A Function for the ψ Subunit in Loading the Escherichia coli DNA Polymerase Sliding Clamp
2007; Elsevier BV; Volume: 282; Issue: 10 Linguagem: Inglês
10.1074/jbc.m610136200
ISSN1083-351X
AutoresStephen G. Anderson, Christopher R. Williams, Mike O’Donnell, Linda B. Bloom,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoCrystal structures of an Escherichia coli clamp loader have provided insight into the mechanism by which this molecular machine assembles ring-shaped sliding clamps onto DNA. The contributions made to the clamp loading reaction by two subunits, χ and ψ, which are not present in the crystal structures, were determined by measuring the activities of three forms of the clamp loader, γ3δδ′, γ3δδ′ψ, and γ3δδ′ψχ. The ψ subunit is important for stabilizing an ATP-induced conformational state with high affinity for DNA, whereas the χ subunit does not contribute directly to clamp loading in our assays lacking single-stranded DNA-binding protein. The ψ subunit also increases the affinity of the clamp loader for the clamp in assays in which ATPγS is substituted for ATP. Interestingly, the affinity of the γ3δδ′ complex for β is no greater in the presence than in the absence of ATPγS. A role for ψ in stabilizing or promoting ATP- and ATPγS-induced conformational changes may explain why large conformational differences were not seen in γ3δδ′ structures with and without bound ATPγS. The β clamp partially compensates for the activity of ψ when this subunit is not present and possibly serves as a scaffold on which the clamp loader adopts the appropriate conformation for DNA binding and clamp loading. Results from our work and others suggest that the ψ subunit may introduce a temporal order to the clamp loading reaction in which clamp binding precedes DNA binding. Crystal structures of an Escherichia coli clamp loader have provided insight into the mechanism by which this molecular machine assembles ring-shaped sliding clamps onto DNA. The contributions made to the clamp loading reaction by two subunits, χ and ψ, which are not present in the crystal structures, were determined by measuring the activities of three forms of the clamp loader, γ3δδ′, γ3δδ′ψ, and γ3δδ′ψχ. The ψ subunit is important for stabilizing an ATP-induced conformational state with high affinity for DNA, whereas the χ subunit does not contribute directly to clamp loading in our assays lacking single-stranded DNA-binding protein. The ψ subunit also increases the affinity of the clamp loader for the clamp in assays in which ATPγS is substituted for ATP. Interestingly, the affinity of the γ3δδ′ complex for β is no greater in the presence than in the absence of ATPγS. A role for ψ in stabilizing or promoting ATP- and ATPγS-induced conformational changes may explain why large conformational differences were not seen in γ3δδ′ structures with and without bound ATPγS. The β clamp partially compensates for the activity of ψ when this subunit is not present and possibly serves as a scaffold on which the clamp loader adopts the appropriate conformation for DNA binding and clamp loading. Results from our work and others suggest that the ψ subunit may introduce a temporal order to the clamp loading reaction in which clamp binding precedes DNA binding. The efficiency of DNA replication is enhanced by processivity factors that enable a DNA polymerase to incorporate thousands of nucleotides in a single DNA-binding event. These processivity factors, a sliding clamp and a clamp loader, are conserved from bacteria to humans (recently reviewed in Refs. 1Zhuang Z. Spiering M.M. Berdis A.J. Trakselis M.A. Benkovic S.J. Nat. Struct. Mol. Biol. 2004; 11: 580-581Crossref PubMed Scopus (3) Google Scholar, 2Indiani C. O′Donnell M. Nat. Rev. Mol. Cell. Biol. 2006; 7: 751-761Crossref PubMed Scopus (144) Google Scholar, 3O′Donnell M. Kuriyan J. Curr. Opin. Struct. Biol. 2006; 16: 35-41Crossref PubMed Scopus (59) Google Scholar, 4O′Donnell M. J. Biol. Chem. 2006; 281: 10653-10656Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Sliding clamps are ring-shaped protein complexes that encircle DNA, and the Escherichia coli clamp consists of two identical arc-shaped β subunits (5Kong X.-P. Onrust R. O′Donnell M. Kuriyan J. Cell. 1992; 69: 425-437Abstract Full Text PDF PubMed Scopus (635) Google Scholar). Clamps slide freely along duplex DNA so that a DNA polymerase bound to a clamp is tethered to the template being copied yet able to move along DNA at rates limited by the rate of nucleotide incorporation (6Stukenberg P.T. Studwell-Vaughan P.S. O′Donnell M. J. Biol. Chem. 1991; 266: 11328-11334Abstract Full Text PDF PubMed Google Scholar, 7Yao N. Turner J. Kelman Z. Stukenberg P.T. Dean F. Shechter D. Pan Z.-Q. Hurwitz J. O′Donnell M. Genes Cells. 1996; 1: 101-113Crossref PubMed Scopus (181) Google Scholar). Clamp loaders catalyze the assembly of sliding clamps on DNA. The complete clamp loader at the E. coli replication fork contains two copies of the τ subunit and one copy each of the γ, δ, δ′, ψ, and χ subunits (8Maki S. Kornberg K. J. Biol. Chem. 1988; 263: 6555-6560Abstract Full Text PDF PubMed Google Scholar, 9Onrust 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, 10Onrust R. Finkelstein J. Turner J. Naktinis V. O′Donnell M. J. Biol. Chem. 1995; 270: 13366-13377Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 11Pritchard A.E. Dallman H.G. Glover B.P. McHenry C.S. EMBO J. 2000; 19: 6536-6545Crossref PubMed Google Scholar). The τ and γ subunits are products of the same gene, dnaX, and the γ subunit is a truncated form produced during translation (12Blinkowa A.L. Walker J.R. Nucleic Acids Res. 1990; 18: 1725-1729Crossref PubMed Scopus (179) Google Scholar, 13Flower A.M. McHenry C.S. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3713-3717Crossref PubMed Scopus (203) Google Scholar, 14Tsuchihashi Z. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2516-2520Crossref PubMed Scopus (224) Google Scholar). Clamp loaders containing three copies of the DnaX protein in any combination are fully active in clamp loading (9Onrust 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), however, the additional C-terminal extension present on τ mediates protein-protein interactions that are required to coordinate other activities at the replication fork (reviewed in Refs. 4O′Donnell M. J. Biol. Chem. 2006; 281: 10653-10656Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 15McHenry C.S. Mol. Microbiol. 2003; 49: 1157-1165Crossref PubMed Scopus (118) Google Scholar, and 16Johnson A. O′Donnell M. Annu. Rev. Biochem. 2005; 74: 283-315Crossref PubMed Scopus (438) Google Scholar). The functional core of clamp loaders is composed of five subunits that are members of the AAA+ family of ATPases (ATPases associated with diverse cellular activities; for recent AAA+ reviews see Refs. 17Ogura T. Whiteheart S.W. Wilkinson A.J. J. Struct. Biol. 2004; 146: 106-112Crossref PubMed Scopus (214) Google Scholar and 18Erzberger J.P. Berger J.M. Annu. Rev. Biophys. Biomol. Struct. 2006; 35: 93-114Crossref PubMed Scopus (585) Google Scholar). 2The abbreviations used are: AAA+, ATPases associated with diverse cellular activities; β-PY, pyrene-labeled β; DCC, 7-diethylaminocoumarin-3-carboxylic acid, succinimidyl ester; γmin, γpsi, and γcom, clamp loaders containing γδδ′, γδδ′ψ, and γδδ′ψχ, subunits, respectively; MDCC, N-(2-(1-maleimidyl)ethyl)-7-(diethylamino)coumarin-3-carboxamide; p/t-DNA, primed template DNA; RhX, X-rhodamine; PBP, phosphate-binding protein; BSA, bovine serum albumin; ATPγS, adenosine 5′-O-(3-thiotriphosphate). Three copies of the γ subunit, and one copy each of the δ and δ′ subunits make up the core of the E. coli clamp loader. This 5-polypeptide complex (γmin) is the minimal assembly of subunits with clamp loading activity (19Onrust R. O′Donnell M. J. Biol. Chem. 1993; 268: 11766-11772Abstract Full Text PDF PubMed Google Scholar, 20Xiao H. Dong Z. O′Donnell M. J. Biol. Chem. 1993; 268: 11779-11784Abstract Full Text PDF PubMed Google Scholar, 21Olson M.W. Dallmann H.G. McHenry C.S. J. Biol. Chem. 1995; 270: 29570-29577Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). A common feature of AAA+ ATPases is that subunits are arranged in a ring in which ATP binding sites located at the interface of adjacent subunits allow dynamic coupling of ATP binding and hydrolysis to conformational changes in the complex. Each of the γmin subunits is composed of three domains, and a ring is formed by a tight interaction of the C-terminal domains (22Jeruzalmi D. O′Donnell M. Kuriyan J. Cell. 2001; 106: 429-441Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 23Kazmirski S.L. Podobnik M. Weitze T.F. O′Donnell M. Kuriyan J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16750-16755Crossref PubMed Scopus (51) Google Scholar). Although the two N-terminal domains of each subunit share homology with AAA+ proteins (22Jeruzalmi D. O′Donnell M. Kuriyan J. Cell. 2001; 106: 429-441Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 24Guenther B. Onrust R. Sali A. O′Donnell M. Kuriyan J. Cell. 1997; 91: 335-345Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 25Podobnik M. Weitze T.F. O′Donnell M. Kuriyan J. Structure (Camb.). 2003; 11: 253-263Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), only the γ subunits bind and hydrolyze ATP. A conserved SRC sequence motif, containing an "Arg finger," is present in the δ′ and γ subunits of the E. coli clamp loader. Subunit-subunit communication of ATP binding and hydrolysis is mediated by these Arg fingers that extend from one subunit toward the ATP site in the neighboring subunit (see Fig. 1). Interaction of an Arg finger with the γ-phosphate of ATP is believed to serve both sensing and catalytic functions, and biochemical studies have shown that ATP-dependent clamp and DNA binding, in addition to ATP hydrolysis, are reduced in Arg finger mutants (26Johnson A. O′Donnell M. J. Biol. Chem. 2003; 278: 14406-14413Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 27Johnson A. Yao N.Y. Bowman G.D. Kuriyan J. O′Donnell M. J. Biol. Chem. 2006; 281: 35531-35543Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 28Snyder A.K. Williams C.R. Johnson A. O′Donnell M. Bloom L.B. J. Biol. Chem. 2004; 279: 4386-4393Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The physical process of clamp loading requires that interactions between the clamp loader and the clamp, and between the clamp loader and DNA, be modulated in a defined temporal order. This affinity modulation is accomplished by ATP binding and hydrolysis sensed by Arg fingers and coupled to conformational changes, in addition to changes in the clamp loader promoted by interactions with the clamp and with DNA. The ATP-bound form of the E. coli clamp loader has a high affinity for both the clamp and DNA and brings these macromolecules together (29Naktinis V. Onrust R. Fang F. O′Donnell M. J. Biol. Chem. 1995; 270: 13358-13365Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 30Hingorani M.M. O′Donnell M. J. Biol. Chem. 1998; 273: 24550-24563Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 31Turner J. Hingorani M.M. Kelman Z. O′Donnell M. EMBO J. 1999; 18: 771-783Crossref PubMed Scopus (157) Google Scholar). Binding to primed template DNA (p/t-DNA) alters interactions within the clamp loader to trigger ATP hydrolysis (32Ason B. Bertram J.G. Hingorani M.M. Beechem J.M. O′Donnell M. Goodman M.F. Bloom L.B. J. Biol. Chem. 2000; 275: 3006-3015Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 33Ason B. Handayani R. Williams C.R. Bertram J.G. Hingorani M.M. O′Donnell M. Goodman M.F. Bloom L.B. J. Biol. Chem. 2003; 278: 10033-10040Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). After hydrolyzing ATP, the affinity for the β clamp and DNA is reduced and the clamp loader releases the clamp on DNA (30Hingorani M.M. O′Donnell M. J. Biol. Chem. 1998; 273: 24550-24563Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 31Turner J. Hingorani M.M. Kelman Z. O′Donnell M. EMBO J. 1999; 18: 771-783Crossref PubMed Scopus (157) Google Scholar, 32Ason B. Bertram J.G. Hingorani M.M. Beechem J.M. O′Donnell M. Goodman M.F. Bloom L.B. J. Biol. Chem. 2000; 275: 3006-3015Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 34Bertram J.G. Bloom L.B. Hingorani M.M. Beechem J.M. O′Donnell M. Goodman M.F. J. Biol. Chem. 2000; 275: 28413-28420Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The crystal structure of γmin, the first clamp loader structure to be solved, has provided tremendous insight into the mechanism of clamp loading (22Jeruzalmi D. O′Donnell M. Kuriyan J. Cell. 2001; 106: 429-441Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). The structure of this E. coli clamp loader is remarkably similar to that of the five-subunit Saccharomyces cerevisiae replication factor C clamp loader (35Bowman G.D. O′Donnell M. Kuriyan J. Nature. 2004; 429: 724-730Crossref PubMed Scopus (341) Google Scholar). The native E. coli clamp loader contains two additional subunits, χ and ψ, that are not present in the crystal structure. Analysis of clamp loader structures does not immediately suggest a function for the χ and ψ subunits. Biochemical studies show that the ψ subunit binds a γ subunit in the clamp loader (9Onrust 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, 20Xiao H. Dong Z. O′Donnell M. J. Biol. Chem. 1993; 268: 11779-11784Abstract Full Text PDF PubMed Google Scholar, 36Glover B.P. McHenry C.S. J. Biol. Chem. 2000; 275: 3017-3020Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 37Gao D. McHenry C.S. J. Biol. Chem. 2001; 276: 4447-4453Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), and χ joins the complex via interactions with ψ (20Xiao H. Dong Z. O′Donnell M. J. Biol. Chem. 1993; 268: 11779-11784Abstract Full Text PDF PubMed Google Scholar). The χ subunit binds single-stranded DNA-binding protein at the replication fork (38Glover B.P. McHenry C.S. J. Biol. Chem. 1998; 273: 23476-23484Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar) and helps coordinate the switch from primase to DNA polymerase (39Yuzhakov A. Kelman Z. O′Donnell M. Cell. 1999; 96: 153-163Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Although the ψ subunit is conserved in bacterial clamp loaders (40Gulbis J.M. Kazmirski S.L. Finkelstein J. Kelman Z. O′Donnell M. Kuriyan J. Eur. J. Biochem. 2004; 271: 439-449Crossref PubMed Scopus (52) Google Scholar, 41Jarvis T.C. Beaudry A.A. Bullard J.M. Ochsner U. Dallmann H.G. McHenry C.S. J. Biol. Chem. 2005; 280: 40465-40473Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar), a function for ψ has not been identified. To determine if χ and ψ make a direct contribution to clamp loading, the activities of three forms of the clamp loader, γmin (γ3δδ′), γpsi (γ3δδ′ψ), and γcom (γ3δδ′ψχ) were characterized (Fig. 1). Nucleotides and Oligonucleotides—Concentrations of ATP (Amersham Biosciences/GE Healthcare) and ATPγS (Roche Applied Science) were determined by measuring the absorbance at 259 nm and using an extinction coefficient of 15,400 m–1 cm–1. [α-32P]ATP (10 mCi/ml) was purchased from Amersham Biosciences/GE Healthcare. Synthetic oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA) and purified by either 12% or 8% denaturing PAGE for the 30-mer or 105-mer, respectively (32Ason B. Bertram J.G. Hingorani M.M. Beechem J.M. O′Donnell M. Goodman M.F. Bloom L.B. J. Biol. Chem. 2000; 275: 3006-3015Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The sequence of the 105-nucleotide template (t1) is 5′-CTG TGC CAC GTA TTC TTA CGC TTT CAG GTC AGA AGG GTT CTA TCT CTG TTG GCC AGA ATC TCC CTT TTA TTA CTG GTC GTG TGA CTG GTG AAT CTG CCA ATG TAA-3′. For anisotropy experiments, an amino linker (5′ Amino Modifier C6) was incorporated at the 5′-end of the 105-mer. This amino linker was covalently labeled with X-rhodamine isothiocyanate (Molecular Probes, Eugene, OR) (42Bloom 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 sequence of the 30-nucleotide primer p1 is 5′-ACG ACC AGT AAT AAA AGG GAC ATT CTG GCC-3′, and primer p2 is 5′-ACA CGA CCA GTA ATA AAA GGG ACA TTC (C6dT)GG-3′ in which C6dT is a T with a C6 amino linker at the 5 position. Both primers are complementary to template t1 and anneal to overlapping sites that differ in position by two nucleotides. Annealing of p1 to t1 creates a 50-nt 5′ template overhang, and annealing p2 to t1 creates a 52-nt 5′ template overhang. Primer p2 was covalently labeled with 7-diethylaminocoumarin-3-carboxylic acid, succinimidyl ester (DCC, Molecular Probes) as described for X-rhodamine (42Bloom 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). Primed-template DNA was annealed by mixing primer and template in 20 mm Tris-HCl, pH 7.5, 50 mm NaCl, heating to 80 °C, and slowly cooling to room temperature. Annealing reactions contained the following molar ratios of primer/template: 1.0/1.1 for p2-DCC/t1, and 1.2/1.0 for p1/t1-RhX and unlabeled p1/t1. Duplex DNA was used without further purification. Buffers—Assay buffer consisted of 20 mm Tris-HCl, pH 7.5, 50 mm NaCl, and 8 mm MgCl2. Protein storage buffer consisted of 20 mm Tris-HCl, pH 7.5, 0.5 mm EDTA, and 10% glycerol. DNA Polymerase III Proteins—DNA polymerase III proteins were purified, and the clamp loader complexes were reconstituted as described previously (5Kong X.-P. Onrust R. O′Donnell M. Kuriyan J. Cell. 1992; 69: 425-437Abstract Full Text PDF PubMed Scopus (635) Google Scholar, 9Onrust 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). Protein concentrations were determined by measuring the absorbance at 280 nm in 6 m guanidine hydrochloride and using calculated extinction coefficients (γcom = 220,050 m–1 cm–1, γpsi = 190,890 m–1 cm–1, γmin = 166,850 m–1 cm–1, β = 14,890 m–1 cm–1). Covalent modification of β on a Cys residue with N-(1-pyrene)maleimide (Molecular Probes) was performed as described (28Snyder A.K. Williams C.R. Johnson A. O′Donnell M. Bloom L.B. J. Biol. Chem. 2004; 279: 4386-4393Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 43Griep M.A. McHenry C.S. Biochemistry. 1988; 27: 5210-5215Crossref PubMed Scopus (17) Google Scholar). The protein concentration of β-PY was determined from the absorbance at 280 nm under nondenaturing conditions after subtracting the contribution due to pyrene absorbance. The pyrene contribution was calculated from the absorbance at 344 nm multiplied by a factor of 0.806. The native extinction coefficient (44Johanson K.O. Haynes T.E. McHenry C.S. J. Biol. Chem. 1986; 261: 11460-11465Abstract Full Text PDF PubMed Google Scholar) for β is 17,900 m–1 cm–1. Equilibrium β Binding Assays—β-PY anisotropy measurements were made using a QuantaMaster QM-1 fluorometer (Photon Technology International) as described (28Snyder A.K. Williams C.R. Johnson A. O′Donnell M. Bloom L.B. J. Biol. Chem. 2004; 279: 4386-4393Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Samples were excited at 345 nm, and emission was measured at 375 nm with excitation and emission slits set at a 5 nm bandpass. Equilibrium DNA Binding Assays—Anisotropy of RhX in p1/t1-DNA-RhX was measured as described previously using a T-format (32Ason B. Bertram J.G. Hingorani M.M. Beechem J.M. O′Donnell M. Goodman M.F. Bloom L.B. J. Biol. Chem. 2000; 275: 3006-3015Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). RhX was excited at 580 nm, and emission was measured at 610 nm using a 6 nm bandpass on both excitation and emission channels. Fluorescence emission spectra of DCC (p2-DCC/t1) were measured using a QuantaMaster QM-1 fluorometer (Photon Technology International) by exciting the sample at 435 nm and measuring the emission from 450 to 550 nm with bandpass set at 4 nm on both excitation and emission channels. Pre-steady-state DNA Binding Assays—Pre-steady-state DCC fluorescence quenching assays were performed at ≈20 °C using an Applied Photophysics SX18MV stopped-flow apparatus configured for sequential mixing and equipped with an excitation monochromator set at 435 nm and a 475 nm cut-on filter in the path of the emission PMT. A solution of clamp loader from one syringe was mixed with a solution of ATP in the presence or absence of β from a second syringe, aged for 1 s, and added to a solution of p2-DCC/t1 from a third syringe. Data were collected for a total of 2 s in intervals of 2 ms. E. coli Phosphate-binding Protein and Pre-steady-state ATPase Assays—PBP was purified and covalently labeled with N-(2-(1-maleimidyl)ethyl)-7-(diethylamino)coumarin-3-carboxamide (MDCC, Molecular Probes) as described (45Williams C.R. Snyder A.K. Kuzmic P. O′Donnell M. Bloom L.B. J. Biol. Chem. 2004; 279: 4376-4385Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). PBP-MDCC-based ATPase assays were performed as described previously (45Williams C.R. Snyder A.K. Kuzmic P. O′Donnell M. Bloom L.B. J. Biol. Chem. 2004; 279: 4376-4385Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) using an Applied Photophysics SX18MV stopped-flow apparatus configured for sequential mixing. MDCC was excited at 425 nm, and emission was measured using a 455 nm cut-on filter. In standard sequential mixing experiments, clamp loaders were preincubated with ATP (±β) for 1 s prior to adding p1/t1 and PBP-MDCC. In Fig. 8, the time that clamp loaders were preincubated with ATP was varied as indicated. Single-turnover ATPase Assays—The 32P-based ATPase assays were performed at room temperature (≈24 °C) by preincubating clamp loader with radiolabeled ATP and β for 5 s prior to initiating hydrolysis by addition of p1/t1 and ATPγS-chase. Final reactions were incubated for 5 s prior to quenching in 95% formamide, 20 mm EDTA. ADP product was then separated from ATP by TLC using polyethyleneimine cellulose F TLC plates (Merck) developed in 0.5 m LiCl and quantitated using a Storm PhosphorImager and ImageQuaNT software (Amersham Biosciences). Binding of the Three Clamp Loaders to the β-Clamp—The goals of this experiment were 1) to assess the contributions of the χ and/or ψ subunits to clamp loader-clamp binding and 2) to provide a measure of the specific activities of the three clamp loader preparations (i.e. β-binding units per mole of complex). A solution-based fluorescence anisotropy assay in which β was covalently labeled with pyrene (PY) was used to measure the formation of clamp loader-β-PY complexes (28Snyder A.K. Williams C.R. Johnson A. O′Donnell M. Bloom L.B. J. Biol. Chem. 2004; 279: 4386-4393Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Thiol-reactive probes, including pyrenylmaleimide selectively label Cys-333 (43Griep M.A. McHenry C.S. Biochemistry. 1988; 27: 5210-5215Crossref PubMed Scopus (17) Google Scholar), which is located on the face of β opposite to that which the clamp loader binds (5Kong X.-P. Onrust R. O′Donnell M. Kuriyan J. Cell. 1992; 69: 425-437Abstract Full Text PDF PubMed Scopus (635) Google Scholar, 46Naktinis V. Turner J. O′Donnell M. Cell. 1996; 84: 137-145Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Labeled β is active in replication assays done in our laboratory 3S. G. Anderson, C. R. Williams, and L. B. Bloom, unpublished data. and by others (43Griep M.A. McHenry C.S. Biochemistry. 1988; 27: 5210-5215Crossref PubMed Scopus (17) Google Scholar, 47Leu F.P. Georgescu R. O′Donnell M. Mol. Cell. 2003; 11: 315-327Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Therefore, the assumption is that PY does not affect interactions with the clamp loader, and this assumption is supported by agreement between Kd values measured in this experiment and in other work (29Naktinis V. Onrust R. Fang F. O′Donnell M. J. Biol. Chem. 1995; 270: 13358-13365Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Binding assays were done by titrating a fixed concentration of β-PY with increasing concentrations of clamp loader. Because the anisotropy of PY is greater for the larger clamp loader-clamp complexes than for free pyrene-labeled β, the observed anisotropy increases as the fraction of β-PY bound increases. Assays contained ATP and were done under stoichiometric binding conditions in which the concentration of β-PY was large (100 nm) relative to the Kd for γcom binding β (3 nm (29Naktinis V. Onrust R. Fang F. O′Donnell M. J. Biol. Chem. 1995; 270: 13358-13365Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar)). Anisotropy values are plotted as a function of clamp loader concentration in Fig. 2 (a–c). Given that one molecule of clamp loader binds each clamp, this 1:1 stoichiometry should be reflected in the binding isotherms if the clamp loader preparations were 100% active. Binding data from three independent titrations were globally fit to a quadratic equation (Equation 1), robs=Kd+aγ+β−(Kd+aγ+β)2−4aγβ2β(rb−rf)+rf(Eq. 1) to calculate dissociation constants (Kd) and the fractions of active clamp loader (a) based on total concentrations of clamp loader (γ) and β-PY (β). Anisotropies for free (rf) and bound (rb) β-PY were also treated as adjustable parameters and globally fit. Calculated dissociation constants were in the low nanomolar range (Fig. 2) for all three complexes demonstrating that the χ and ψ subunits are not required for high affinity binding to β in assays containing ATP. For each clamp loader preparation, saturation in β-PY binding was observed at a ratio greater than one clamp loader per clamp. This result was interpreted as reflecting a population of inactive clamp loader that contributes to the total protein concentration. To quantitatively compare the activities of the three clamp loaders, the concentration of active clamp loader, as determined in this β-binding assay, was used in all subsequent experiments. We recognize that the β-binding activity is not necessarily equivalent to other activities such as ATP hydrolysis, however, this standardization of concentrations gave consistent results from preparation to preparation, and our major conclusions do not depend on this normalization. Structural data for γmin containing bound ATPγS suggested that productive binding to the β-clamp would not be possible given the conformation of the clamp loader (23Kazmirski S.L. Podobnik M. Weitze T.F. O′Donnell M. Kuriyan J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16750-16755Crossref PubMed Scopus (51) Google Scholar). To determine whether this observation would be reflected in solution, β-PY was titrated with the three clamp loaders in assays containing ATPγS (Fig. 2d). Binding of γmin to β-PY was reduced by ∼100-fold in assays containing ATPγS compared with ATP. Notably, the affinity of γmin for β in assays with ATPγS(Kd = 103 ± 24 nm) was not significantly greater than those for γcom (45Williams C.R. Snyder A.K. Kuzmic P. O′Donnell M. Bloom L.B. J. Biol. Chem. 2004; 279: 4376-4385Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) and γmin (48Williams C.R. The Clamp Loader of Eschesichia coli DNA Polymerase III. Kinetics of the ATP-dependent Steps in the Sliding-clamp Loading Reaction. University of Florida, Gainesville, FL2003Google Scholar) binding β-PY in the absence of nucleotide (Kd ≈ 150–200 nm). In other words, an ATP-bound conformational state with high affinity for the clamp was not formed by γmin to a large extent in assays with ATPγS. The affinities of γpsi and γcom for β (Kd values of 13 ± 4 nm and 22 ± 6nm, respectively) were decreased by a factor of ∼10 (Fig. 2d) showing that the presence of the ψ subunit reduces the effect of substituting ATPγS for ATP on β binding activity. Equilibrium DNA-binding Activities of the Three Clamp Loaders—A second fluorescence anisotropy-based assay was used to determine whether the χ and/or ψ subunits contribute to the DNA-binding activity of the E. coli clamp loader. Primed template DNA was covalently labeled with X-rhodamine (RhX) on the 5′-template end. The anisotropy of RhX increases when γcom binds p/t-DNA-RhX (42Bloom 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). Solutions of p/t-DNA-RhX, clamp loader, and the β-clamp were sequentially added to a cuvette containing a solution of ATPγS, and polarized intensities were measured after each addition (Fig. 3). ATPγS was added instead of ATP to measure DNA binding in the absence of appreciable DNA-dependent ATP hydrolysis. Anisotropy values were calculated for free p/t-DNA-RhX and for clamp loaders binding p/t-DNA-RhX in the absence and presence of β. Addition of γmin to p/t-DNA-RhX (Fig. 3a, black versus striped bars) had very little affect on the anisotropy of the probe, whereas addition of γpsi (Fig. 3b, black versus striped bars) or γcom (Fig. 3c, black versus striped bars) increased the anisotropy in a concentration-dependent manner. Weaker p/t-DNA binding by γmin compared with γpsi and γcom suggests a role for the ψ subunit in DNA binding. The ψ subunit may bind DNA directly, or alternatively, act indirectly by stabilizing a conformational state of the clamp loader with high affinity for DNA. DNA binding was not
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