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• W2085065737 abstract "The β protein, a dimeric ring-shaped clamp essential for processive DNA replication by Escherichia coli DNA polymerase III holoenzyme, is assembled onto DNA by the γ complex. This study examines the clamp loading pathway in real time, using pre-steady state fluorescent depolarization measurements to investigate the loading reaction and ATP requirements for the assembly of β onto DNA. Two β dimer interface mutants, L273A and L108A, and a nonhydrolyzable ATP analog, adenosine 5′-O-(3-thiotriphosphate) (ATPγS), have been used to show that ATP binding is required for γ complex and β to associate with DNA, but that a γ complex-catalyzed ATP hydrolysis is required for γ complex to release the β·DNA complex and complete the reaction. In the presence of ATP and γ complex, the β mutants associate with DNA as efficiently as wild type β. However, completion of the reaction is much slower with the β mutants because of decreased ATP hydrolysis by the γ complex, resulting in a much slower release of the mutants onto DNA. The effects of mutations in the dimer interface were similar to the effects of replacing ATP with ATPγS in reactions using wild type β. Thus, the assembly of β around DNA is coupled tightly to the ATPase activity of the γ complex, and completion of the assembly process requires ATP hydrolysis for turnover of the catalytic clamp loader. The β protein, a dimeric ring-shaped clamp essential for processive DNA replication by Escherichia coli DNA polymerase III holoenzyme, is assembled onto DNA by the γ complex. This study examines the clamp loading pathway in real time, using pre-steady state fluorescent depolarization measurements to investigate the loading reaction and ATP requirements for the assembly of β onto DNA. Two β dimer interface mutants, L273A and L108A, and a nonhydrolyzable ATP analog, adenosine 5′-O-(3-thiotriphosphate) (ATPγS), have been used to show that ATP binding is required for γ complex and β to associate with DNA, but that a γ complex-catalyzed ATP hydrolysis is required for γ complex to release the β·DNA complex and complete the reaction. In the presence of ATP and γ complex, the β mutants associate with DNA as efficiently as wild type β. However, completion of the reaction is much slower with the β mutants because of decreased ATP hydrolysis by the γ complex, resulting in a much slower release of the mutants onto DNA. The effects of mutations in the dimer interface were similar to the effects of replacing ATP with ATPγS in reactions using wild type β. Thus, the assembly of β around DNA is coupled tightly to the ATPase activity of the γ complex, and completion of the assembly process requires ATP hydrolysis for turnover of the catalytic clamp loader. primer/template adenosine diphosphate adenosine 5′-O-(3-thiotriphosphate) wild type. Replication of genomic DNA in Escherichia coli involves the assembly of a multisubunit enzyme complex on the DNA molecule. These proteins converge in such a way as to promote processive synthesis in both leading and lagging strands. In organisms as diverse as bacteriophages, bacteria, yeasts, and humans, the components that characterize a processive replicating machine are quite similar. In the case of E. coli, γ complex, using ATP, loads β subunit onto DNA. The β clamp, fully encircling the DNA molecule, associates with core and holds it firmly in place so extension of the primer can proceed. Because lagging strand synthesis is discontinuous, a cycling of the proteins upon completion of each Okazaki fragment to a new primer is required; therefore, periodic loading of β is essential for replication of the genome to continue. Duplication of genomic DNA in E. coli requires processive DNA replication activity of the 10-subunit DNA polymerase III holoenzyme. The E. coli DNA polymerase III holoenzyme consists of three main functional units: the core polymerase, the β sliding clamp, and the γ complex clamp loader (reviewed in Refs. 1McHenry C.S. J. Biol. Chem. 1991; 266: 19127-19130Abstract Full Text PDF PubMed Google Scholarand 2Kelman Z. O'Donnell M. Annu. Rev. Biochem. 1995; 64: 171-200Crossref PubMed Scopus (361) Google Scholar). The three subunits distinctive to E. coli DNA polymerase III core (3McHenry C.S. Crow W. J. Biol. Chem. 1979; 254: 1748-1753Abstract Full Text PDF PubMed Google Scholar) are α, which has DNA polymerase activity (4Maki H. Kornberg A. J. Biol. Chem. 1985; 260: 12987-12992Abstract Full Text PDF PubMed Google Scholar); ε, which has a 3′ to 5′ exonuclease activity (5Scheuermann R. Tam S. Burgers P.M. Lu C. Echols H. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 7085-7089Crossref PubMed Scopus (146) Google Scholar, 6DiFrancesco R. Bhatnagar S.K. Brown A. Bessman M.J. J. Biol. Chem. 1984; 259: 5567-5573Abstract Full Text PDF PubMed Google Scholar); and θ, which, at this time, lacks a well defined function (3McHenry C.S. Crow W. J. Biol. Chem. 1979; 254: 1748-1753Abstract Full Text PDF PubMed Google Scholar, 7Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1993; 268: 11785-11791Abstract Full Text PDF PubMed Google Scholar). The core, by itself, is not processive. It is capable of synthesizing short segments of DNA but dissociates readily from the primer/template, extending the primer only 10–20 nucleotides per binding event in vitro(8Fay P.J. Johanson K.O. McHenry C.S. Bambara R.A. J. Biol. Chem. 1981; 256: 976-983Abstract Full Text PDF PubMed Google Scholar). The β sliding clamp and the γ complex are accessory proteins, the presence of which converts this nonprocessive component into an apparatus that synthesizes thousands of nucleotides per binding event (4Maki H. Kornberg A. J. Biol. Chem. 1985; 260: 12987-12992Abstract Full Text PDF PubMed Google Scholar, 9Stukenberg P.T. Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1991; 266: 11328-11334Abstract Full Text PDF PubMed Google Scholar). Analysis by x-ray diffraction has shown that the β subunit is a ring-shaped dimer that has a central hole, with an inner diameter of about 35 Å, large enough to encircle duplex DNA (10Kong X.-P. Onrust R. O'Donnell M. Kuriyan J. Cell. 1992; 69: 425-437Abstract Full Text PDF PubMed Scopus (636) Google Scholar). The γ complex is a multisubunit clamp loader composed of two γ subunits and one each of the δ, δ′, χ, and ψ subunits (11Maki S. Kornberg K. J. Biol. Chem. 1988; 263: 6555-6560Abstract Full Text PDF PubMed Google Scholar, 12Onrust R. Finkelstein J. Naktinis V. Turner J. Fang L. O'Donnell M. J. Biol. Chem. 1995; 270: 13348-13357Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Biochemical evidence has established a mechanism by which the γ complex acts to assemble β onto DNA, wherein β acts as a sliding clamp, tethering the core polymerase to the DNA molecule. Techniques for studying the mechanism of the γ complex-catalyzed reaction have been confined to observing their assembly on large circular single-stranded DNA, such as bacteriophage templates, or by combining the components and observing their association using gel filtration and SDS-polyacrylamide gel electrophoresis. To further characterize the pathway of clamp assembly, we used fluorescence depolarization to measure the change in anisotropy of a short primer/template labeled with a fluorescent probe, rhodamine-x. The rotational motion of the DNA is dependent on its molecular weight and is slowed when proteins are bound; therefore, the change in fluorescence anisotropy of the labeled p/t1 DNA is a measure of the interactions between the γ complex, β, and DNA. The β clamp is a stable dimer in solution due to the presence of several hydrogen bonds and hydrophobic contacts at the dimer interface between the two monomers. The γ complex must break these contacts to open the ring, and therefore, the nature of the β dimer interface likely plays an important role in the ATP-coupled process of clamp assembly on DNA. We have found that mutation of the hydrophobic residue Leu273 to Ala or mutation of Leu108 to Ala at the dimer interface yields β clamps that can assemble on DNA but inhibit the DNA-dependent ATPase activity of the γ complex. Steady state and pre-steady state measurements of the protein-DNA interactions indicate a corresponding inhibition in the dissociation of γ complex from the β·DNA complex. Results with ATPγS, using wt β, substantiate the β mutant study, indicating that in the absence of ATP hydrolysis, release of γ complex from β·DNA is blocked. Thus, we show that β can be loaded onto DNA in the presence of ATP; however, ATP hydrolysis is required to release the clamp loader from the clamp completing the clamp loading process. Subunits of the γ complex were overexpressed and purified as described: γ (13Studwell P.S. O'Donnell M. J. Biol. Chem. 1990; 265: 1171-1178Abstract Full Text PDF PubMed Google Scholar), δ, δ′ (14Dong Z. Onrust R. Skangalis M. O'Donnell M. J. Biol. Chem. 1993; 268: 11758-11765Abstract Full Text PDF PubMed Google Scholar), χ, and ψ (15Xiao H. Crombie R. Dong Z. Onrust R. O'Donnell M. J. Biol. Chem. 1993; 268: 11773-11778Abstract Full Text PDF PubMed Google Scholar). The γ complex was reconstituted from purified subunits as described (12Onrust R. Finkelstein J. Naktinis V. Turner J. Fang L. O'Donnell M. J. Biol. Chem. 1995; 270: 13348-13357Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Site-directed mutagenesis of the dnaN gene was achieved producing an L273A mutant β protein and an L108A mutant β protein. Wild type β and β mutants were overexpressed and purified as described (16Onrust R. O'Donnell M. J. Biol. Chem. 1993; 268: 11766-11772Abstract Full Text PDF PubMed Google Scholar). The enzyme reaction buffer contained 20 mmTris-HCl, pH 7.5, 40 μg/ml bovine serum albumin, 5 mmdithiothreitol, 8 mm MgCl2, and 50 mm NaCl. Oligonucleotides were synthesized using β-cyanoethyl phosphoramidite chemistry on an Applied Biosystems synthesizer (model 392 RNA/DNA) and purified by denaturing polyacrylamide gel electrophoresis. Fluorescent measurements were done using the following primer/template sequence:5′ CT GGT AAT ATC CAG3′ TCT TCT TGA GTT TGA TAG CCG GAACGA CCA TTA TAG GTCAAC AAT ATT ACC GCC A 3′TTG TTA TAA TGG CGG TCG GTA ACG TTG TCC TTT TTG CGA GT 5′SEQUENCE 1 The 80-mer template was labeled at the 5′-end with rhodamine-x (Molecular probes, catalog no. X-491) to which the 30-mer primer was annealed (17Bloom L.B. Turner J. Kelman Z. Beechem J.M. O'Donnell M. Goodman M.F. J. Biol. Chem. 1996; 271: 30699-30708Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). A QuantaMaster QM-1 fluorometer (Photon Technology International) with a single emission channel (L format) was used to measure steady state anisotropy. Samples were excited with vertically polarized light (Oriel dichroic sheet polarizer) at 580 nm (8-nm band pass), and both vertical and horizontal emission was monitored at 610 nm (8-nm band pass). Three individual anisotropy measurements were taken for each sample, and the values were averaged to compensate for manual rotation of the polarizer. The G factor was determined, and its value was used to calculate anisotropy. Steady state experiments were performed in a 1-cm path cuvette by measuring the initial anisotropy of 54 nm p/t DNA (labeled with rhodamine-x) in 186 μl of reaction buffer containing ATP. A 4-μl aliquot of 20 μm β dimer and a 10-μl aliquot of 4 μm γ complex were added, and anisotropy measurements were taken after each addition of protein. The final concentrations were 50 nm p/t DNA, 400 nm β dimer, 200 nm γ complex, and 500 μm ATP. The mixture was allowed to stand for 15 min (>15 min for mutants) at room temperature after which the anisotropy was measured again. One final anisotropy measurement was taken after the addition of a 2-μl portion of 45 mm ATP. The procedure was performed a second time; however, the γ complex and β dimer were added in reverse order. A mixture of 66 nmp/t DNA, 533 nm β dimer, and 666 μm[α-32P]ATP was incubated in a 45-μl total volume of reaction buffer. In an additional tube, an 800 nm mixture of γ complex in reaction buffer was prepared. The solutions were incubated at 37 °C for 2 min. Reactions were initiated by adding a 15-μl aliquot of the γ complex solution to the 45-μl tube containing β, p/t DNA, and [α-32P]ATP. Final concentrations were 50 nm p/t DNA, 400 nm β dimer, 200 nm γ complex, and 500 μm[α-32P]ATP. An 8-μl aliquot was removed at 1, 5, 10, 20, 40, and 80 min and quenched with an 8-μl portion of a 10% SDS, 40 mm EDTA mixture. Labeled ATP was separated from labeled ADP by spotting 1-μl aliquots on silica gel sheets (Kodak chromagram) and developing with an 11:7:2 NH4OH:isopropanol:water solution. The radiolabeled reactants and products were detected and quantitated on a PhosphorImager (Molecular Dynamics). Stopped-flow anisotropy measurements were performed using T-format instrumentation as described (18Otto M.R. Lillo M.P. Beechem J.M. Biophys. J. 1994; 67: 2511-2521Abstract Full Text PDF PubMed Scopus (65) Google Scholar). Three syringes on the stopped-flow apparatus were filled with the appropriate component. Typically, one syringe contained protein in reaction buffer, the second syringe contained DNA in reaction buffer, and the third syringe contained reaction buffer alone. Reactions were initiated by combining a 100-μl aliquot of protein solution with a 100-μl aliquot of DNA solution in the mixing chamber. Fluorescence measurements were made by exciting the sample at 580 nm with vertically polarized light and simultaneously collecting the vertically and horizontally polarized emission components through a 600-nm cut on filter. To increase the signal to noise ratio, multiple runs (5Scheuermann R. Tam S. Burgers P.M. Lu C. Echols H. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 7085-7089Crossref PubMed Scopus (146) Google Scholar, 6DiFrancesco R. Bhatnagar S.K. Brown A. Bessman M.J. J. Biol. Chem. 1984; 259: 5567-5573Abstract Full Text PDF PubMed Google Scholar, 7Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1993; 268: 11785-11791Abstract Full Text PDF PubMed Google Scholar, 8Fay P.J. Johanson K.O. McHenry C.S. Bambara R.A. J. Biol. Chem. 1981; 256: 976-983Abstract Full Text PDF PubMed Google Scholar, 9Stukenberg P.T. Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1991; 266: 11328-11334Abstract Full Text PDF PubMed Google Scholar, 10Kong X.-P. Onrust R. O'Donnell M. Kuriyan J. Cell. 1992; 69: 425-437Abstract Full Text PDF PubMed Scopus (636) Google Scholar, 11Maki S. Kornberg K. J. Biol. Chem. 1988; 263: 6555-6560Abstract Full Text PDF PubMed Google Scholar, 12Onrust R. Finkelstein J. Naktinis V. Turner J. Fang L. O'Donnell M. J. Biol. Chem. 1995; 270: 13348-13357Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) were averaged. The anisotropy of control preshot reactions was determined by mixing 100 μl of DNA with 100 μl of reaction buffer. Two reactions were performed. In the first reaction, 100 nm p/t DNA, 800 nm β dimer (or β mutants), and 500 μm ATP in reaction buffer were placed in one syringe. The second syringe contained 600 nm γ complex and 500 μm ATP in reaction buffer. Control preshots contained 50 nm p/t DNA, 400 nm β (or β mutants), and 250 μm ATP. In the second reaction, syringe 1 contained 100 nm p/t DNA and 500 μm ATP in reaction buffer, and syringe 2 contained 800 nm β (or β mutants), 600 nm γ complex, and 500 μm ATP in reaction buffer. Control preshots contained 50 nm p/t DNA and 250 μm ATP in reaction buffer. The first syringe contained 100 nm p/t DNA, 800 nmβ dimer, 600 nm γ complex, and 2 mm ATP in reaction buffer. The second syringe contained 500 nm M13 “trap” DNA in reaction buffer. M13 trap DNA consisted of single-stranded DNA from wild type M13 with two 30-nucleotide primers annealed at different sites. The pre-steady state experiments were performed as described above except that ATPγS was used in place of ATP. In the first experiment, one syringe contained 100 nm p/t DNA, 800 nm β dimer, and 500 μm ATPγS in reaction buffer, and the second syringe contained 600 nm γ complex and 500 μm ATPγS in reaction buffer. In the next experiment, the first syringe was filled with 800 nm β dimer, 600 nm γ complex, and 500 μm ATPγS in reaction buffer. The second syringe was filled with 100 nmp/t DNA and 500 μm ATPγS in reaction buffer. Two different chase experiments were performed. In the first experiment, one syringe was filled with 400 nm β dimer, 300 nm γ complex, 50 nm p/t DNA, and 500 μm ATPγS in reaction buffer, and the second syringe contained 5 mm ATP in reaction buffer. In the second experiment, one syringe was filled with 400 nm β dimer, 300 nm γ complex, 50 nm p/t DNA, and 500 μm ATP in reaction buffer. The second syringe contained 5 mm ATPγS in reaction buffer. The γ complex-catalyzed reaction of loading β onto DNA was analyzed directly in solution using steady state fluorescence depolarization, a technique measuring the rotational diffusion of a fluorophore (19Lakowicz J.R. Principles of Fluorescence Spectroscopy. Plenum Press, New York1983: 145-147Google Scholar). In these experiments a fluorescent probe, rhodamine-x, was covalently attached to DNA, and the fluorescence anisotropy of the probe on DNA was measured. Because the diffusion rate of free DNA is greater than that of DNA bound by proteins, the fluorescence anisotropy of the DNA probe will directly report on the association of proteins with DNA (20LeTilly V. Royer C.A. Biochemistry. 1993; 32: 7753-7758Crossref PubMed Scopus (149) Google Scholar, 21Perez-Howard G.M. Weil P.A. Beechem J.M. Biochemistry. 1995; 34: 8005-8017Crossref PubMed Scopus (131) Google Scholar). A decrease in the rate of rotation, due to protein binding, will result in an overall increase in steady state anisotropy. In all equilibrium and pre-steady state anisotropy experiments, the total intensity signal was examined and found to be absolutely invariant under all experimental conditions (data not shown). Hence, all observed anisotropy changes reflect the rotational property of p/t DNA and not any other photophysical effect. These results are consistent with our previous time-resolved fluorescence measurements using the same system (17Bloom L.B. Turner J. Kelman Z. Beechem J.M. O'Donnell M. Goodman M.F. J. Biol. Chem. 1996; 271: 30699-30708Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). For a recent review on the use of fluorescence anisotropy for protein-DNA interactions, see Heyduk et al. (22Heyduk T. Ma Y. Tang H. Ebright R.H. Methods Enzymol. 1996; 274: 492-503Crossref PubMed Scopus (139) Google Scholar). Interactions between proteins and DNA were analyzed under steady state conditions for clamp loading activity and also under pre-steady state conditions to observe formation of the protein-DNA complex in real time. For anisotropy measurements, a 30-nucleotide primer was annealed to an 80-nucleotide template labeled at the 5′-end with a fluorescent probe, rhodamine-x. The primer was placed in the center of the 80-mer, leaving single-stranded segments, each 25 nucleotides in length, at both ends of the template. This 30/80-nucleotide primer/template combination was shown to be the minimum size for DNA to still support processive synthesis by the core polymerase in the presence of both the γ complex and the β dimer (17Bloom L.B. Turner J. Kelman Z. Beechem J.M. O'Donnell M. Goodman M.F. J. Biol. Chem. 1996; 271: 30699-30708Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). The loading of wt β and β mutants onto DNA was measured under steady state conditions using titration experiments. The proteins were added to DNA in a specific order to determine how each constituent affects the fluorescence anisotropy of the DNA probe. Labeled p/t DNA (50 nm), ATP (500 μm), and buffer containing Mg2+ were placed in a cuvette, and the anisotropy of the p/t DNA was determined. Two types of titration experiments were performed. In the first (Figs. 1 A and 2, A and C) β (400 nm) was added to p/t DNA prior to the addition of γ complex (200 nm), and in the second (Figs. 1 Band 2, B and D) γ complex was added to DNA prior to β. Anisotropy measurements were taken following the addition of each protein. The results of these experiments were previously shown for wt β in a publication by Bloom et al. (17Bloom L.B. Turner J. Kelman Z. Beechem J.M. O'Donnell M. Goodman M.F. J. Biol. Chem. 1996; 271: 30699-30708Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) but were repeated in this work to compare the results of wt β with the two β mutants.Figure 2Increase in steady state anisotropy for rhodamine-x-labeled DNA when γ complex loads β mutants on DNA.A reaction identical to that outlined by the sketch at the top of Fig. 1 was followed to measure steady state anisotropy for the mutants except wt β was replaced with L273A mutant β and L108A mutant β.A, the anisotropy of rhodamine-x-labeled p/t DNA (DNA) was measured. The anisotropy of this DNA solution was measured again after adding a small aliquot of L273A mutant β (Beta). The γ complex (Gamma) was then added, and the anisotropy was again measured. The anisotropy measurement was repeated after 15, 45, 60, 75, 90, and 105 min followed by one additional reading after the addition of ATP (ATP).B, conditions were identical to those in A except that γ complex was added before L273A mutant β. C, the steady state anisotropy of p/t DNA (DNA) was measured. A small aliquot of L108A mutant β (Beta) was added, and the anisotropy was again measured. An anisotropy measurement was again taken after the addition of γ complex (Gamma). The reaction was allowed to stand for 15, 45, and 60 min, and the anisotropy was measured after each period of time. Following the addition of ATP, the anisotropy measurement was repeated. D,conditions were identical to those in C except that γ complex was added before L018A mutant β. The final concentrations in each reaction described above were 50 nm p/t DNA, 400 nm β, 200 nm γ complex, and 500 μm ATP. In either case, both γ complex and β are required to increase the anisotropy levels to 0.26 and 0.28 for the L273A mutant β and the L108A mutant β, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) When wt β or β mutants were added to p/t DNA, the anisotropy did not increase significantly over the initial value of approximately 0.16 (Figs. 1 A and 2, A and C). A substantial transition occurred, however, when γ complex was added to the reaction, increasing the anisotropy values from 0.16 to 0.25 for wt β, 0.26 for L273A mutant β and to 0.28 for L108A mutant β (Figs. 1 A and 2, A and C). To determine whether the increases in anisotropy were caused by the association of γ complex with p/t DNA, the order in which the β dimer and γ complex were added was reversed. Addition of the γ complex to p/t DNA resulted in a small increase in anisotropy from 0.16 to 0.17 (Figs. 1 B and 2, B and D), but the large anisotropy increases did not occur until either wt β or the β mutants were added (Figs. 1 B and 2, B and D). These data demonstrate that an increase in anisotropy for rhodamine-x-labeled p/t DNA requires the presence of both the γ complex and the β dimer, but they do not answer the question of which proteins are bound to DNA, β by itself or the γ complex and β. It has been shown, however, that after the γ complex loads β onto DNA, β alone remains bound to the DNA molecule (9Stukenberg P.T. Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1991; 266: 11328-11334Abstract Full Text PDF PubMed Google Scholar). These initial results indicate that the β mutants are capable of interacting with the γ complex and p/t DNA in the same manner as wt β. One noticeable observation is that anisotropy increases for rhodamine-x-labeled p/t DNA in the presence of the γ complex and β mutants were slightly larger (0.26 for L273A and 0.28 for L108A) than when in the presence of γ complex and wt β (0.25). Following the addition of γ complex and wt or mutant β to p/t DNA, the reactions were incubated for 15 min. During this 15 min incubation, the anisotropy of wt β dropped to a value of 0.17, a value close to that of free p/t DNA (Fig. 1, A and B, 15 min). In contrast, when reactions with β mutants were incubated for 15 min essentially no decreases in anisotropy were observed (Fig. 2,A–D, 15 min). For the L273A mutant, the anisotropy remained at 0.26 for approximately 60 min and decreased to a value of only 0.24 after 105 min. This value is still well above the level of 0.16 for free p/t DNA (Fig. 2, A and B). For L108A mutant β, it took approximately 1 h for the anisotropy to decrease to a value of 0.21 (Fig. 2, C and D). In reactions with both the wt and mutant βs, when additional ATP was added, the anisotropy values once again increased (Figs. 1 and 2, ATP). The fact that addition of fresh ATP restored high anisotropy values indicates that the fraction of p/t DNA bound by protein is due to the utilization of ATP and can be restored if ATP is replenished to a depleted reaction. In the absence of ATP, there was no association of β or γ complex with p/t DNA (data not shown). For reactions with wt β, the increased anisotropy values following the addition of β and γ complex to p/t DNA actually represent a steady state population of bound protein that is cycling on and off the DNA (see kinetic experiments described below). In each cycle, β is loaded onto DNA in a reaction that requires hydrolysis of ATP; it then dissociates from DNA or is removed by the γ complex to once again be loaded at the expense of more ATP. For reactions with the β mutants, a much longer period of time is required before depletion of ATP, suggesting that this cycling reaction is significantly slower than that for wt β. To investigate whether ATP was being hydrolyzed by the γ complex at slower rates in the presence of the β mutants than with wt β, ATPase assays were performed. Rates of ATP hydrolysis were measured using [α-32P]ATP, the product of which, after hydrolysis, was analyzed by separating hydrolyzed ADP from ATP using thin layer chromatography and quantitating the separated fractions on a PhosphorImager. The β dimer (400 nm), p/t DNA (50 nm), [α-32P]ATP (500 μm), and buffer with Mg2+ were combined. The reaction was initiated by adding the γ complex (200 nm). Aliquots were removed at 1, 5, 10, 20, 40, and 80 min and then quenched using a solution of SDS and EDTA. Previous studies have shown that γ complex ATPase activity is stimulated by DNA and is stimulated further in the presence of both DNA and β (23Burgers P.M.J. Kornberg A. J. Biol. Chem. 1982; 257: 11468-11473Abstract Full Text PDF PubMed Google Scholar, 24Burgers P.M.J. Kornberg A. J. Biol. Chem. 1982; 257: 11474-11478Abstract Full Text PDF PubMed Google Scholar, 25Onrust R. Stukenberg P.T. O'Donnell M. J. Biol. Chem. 1991; 266: 21681-21686Abstract Full Text PDF PubMed Google Scholar). Here we have performed ATPase assays under conditions identical to the DNA binding anisotropy experiments. Rates of ATP hydrolysis were measured in reactions with γ complex alone; γ complex and p/t DNA; and γ complex, p/t DNA, and wt β. The rate of ATP hydrolysis was very slow for γ complex alone, 0.4 μmol-min−1 (Fig. 3). The ATPase rate was stimulated 10-fold by addition of DNA into the reaction (4.3 μmol-min−1), indicating an interaction between γ complex and DNA (Fig. 3); and finally, addition of β further stimulated the rate of ATP hydrolysis 30-fold over the basal ATPase rate of γ complex alone (12 μmol-min−1) (Fig. 3). These data demonstrate that γ complex, β, and DNA participate in an ATP-utilizing process. The ATP consumption for β alone was negligible (data not shown). The DNA-dependent steady state rates of ATP hydrolysis by γ complex were 1.5 μmol-min−1 and 6.2 μmol-min−1 in the presence of the L273A mutant β and L108A mutant β, respectively (Fig. 4). The DNA-dependent γ complex ATPase rate is 8-fold faster when in the presence of wt β than when in the presence of L273A mutant β and 2-fold faster than when in the presence of L108A mutant β. In fact, the DNA-dependent ATP hydrolysis rate for γ complex alone is 3-fold higher than when L273A mutant β is added to the reaction. These results correlate well with anisotropy data, which suggest that ATP consumption is much slower in reactions with β mutants than in reactions with wt β (Fig. 2). Real time measurements of γ complex-catalyzed loading of wt β and β mutants onto p/t DNA were obtained using stopped-flow fluorescence anisotropy. In these experiments, real time changes in the steady state anisotropy of rhodamine-x-labeled p/t DNA were measured during the rapid mixing of DNA and proteins. For the reaction shown in Scheme 1 (Fig. 5), one stopped-flow syringe was loaded with wt or mutant β (800 nm), p/t DNA (100 nm), ATP (500 μm), and buffer containing Mg2+. A second stopped-flow syringe was loaded with γ complex (600 nm), ATP (500 μm), and the same buffer with Mg2+. Reactions were initiated by delivering equal volumes of the contents of the two syringes to a cuvette, in which fluorescence was measured. For the reaction shown in Scheme 2 (Fig. 5), the first syringe was loaded with p/t DNA (100 nm), ATP (500 μm), and buffer with Mg2+, and the second syringe was loaded with β (800 nm), γ complex (600 nm), ATP (500 μm), and buffer with Mg2+. Reactions were initiated by mixing equal volumes of reagents from each syringe. For both reactions, multiple runs (at least five) were summed to increase the signal to noise ratio, and the raw anisotropy data were fit to a sum of exponential terms. The total intensity of signals during these reactions was completely invariant (data not shown). Reaction time courses for stopped-flow anisotropy measurements of γ" @default.
• W2085065737 created "2016-06-24" @default.
• W2085065737 creator A5002499771 @default.
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• W2085065737 creator A5046396825 @default.
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• W2085065737 date "1998-09-01" @default.
• W2085065737 modified "2023-09-30" @default.
• W2085065737 title "Pre-steady State Analysis of the Assembly of Wild Type and Mutant Circular Clamps of Escherichia coli DNA Polymerase III onto DNA" @default.
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• W2085065737 doi "https://doi.org/10.1074/jbc.273.38.24564" @default.
• W2085065737 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9733751" @default.
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