emphasize that for the p/t remained for 15 min at room temperature (Fig. 2). The anisot- DNA, the increase in anisotropy with increasing concentration ropy regained its former level in both experiments when a fresh of g complex is most likely caused by loading of b onto DNA. aliquot of ATP was added (Fig. 2). These results demonstrate The replication assays (Fig. 1) show that a processive replica- that the interactions of b and g complex with DNA required tion complex can be formed on this p/t, demonstrating that g hydrolysis of ATP and that the ATP initially present was con- complex is fully capable of loading b on this p/t in a biologically sumed during the 15-min incubation. The g complex has been relevant manner. shown to be a DNA-dependent ATPase, and ATP is required for The association of g complex with either single-stranded 3 32 g complex to load b on DNA (27). Hydrolysis of a- P-labeled DNA or p/t DNA required ATP hydrolysis. In the presence of ATP was measured in solutions containing an unlabeled p/t, b, the nonhydrolyzable ATP analog, AMPPNP, g complex alone and g complex (data not shown). ATP was converted to ADP and a bzg complex did not associate with either single-stranded under these conditions. Loading of b by the g complex did not 80-mer or with 30-mer/80-mer p/t DNA (data not shown). Since take place when ATP was replaced in the reaction with either g complex was observed to bind ssDNA and p/t DNA, in the UTP or by the nonhydrolyzable ATP analog AMPPNP (data absence of b in an ATP-dependent reaction, a preliminary not shown). experiment was carried out to see if binding of individual Binding of g Complex to DNA—Association of g complex and subunits of g complex to ssDNA could also take place in the loading of b on DNA by g complex are not simple equilibrium presence of ATP. No measurable binding to either a single- protein-DNA binding interactions. Association of g complex stranded 30-mer or 80-mer was observed for g, d, d9,or xzc at with DNA is dependent on ATP hydrolysis, and loading of b concentrations of at least 600 nM protein and 50 nM DNA onto DNA depends on both the catalytic activity of g complex (data not shown). and the hydrolysis of ATP. Association constants for these Time-resolved Anisotropy Measurements—Time-resolved in- proteins to DNA were not measured under true equilibrium tensity and anisotropy measurements (ns time scale) were binding conditions; instead, association was measured as a made on p/t DNA containing a rhodamine-labeled primer-59- function of protein concentration to give apparent binding af- terminus. These measurements were made in the presence and finities under steady state conditions (Fig. 3). The binding of g absence of b and g complex. When b and g complex are present, complex to different DNA substrates, single-stranded 30-nt the system is dynamic, i.e. b and g complex continuously asso- ciate and dissociate from the DNA in an ATP-dependent reac- J. Turner and M. O’Donnell, unpublished results. tion on a ms time scale. The time-resolved measurements re- 30704 pol III Accessory Proteins b and g Complex FIG.5. Reaction time courses for loading b on p/t DNA: the addition of g complex to b and X-rhodamine-labeled DNA. Four separate time course reactions are shown. Each reaction contained 50 nM p/t (X-rhodamine label on 59-template end), 400 nM b as dimer, and 0.5 mM ATP as described under “Experimental Procedures.” Reactions were initiated by adding solutions differing in the concentration of g complex to a solution of b, DNA, and ATP. Final concentrations of g complex were 0 (showing a constant anisotropy), 100, 200, and 400 nM. The anisotropy increased upon the addition of g complex. The magni- tude of the anisotropy change increased with increasing concentration of g complex, while the observed rates (12 s ) of increase in anisotropy FIG.4. Time-resolved decays of total intensity and anisotropy remained constant. A sketch of the reaction scheme is drawn at the top. for rhodamine-labeled DNA in the presence and absence of b and g complex. A, time-resolved decay of total intensity for 25 nM p/t with an X-rhodamine label on the 59-primer terminus in the presence and absence of 200 nM g complex and 630 nM b dimer. Both reactions reflective of the rotational diffusion of the protein-DNA contained 20 mM TriszHCl, pH 7.5, 50 mM NaCl, 8 mM MgCl ,2mM ATP, complex. and less than 1% glycerol as described under “Experimental Proce- Pre-steady State Association of b and g Complex with p/t dures.” Data were collected over 30 min. The concentration of ATP was increased from 0.5 to 2 mM so that ATP would not be completely DNA Observed in Real Time—The kinetics of loading the b consumed during the time course of the measurement. The presence of sliding clamp onto p/t DNA by the g complex were observed in b and g complex does not affect the lifetime of the X-rhodamine probe; real time by measuring the steady state anisotropy of rhoda- the decay curves overlap. B, time-resolved decays of anisotropy for the mine-labeled p/t in stopped-flow reactions (Fig. 5). Note that same samples. The upper data curve shows the slower rate of anisotropy the protein-DNA association kinetics occurring on a pre-steady decay for the p/t in the presence of b and g complex. The lower data curve shows the more rapid rate of anisotropy decay for the p/t alone. state (ms) time scale are detected by changes in the steady state rotational anisotropy. Four curves are shown in Fig. 5. flect the steady state populations of bound and free DNA. The lower horizontal curve shows a constant value for the The decay in fluorescence intensity, i.e. the fluorescence life- steady state anisotropy of p/t DNA in the presence of b, which time, of the rhodamine-labeled p/t was the same for the naked does not interact with p/t under these conditions (Fig. 2). The DNA and when the b and g complex were present (Fig. 4A). The three upper curves illustrate loading of a constant amount of b fluorescence lifetimes for the p/t with 59-template label were (400 nM dimer) using three levels of g complex (100, 200, or 400 also the same in the presence and absence of b and g complex nM). The concentrations of b, g complex, p/t, and ATP for the (data not shown). Time-resolved decays in anisotropy showed reaction containing 200 nM g complex were the same as in the an increase in the rotational correlation time for the rhodamine steady state experiment in Fig. 2. probe in the presence of b and g complex, demonstrating that b The reaction was carried out using the following scheme (Fig. and/or g complex is associated with the p/t (Fig. 4B). 5, sketch of reaction). b (800 nM b ) was present in one syringe Steady state anisotropy values (Fig. 2) are a function of both along with p/t DNA (100 nM), ATP (1 mM), and buffer contain- the lifetime and rotational correlation time of a fluorophore (23, ing Mg . The g complex was present in a second syringe in the 24). Since the fluorescent lifetime of the rhodamine probe was same buffer with Mg but without ATP. The X-rhodamine the same in the presence and absence of b and g complex, the label was present at the 80-mer template-59-terminus. The changes in steady state anisotropy that we measured using contents of the two syringes were delivered to the reaction rhodamine-labeled DNA are the result of changes in rotational chamber, and anisotropy data points were collected at 2-ms motion of the probe only. Therefore, the signal in Fig. 2 is truly intervals (see “Experimental Procedures”). Multiple runs were pol III Accessory Proteins b and g Complex 30705 FIG.6. Reaction time courses for loading b on p/t DNA: different orders of the addition of g complex and b to X-rhodamine-labeled DNA. A, reaction time course for the addition of a solution of g complex and ATP to a solution of b, p/t DNA (X-rhodamine label on the 59-primer end), and ATP. Final concentrations were 50 nM p/t, 300 nM g complex, 400 nM b dimer, and 0.5 mM ATP as described under “Experimental Procedures.” The curve showing a constant anisotropy is a control reaction without g complex where reaction buffer only was added to the solution of b, p/t DNA, and ATP. B, reaction time course where a solution of b, g complex, and ATP was added to a solution of p/t DNA and ATP. Final reaction conditions in both panels are the same. The curve with constant anisotropy shows the addition of reaction buffer to DNA and ATP. When the reaction was initiated by the addition of b, g complex, and ATP to p/t, the observed increase in anisotropy was more rapid (much of the reaction is missed in the dead time of the instrument) than when the reaction was initiated by the addition of g complex and ATP to b, p/t DNA, and ATP. A sketch of the reaction scheme is drawn at the top. summed to increase the signal:noise ratio, and the data shown loading it on DNA (9, 16). A third possibility is that g complex were run-averaged over 5 points, while the raw data were fit by initially binds the p/t at multiple sites. A nonproductively a double exponential (Fig. 5, solid curves). bound g complex may have to dissociate or may have to be The data showed an increase in anisotropy for the labeled p/t displaced before productive loading of b onto the p/t can occur with an observed rate of 12 6 2s , resulting from association to achieve steady state loading and dissociation. A fourth, of b and g complex with p/t DNA. In this experiment, b was much more interesting, possibility might be a dramatic confor- either equal to or in excess of g complex, and both were in mational change in the bzg complex that affects the rotational excess of p/t DNA. The amplitudes of the reaction time courses mobility of the protein-DNA complex. increased with an increase in g complex and reflect an increase Stopped-flow anisotropy measurements were made using dif- in the total amount of protein bound to DNA. Although more ferent orders of the addition of b and g complex to 30-mer/80- protein bound to p/t DNA as the concentration of g complex was mer p/t DNA (Fig. 6). Depending on which interactions or steps increased, the observed reaction rate remained constant. Each are rate-limiting, the order of the addition of these components protein-bound DNA curve exhibited a small, but significant, to one another should affect the observed reaction rate. In these decrease between 250 and 350 ms prior to reaching steady state reactions, the 59-primer terminus, rather than the template, (t . 500 ms). was labeled with X-rhodamine. Since the rate of association of There are several possible explanations for this small de- b and g complex with DNA was not affected by the concentra- crease in anisotropy. First, the dip could reflect an initial tion of g complex, but the magnitude of the anisotropy increase sliding off of b from the relatively short 30-mer/80-mer p/t DNA was a function of the g complex concentration (Fig. 5), a con- in the absence of SSB, relaxing to a steady state characterized centration of 300 nM g complex was used in these experiments by repeated sliding off and reloading of b onto p/t DNA. Note to give a large anisotropy signal change during the time course that SSB which was present in the polymerization reactions to of these reactions. The concentrations of b, DNA, and ATP were keep the b dimer from sliding off the p/t DNA was not present the same as in Fig. 5. during the anisotropy measurements because the presence of In Fig. 6A, b (800 nM dimer) was preincubated with p/t DNA SSB bound to the DNA would limit the increase in anisotropy (100 nM) and ATP in one syringe, and g complex (600 nM) and that would be observed when b and g complex bind. A second ATP were in the second syringe. In Fig. 6B, b (800 nM), g explanation is that the decreased anisotropy reflects dissocia- complex (600 nM), and ATP were present together in one sy- tion of some fraction of g complex from the protein-DNA com- ringe, and p/t DNA (100 nM) and ATP were in the second plex. g complex has been shown to dissociate from b after syringe. There was a much more rapid increase in anisotropy 30706 pol III Accessory Proteins b and g Complex when the solution of b and g complex were added to p/t DNA (Fig. 6B) than when g complex was added to b and DNA (Fig. 6A). An estimate of the loading rate of b following preincuba- tion with g complex is 70 s , which is at least 6 times faster than the rate of loading b in the absence of preincubation with g complex (Figs. 5 and 6A). Inclusion of ATP along with the g complex prior to encountering b and DNA resulted in the same observed rate of increase in anisotropy, 12 s , as in reactions where ATP was added to g complex at the same time as b and DNA (Fig. 5). Thus, the rate-determining step appears to be different for reactions where b, g complex, and ATP were pre- incubated prior to the addition of the p/t in comparison with reactions where either g complex alone or g complex and ATP were added to a solution of b and p/t DNA. The decrease in anisotropy observed between 250 and 350 ms appears to be more pronounced in the reaction in Fig. 6B than in Fig. 6A. Both data sets were fit to the sum of an exponential increase and an exponential decrease in anisotropy. Since the rate of the increase in Fig. 6B is more rapid, more of the decrease in anisotropy is observed. In a similar experiment, a solution of b and g complex in the absence of ATP was added to a solution of p/t and ATP (data not shown). The observed rate of increase in anisotropy for this reaction was 11 s , indicating that this combination is not sufficient to bypass the rate-limiting step. These results are consistent with previous data showing that a stable multipro- tein complex consisting of b and g complex forms in an ATP- FIG.7. Reaction time course for the dissociation of b or b and dependent reaction in the absence of DNA (28). g complex from X-rhodamine-labeled DNA. The bottom trace shows Dissociation of b or bzg Complex from p/t DNA in Real a reaction initiated by the addition of p/t DNA (X-rhodamine label on Time—In steady state reactions (Fig. 2), ATP was rapidly hy- 59-primer end), b, g complex, and ATP to a solution of M13 trap DNA as drolyzed, most likely by repeated loading of b, which rapidly described under “Experimental Procedures.” Final concentrations were 50 nM p/t, 400 nM b dimer, 200 nM g complex, and 1 mM ATP. The dissociated from the short synthetic p/t DNA. Under our reac- concentrations of b, g complex, and p/t are the same as in Figs. 2 and 5. tion conditions, two possible dissociation pathways may exist: a The concentration of ATP was increased from 0.5 to 1 mM to ensure that two-step dissociation process where g complex dissociates from ATP was not completely consumed during the preincubation of b, g b and DNA after loading b and then b diffuses off the p/t, or a complex, and p/t. The observed rate of dissociation of proteins from one-step process where a bzg complex dissociates from the DNA was 6.4 s . The top trace shows constant anisotropy for a control reaction where a solution of p/t DNA, b, g complex, and ATP is added to DNA. The rate of dissociation of b or bzg complex from the reaction buffer rather than to trap DNA. A sketch of the reaction 30-mer/80-mer p/t DNA (depending on which pathway occurs) scheme is drawn at the top. was measured by preincubating b , g complex, ATP, and DNA in one syringe, while in the other syringe was placed an excess loading reaction carried out by the five-protein g complex can of single-stranded M13 DNA with two 30-nt primers. When the be detected, with high sensitivity, by measuring changes in the contents of the two syringes were mixed together in the steady state rotational anisotropy of fluorescence-tagged DNA. stopped-flow reaction chamber, any b and g complex that had A binding analysis, using stopped-flow techniques, has allowed either dissociated from the p/t DNA or had never been bound to us to visualize the clamp-loading reaction in real time. the p/t became trapped by the M13 DNA so that it could not Loading the b clamp onto DNA by the g complex is a prereq- reload onto the 30-mer/80-mer p/t. uisite to achieving processive leading and lagging strand DNA The rotational anisotropy remained essentially unchanged synthesis in E. coli. To study the reaction steps that involve on a 1-s time scale in the absence of trap DNA (Fig. 7, top trace). loading of the b sliding clamp onto p/t DNA, we have designed A reduction in the steady state rotational anisotropy reflects an a simple model system that supports processive synthesis by increase in the rotational motion of the p/t DNA as b (or bzg the pol III core polymerase in the presence of b, g complex, and complex) dissociated from the p/t, presumably by sliding off SSB. We find that processive synthesis is supported by a 30-nt over the end (Fig. 7, bottom trace). Dissociation was rapid, with primer annealed to the central region of an 80-nt template, so a first order off-rate constant of 6.4 s . The data can be that 25-nt single-stranded regions of DNA exist on both the 59- represented by a model in which b rapidly slides off the p/t and 39-ends of the template. This 30-mer/80-mer p/t DNA sys- DNA after being loaded. A cycle of repeated loading of b that tem is convenient for studying the binding of accessory proteins requires ATP hydrolysis by g complex, followed by rapid loss of (this report) and will also make possible study of the effects of b, would lead to consumption of ATP with time. A rapid disso- processivity on the fidelity of DNA synthesis. ciation of b would explain the requirement for SSB in primer Extensions of ssDNA of at least 20 nt were necessary on both extension assays to trap b on a linear p/t for enough time for the ends of the p/t DNA, in order to support processive synthesis by core to associate with b (Fig. 1). pol III core polymerase in the presence of b and g complex (Fig. DISCUSSION 1). Processive synthesis was observed only when SSB was Here we have shown that a simple DNA oligonucleotide included in reactions with core, b, and g complex. These single- primer-template that supports processive DNA synthesis can stranded regions are most likely needed to bind SSB efficiently. be used as a model system for analyzing interactions between SSB inhibited synthesis in reactions by core alone except when pol III accessory proteins and core pol III with DNA. In the the single-stranded region of DNA on the 59-template end was present study, we have demonstrated that the b sliding clamp- only 10 nt long, suggesting that SSB cannot bind to this short pol III Accessory Proteins b and g Complex 30707 FIG.8. Model showing g complex loading of b clamp onto DNA for lag- ging strand DNA synthesis. g complex binds b clamp in an ATP-dependent reac- tion. A rate-limiting step of about 12 s may possibly reflect opening of the toroi- dal b dimer clamp prior to loading onto p/t DNA. The last step shows dissociation of the complex from p/t DNA, which initiates the next clamp-loading cycle. region of ssDNA. Binding of SSB to single-stranded regions of was independent of the concentration of g complex. Instead, the the template on both sides of the primer may help stabilize b on slow step is more likely to be intramolecular in nature, because short p/t DNA long enough for a complex between b and core to it does not depend on the concentration of g complex. Perhaps assemble. a conformational change within b or g complex may be limiting The 30-mer/80-mer p/t DNA was labeled either at the 59- the rate of loading b. Two prominent possibilities for this step primer terminus or the 59-template terminus with a fluorescent are a conformational change in g complex or a conformational probe, X-rhodamine, and the fluorescence anisotropy of this change in b, such as “opening” of the ring prior to placement on probe was then used to detect interactions with the pol III DNA. accessory proteins, b and g complex. Association of b and g The pol III holoenzyme contains two DNA polymerase cores complex with the labeled p/t DNA decreased the rate of rota- (for simultaneous replication of both strands of a duplex chro- tional motion of the DNA and thus increased the steady state mosome) and only one g complex, all connected together by a anisotropy of the probe. When b and g complex were added to dimer of the t subunit (15). The g complex acts catalytically labeled p/t DNA, the anisotropy increased, demonstrating load- during lagging strand replication to load b clamps onto RNA ing of b by g complex. In contrast to the requirement for SSB to primers as they are produced by the helicase/primase (29). The obtain processive synthesis on the short p/t DNA (Fig. 1), SSB lagging polymerase, upon finishing a fragment, rapidly disen- was not required for b loading, further supporting the idea that gages from its b clamp, leaving it behind on DNA, and cycles SSB can provide a block keeping b from sliding off the ends of back to the newly assembled b clamp on the upstream primer the primer. Consistent with the idea that SSB serves as a block to extend the next lagging strand fragment (Fig. 8). Since the to stabilize b on linear DNA, a rapid rate of dissociation of b replication fork moves at a rate of approximately 1 kilobase/s was observed in the absence of SSB (Fig. 7). In these experi- and lagging strand fragments average 1–2 kilobase in length, a ments, a block is not necessary for loading per se, but rather to new fragment is produced every 1 or 2 s. Thus, the g complex keep b on the template so that core can bind to it and harness must be capable of clamping b onto primers within 1 s. The its ability to serve as a processivity factor. speed of clamp assembly observed in this report (12 s , t1 5 We investigated binding of g complex to individual ssDNA 58 ms) is well within the speed required for g complex action on and p/t DNA components. In the absence of b clamp, g complex the lagging strand. Perhaps even more relevant is that the rate bound extremely weakly to 30-mer ssDNA and 30-mer/80-mer of clamp assembly when b and g complex were preincubated p/t DNA (Fig. 3A). A somewhat higher affinity binding was together was about 70 s (t1 ' 10 ms), which is much faster observed with 80-mer ssDNA (Fig. 3A). Binding to each of the than required for recycling b clamp onto the lagging strand for forms of DNA was significantly enhanced in the presence of b rapid synthesis of Okazaki fragments. It is reasonable to sup- clamp (Fig. 3B). In preliminary experiments with the individ- pose that b clamp and g complex are, in fact, “preincubated” in ual subunits comprising g complex, we were unable to detect vivo, prior to the occurrence of the clamp-loading reaction. In binding of g, d, d9,or xzc subunits to the single-stranded 30-mer vivo, g complex loads b onto a template primed by primase with or 80-mer (data not shown). RNA. Although the rate of loading b onto a template primed Stopped-flow fluorescence anisotropy was used to measure with DNA was rapid enough to be consistent with loading rates the association of b and g complex with p/t DNA, on a ms time required during replication, the efficiency of loading b on a scale, in real time. Three different molecules, b, g complex, and template primed with RNA might be even greater. DNA, come together in these loading reactions, and g complex It has been shown previously that the g complex undergoes a hydrolyzes ATP to load b on DNA. Three types of loading structural alteration in the presence of ATP (28). It is believed reactions were performed in which the order of the addition of that this conformational change is necessary for g complex to these components to one another was varied. These experi- bind b, because ATP is required to isolate a b-g complex inter- ments demonstrated that preincubating g complex, ATP, and b action in vitro. However, preincubation of g complex with ATP caused b to load onto DNA at a rate in excess of 70 s (Fig. 6B), did not result in an increase in the rate of b loading, suggesting which appeared to bypass the rate-limiting step in the overall that the ATP-dependent conformational change in g complex loading reaction, which occurred at a rate of 12 s (Figs. 5 may not be rate-limiting during the process of b loading. There- and 6A). fore, the rate-limiting step is most likely an intramolecular The rate-limiting step is not likely to be the association of g event that occurs upon the interaction of b with g complex complex with b, because in reactions where the concentration (which has itself already changed its conformation upon inter- of g complex was varied (Fig. 5), the observed association rate action with ATP). The most attractive possibility then is that 30708 pol III Accessory Proteins b and g Complex 11785–11791 the rate-limiting step is opening of the toroidal b dimer. To 8. Fay, P. J., Johanson, K. O., McHenry, C. S., and Bambara, R. A. (1981) J. Biol. determine if this explanation is correct, we will investigate b Chem. 256, 976–983 mutant proteins in this assay that have different oligomeric 9. Stukenberg, P. T., Studwell-Vaughan, P. S., and O’Donnell, M. (1991) J. Biol. Chem. 266, 11328–11334 stability, the prediction being that a tighter dimer interface 10. Maki, S., and Kornberg, A. (1988) J. Biol. Chem. 263, 6555–6560 would decrease the rate of loading, while a weaker interface 11. Onrust, R., Finkelstein, J., Naktinis, V., Turner, J., Fang, L., and O’Donnell, might result in a faster rate. M. (1995) J. Biol. Chem. 270, 13348–13357 12. Kong, X.-P., Onrust, R., O’Donnell, M., and Kuriyan, J. (1992) Cell 69, In summary, we have shown that a simple DNA oligonucleo- 425–437 tide primer-template that supports processive DNA synthesis 13. McHenry, C. S. (1991) J. Biol. Chem. 266, 19127–19130 can be used as a model system for analyzing interactions be- 14. Studwell-Vaughan, P. S., and O’Donnell, M. (1991) J. Biol. Chem. 266, 19833–19841 tween pol III accessory proteins and core pol III with DNA. We 15. Onrust, R., Finkelstein, J., Turner, J., Naktinis, V., and O’Donnell, M. (1995) have demonstrated that the b sliding clamp-loading reaction J. Biol. Chem. 270, 13366–13377 16. Stukenberg, P. T., and O’Donnell, M. (1995) J. Biol. 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Published: Nov 1, 1996

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