Mechanism of β Clamp Opening by the δ Subunit ofEscherichia coli DNA Polymerase III Holoenzyme
2001; Elsevier BV; Volume: 276; Issue: 22 Linguagem: Inglês
10.1074/jbc.m100592200
ISSN1083-351X
AutoresJelena Stewart, Manju Hingorani, Zvi Kelman, Mike O’Donnell,
Tópico(s)Escherichia coli research studies
ResumoThe β sliding clamp encircles the primer-template and tethers DNA polymerase III holoenzyme to DNA for processive replication of the Escherichia coli genome. The clamp is formed via hydrophobic and ionic interactions between two semicircular β monomers. This report demonstrates that the β dimer is a stable closed ring and is not monomerized when the γ complex clamp loader (γ3δ1δ1χ1ψ1) assembles the β ring around DNA. δ is the subunit of the γ complex that binds β and opens the ring; it also does not appear to monomerize β. Point mutations were introduced at the β dimer interface to test its structural integrity and gain insight into its interaction with δ. Mutation of two residues at the dimer interface of β, I272A/L273A, yields a stable β monomer. We find that δ binds the β monomer mutant at least 50-fold tighter than the β dimer. These findings suggest that when δ interacts with the β clamp, it binds one β subunit with high affinity and utilizes some of that binding energy to perform work on the dimeric clamp, probably cracking one dimer interface open. The β sliding clamp encircles the primer-template and tethers DNA polymerase III holoenzyme to DNA for processive replication of the Escherichia coli genome. The clamp is formed via hydrophobic and ionic interactions between two semicircular β monomers. This report demonstrates that the β dimer is a stable closed ring and is not monomerized when the γ complex clamp loader (γ3δ1δ1χ1ψ1) assembles the β ring around DNA. δ is the subunit of the γ complex that binds β and opens the ring; it also does not appear to monomerize β. Point mutations were introduced at the β dimer interface to test its structural integrity and gain insight into its interaction with δ. Mutation of two residues at the dimer interface of β, I272A/L273A, yields a stable β monomer. We find that δ binds the β monomer mutant at least 50-fold tighter than the β dimer. These findings suggest that when δ interacts with the β clamp, it binds one β subunit with high affinity and utilizes some of that binding energy to perform work on the dimeric clamp, probably cracking one dimer interface open. DNA polymerase III proliferating cell nuclear antigen single-stranded DNA surface plasmon resonance hemagglutinin 3H- and32P-labeled β subunit, respectively subunit β with HA epitope tag and protein kinase tag, respectively replication factor C The DNA polymerase III (Pol III)1 holoenzyme is primarily responsible for replicating the 4.4-megabase Escherichia coli genome (1Kornberg A. Baker T. DNA Replication. 2nd Ed. W. H. Freeman and Co., New York, NY1992Google Scholar, 2Kelman Z. O'Donnell M. Annu. Rev. Biochem. 1995; 64: 171-200Crossref PubMed Scopus (361) Google Scholar). Pol III holoenzyme performs this task with high speed and accuracy with the help of ten component subunits. These are α (the DNA polymerase (3Maki H. Kornberg A. J. Biol. Chem. 1985; 260: 12987-12992Abstract Full Text PDF PubMed Google Scholar)), ε (the proofreading 3′-5′ exonuclease (4Scheuermann R.H. Echols H. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7747-7751Crossref PubMed Scopus (169) Google Scholar)), and θ (unknown function) that form the DNA polymerase III core (5McHenry C.S. Crow W. J. Biol. Chem. 1979; 252: 6478-6484Abstract Full Text PDF Google Scholar); β (the sliding clamp (6Stukenberg P.T. Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1991; 266: 11328-11334Abstract Full Text PDF PubMed Google Scholar, 7Kong X.P. Onrust R. O'Donnell M. Kuriyan J. Cell. 1992; 69: 425-437Abstract Full Text PDF PubMed Scopus (638) Google Scholar)); and the multisubunit DnaX complex (γτδδ′χψ) that functions as the clamp loader (8Maki S. Kornberg A. 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, 10Dallmann H.G. McHenry C.S. J. Biol. Chem. 1995; 270: 29563-29569Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) and contains at least two subunits of the τ "organizer"; that binds two core polymerases (11McHenry C.S. J. Biol. Chem. 1982; 257: 2657-2663Abstract Full Text PDF PubMed Google Scholar, 12Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1991; 266: 19833-19841Abstract Full Text PDF PubMed Google Scholar, 13Onrust R. Finkelstein J. Turner J. Naktinis V. O'Donnell M. J. Biol. Chem. 1995; 270: 13366-13377Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) and connects to the DnaB helicase at the replication fork (14Kim S. Dallmann H.G. McHenry C.S. Marians K.J. Cell. 1996; 84: 643-650Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 15Yuzhakov A. Turner J. O'Donnell M. Cell. 1996; 86: 877-886Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Rapid and processive DNA synthesis by Pol III holoenzyme is dependent on the interaction between the α subunit of the core polymerase and the β clamp (6Stukenberg P.T. Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1991; 266: 11328-11334Abstract Full Text PDF PubMed Google Scholar). β is a ring-shaped protein that encircles double-stranded DNA and can slide freely along its length (6Stukenberg P.T. Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1991; 266: 11328-11334Abstract Full Text PDF PubMed Google Scholar, 7Kong X.P. Onrust R. O'Donnell M. Kuriyan J. Cell. 1992; 69: 425-437Abstract Full Text PDF PubMed Scopus (638) Google Scholar). By itself, core polymerase can extend a primer by only a few nucleotides before dissociating from DNA (16Fay P.J. Johanson K.O. McHenry C.S. Bambara R.A. J. Biol. Chem. 1981; 256: 976-983Abstract Full Text PDF PubMed Google Scholar). When β is bound to the polymerase and topologicaly linked to the primer-template, it serves as a mobile tether to keep the enzyme associated with DNA, facilitating replication of several thousand nucleotides at a time. Similar mechanisms for processive DNA synthesis by replicative polymerases have been discovered in a variety of other organisms (reviewed in Refs. 2Kelman Z. O'Donnell M. Annu. Rev. Biochem. 1995; 64: 171-200Crossref PubMed Scopus (361) Google Scholar, 17Baker T.A. Bell S.P. Cell. 1998; 92: 295-305Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar,18Hingorani M.M. O'Donnell M. Curr. Biol. 2000; 10: 25-29Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, and 19Kuriyan J. O'Donnell M. J. Mol. Biol. 1993; 234: 915-925Crossref PubMed Scopus (203) Google Scholar), including eukaryotic DNA polymerase δ (tethered to DNA by the PCNA sliding clamp (20Gulbis J.M. Kelman Z. Hurwitz J. O'Donnell M. Kuriyan J. Cell. 1996; 87: 297-306Abstract Full Text Full Text PDF PubMed Scopus (647) Google Scholar, 21Krishna T.S. Kong X.P. Gary S. Burgers P.M. Kuriyan J. Cell. 1994; 79: 1233-1243Abstract Full Text PDF PubMed Scopus (755) Google Scholar)) and bacteriophage T4 DNA polymerase, gp43 (tethered by the gp45 sliding clamp (22Moarefi I. Jeruzalmi D. Turner J. O'Donnell M. Kuriyan J. J. Mol. Biol. 2000; 296: 1215-1223Crossref PubMed Scopus (138) Google Scholar)). The crystal structure of β shows it to be a ring-shaped dimer formed by the head-to-tail interaction of two semicircle-shaped monomers (7Kong X.P. Onrust R. O'Donnell M. Kuriyan J. Cell. 1992; 69: 425-437Abstract Full Text PDF PubMed Scopus (638) Google Scholar). A continuous β-sheet forms a scaffold around the outer surface of the ring that supports 12 α-helices lining the inside of the ring. The central cavity is about 35 Å in diameter, which is large enough to encircle double-stranded DNA as well as one or two layers of water molecules. Moreover, although the inside of the β ring is positively charged, it lacks specific contact with DNA, allowing β to form a stable topological link with the DNA and yet slide freely along the duplex. At the two identical dimer interfaces, a continuous β-sheet formed by hydrogen bonding between β strands from each monomer stabilizes the ring structure in addition to a small hydrophobic core formed by packing of Ile272 and Leu273 of one monomer with Phe106 and Leu108 on the other monomer. Charged amino acids at the interface are also in position to form six ion pairs (these interactions are detailed in Fig. 4). These numerous and potentially strong interactions between the two β subunits presumably underlie the highly stable dimeric structure of β and its ability to remain bound to DNA with a half-life of over 100 min (23Yao 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 (180) Google Scholar, 24Leu F.P. Hingorani M.M. Turner J. O'Donnell M.E. J. Biol. Chem. 2000; 275: 34609-34618Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Yet the closed circular clamp must be opened frequently during DNA replication for assembly on DNA to initiate processive replication as well as for disassembly of the β ring from DNA when replication is complete. The γ complex clamp loader (γδδ′χψ) assembles β clamps on primer-template DNA (where they can be used by the polymerase) and can also remove clamps from DNA when necessary (23Yao 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 (180) Google Scholar, 24Leu F.P. Hingorani M.M. Turner J. O'Donnell M.E. J. Biol. Chem. 2000; 275: 34609-34618Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 25Stukenberg P.T. Turner J. O'Donnell M. Cell. 1994; 73: 877-887Abstract Full Text PDF Scopus (148) Google Scholar, 26Marians K.J. Annu. Rev. Biochem. 1992; 61: 673-719Crossref PubMed Google Scholar). The process of clamp assembly requires that the γ complex open the β clamp, guide DNA into the central cavity, and facilitate closure of the clamp around DNA. Crystal structure analysis, 2The crystal structure of γδδ′ complex has been solved (D. Jeruzalmi and J. Kuriyan, personal communication). The stoichiometry is γ3δ1δ′1, and the five subunits form a pentameric ring. and a recent biochemical study (27Pritchard A.E. Dallmann H.G. Glover B.P. McHenry C.S. EMBO J. 2000; 19: 6536-6545Crossref PubMed Google Scholar) reveals that the γ complex contains three copies of γ; the other subunits (δ, δ′, χ, ψ) are each present in a single copy (10Dallmann H.G. McHenry C.S. J. Biol. Chem. 1995; 270: 29563-29569Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 13Onrust R. Finkelstein J. Turner J. Naktinis V. O'Donnell M. J. Biol. Chem. 1995; 270: 13366-13377Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The δ subunit of γ complex binds to β and destabilizes or opens the dimer interface (28Naktinis V. Onrust R. Fang L. O'Donnell M. J. Biol. Chem. 1995; 270: 13358-13365Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 29Turner J. Hingorani M.M. Kelman Z. O'Donnell M. EMBO J. 1999; 18: 771-783Crossref PubMed Scopus (157) Google Scholar). The γ subunits are the only ones that hydrolyze ATP (30Lee S.H. Walker J.R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2713-2717Crossref PubMed Scopus (25) Google Scholar, 31Tsuchihashi Z. Kornberg A. J. Biol. Chem. 1989; 264: 17790-17795Abstract Full Text PDF PubMed Google Scholar, 32Hingorani M.M. O'Donnell M. J. Biol. Chem. 1998; 273: 24550-24563Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). The δ′ subunit is homologous to γ and appears to play a role in modulating the access of δ to β (10Dallmann H.G. McHenry C.S. J. Biol. Chem. 1995; 270: 29563-29569Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 33Dong Z. Onrust R. Skangalis M. O'Donnell M. J. Biol. Chem. 1993; 268: 11758-11765Abstract Full Text PDF PubMed Google Scholar, 34O'Donnell M. Onrust R. Dean F.B. Chen M. Hurwitz J. Nucleic Acids Res. 1993; 21: 1-3Crossref PubMed Scopus (144) Google Scholar). In the absence of ATP, the affinity between the γ complex and β is low compared with the affinity between the δ subunit and β (28Naktinis V. Onrust R. Fang L. O'Donnell M. J. Biol. Chem. 1995; 270: 13358-13365Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Clamp assembly initiates when ATP binds the γ subunits and induces a change in conformation of the γ complex that results in ability of δ to bind β (28Naktinis V. Onrust R. Fang L. O'Donnell M. J. Biol. Chem. 1995; 270: 13358-13365Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 29Turner J. Hingorani M.M. Kelman Z. O'Donnell M. EMBO J. 1999; 18: 771-783Crossref PubMed Scopus (157) Google Scholar,32Hingorani M.M. O'Donnell M. J. Biol. Chem. 1998; 273: 24550-24563Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). The δ′ subunit appears at least partially responsible for modulating the access of δ to β, since a previous study indicated that δ′ and β compete for interaction with δ (29Turner J. Hingorani M.M. Kelman Z. O'Donnell M. EMBO J. 1999; 18: 771-783Crossref PubMed Scopus (157) Google Scholar). The ATP-induced conformational change of γ complex may entail removing a surface of δ′ from δ, allowing δ to bind and open the β clamp. In the presence of a nonhydrolyzable ATP analogue, the clamp loader-β complex binds primer-template DNA with high affinity (32Hingorani M.M. O'Donnell M. J. Biol. Chem. 1998; 273: 24550-24563Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 36Bertram J.G. Bloom L.B. Turner J. O'Donnell M. Beechem J.M. Goodman M.F. J. Biol. Chem. 1998; 273: 24564-24574Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Interaction of γ complex with DNA, especially primed template, triggers ATP hydrolysis and is stimulated by the presence of β (29Turner J. Hingorani M.M. Kelman Z. O'Donnell M. EMBO J. 1999; 18: 771-783Crossref PubMed Scopus (157) Google Scholar,32Hingorani M.M. O'Donnell M. J. Biol. Chem. 1998; 273: 24550-24563Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 36Bertram J.G. Bloom L.B. Turner J. O'Donnell M. Beechem J.M. Goodman M.F. J. Biol. Chem. 1998; 273: 24564-24574Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 37Onrust R. Stukenberg P.T. O'Donnell M. J. Biol. Chem. 1991; 266: 21681-21686Abstract Full Text PDF PubMed Google Scholar). ATP hydrolysis is coupled to closure of the clamp around DNA and γ complex turnover. The χ subunit of γ complex binds to SSB and helps coordinate the switch between the primase, clamp loader, and polymerase proteins at the primer template (38Kelman Z. Yuzhakov A. Andjelkovic J. O'Donnell M. EMBO J. 1998; 17: 2436-2449Crossref PubMed Scopus (156) Google Scholar, 39Yuzhakov A. Kelman Z. O'Donnell M. Cell. 1999; 96: 153-163Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), and ψ enhances the stability of the γ complex; however, these two proteins are not absolutely essential for clamp assembly (40O'Donnell M. Studwell P.S. J. Biol. Chem. 1990; 265: 1179-1187Abstract Full Text PDF PubMed Google Scholar, 41Xiao H. Crombie R. Dong Z. Onrust R. O'Donnell M. J. Biol. Chem. 1993; 268: 11773-11778Abstract Full Text PDF PubMed Google Scholar, 42Olson M. Dallmann H.G. McHenry C.S. J. Biol. Chem. 1995; 270: 29570-29577Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 43Glover B.P. McHenry C.S. J. Biol. Chem. 1998; 273: 23476-23484Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Although all three subunits, γ, δ, and δ′, are required for loading β onto DNA, the single δ subunit appears to be the predominant contact between β and the γ complex (28Naktinis V. Onrust R. Fang L. O'Donnell M. J. Biol. Chem. 1995; 270: 13358-13365Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). It remains possible that weaker interactions between β and the other γ complex subunits exist. 3Weak interaction between γ and β and between χ and β can be detected by surface plasmon resonance (A. Yuzhakov and M. O'Donnell, unpublished observations). However, our previous studies demonstrated that δ alone can open and remove β clamps from circular DNA molecules with nearly the same efficiency as γ complex (k unloading γ− complex = 0.015 s−1; k δ unloading = 0.011 s−1) (24Leu F.P. Hingorani M.M. Turner J. O'Donnell M.E. J. Biol. Chem. 2000; 275: 34609-34618Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). We were therefore curious as to how the δ subunit generates the leverage required to part the apparently tightly closed β dimer interfaces. Previous studies indicate that β opening at just one interface is sufficient to allow passage of DNA into (or out of) the central cavity (29Turner J. Hingorani M.M. Kelman Z. O'Donnell M. EMBO J. 1999; 18: 771-783Crossref PubMed Scopus (157) Google Scholar). Experiments herein measure the exchange of labeled β subunits as they are utilized by the γ complex, and the results support the conclusion that the dimeric clamp is not split apart into monomers but rather stays intact during clamp assembly, presumably opening at only one interface for entry of DNA. In the simplest possible mechanism, the clamp loader could prompt clamp opening merely by perturbing one of the dimer interfaces and transiently reducing its stability. Study of the δ-β interaction in this report provides insight into how the δ and γ complex might open the β ring. We demonstrate here that the β ring retains its dimeric structure when bound by one δ subunit. Furthermore, we have mutated two hydrophobic residues in the β dimer interface to produce a stable monomeric version of β. Only one δ subunit binds the β monomer, which is surprising, given the one δ/two β stoichiometry of the wild type δ-β complex. This suggests that the binding site of δ on the β ring is located primarily on one of the two β subunits. The affinity of δ for the β monomer mutant is about 50-fold greater than for the β dimer, implying that the binding energy of δ to a single β subunit of the dimer is harnessed to perform work, namely to force open one of the dimer interfaces. The δ subunit binds β at the carboxyl terminus, which lies in the vicinity of the dimer interface (44Naktinis V. Turner J. O'Donnell M. Cell. 1996; 84: 137-145Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Therefore, it is conceivable that δ binding to one β protomer disrupts the contacts in a nearby dimer interface that hold the ring closed. Radioactive nucleotides were purchased from PerkinElmer Life Sciences. Unlabeled nucleotides were purchased from Amersham Pharmacia Biotech. M13mp18 ssDNA was prepared by phenol extraction of purified M13mp18 phage that had been banded twice in CsCl gradients (45Turner J. O'Donnell M. Methods Enzymol. 1995; 262: 442-449Crossref PubMed Scopus (27) Google Scholar) and primed with a 30-nucleotide primer (Life Technologies, Inc.) as described (46Studwell P.S. O'Donnell M. J. Biol. Chem. 1990; 265: 1171-1178Abstract Full Text PDF PubMed Google Scholar). Buffer A contained 20 mm Tris-HCl (pH 7.5), 0.5 mm EDTA (pH 8.0), 100 mm NaCl, and 10% glycerol. DNA replication buffer contained 20 mm Tris-HCl (pH 7.5), 0.1 mm EDTA, 40 μg/ml bovine serum albumin, 5 mm dithiothreitol, 8 mm MgCl2, 4% glycerol, 0.5 mm ATP, 60 μm dGTP, and 60 μm dCTP. Surface plasmon resonance (SPR) buffer contained 10 mm Hepes-NaOH (pH 7.4), 150 mm NaCl, 3.4 mm EDTA, and 0.005% Tween 20. Proteins were purified as described: α, ε, γ (46Studwell P.S. O'Donnell M. J. Biol. Chem. 1990; 265: 1171-1178Abstract Full Text PDF PubMed Google Scholar), δ, δ′, χ, ψ (33Dong Z. Onrust R. Skangalis M. O'Donnell M. J. Biol. Chem. 1993; 268: 11758-11765Abstract Full Text PDF PubMed Google Scholar), θ (47Studwell-Vaughan P.S. O'Donnell M. J. Biol. Chem. 1993; 268: 11785-11791Abstract Full Text PDF PubMed Google Scholar), and SSB (46Studwell P.S. O'Donnell M. J. Biol. Chem. 1990; 265: 1171-1178Abstract Full Text PDF PubMed Google Scholar). γ complex and Pol III* (a subcomplex of Pol III holoenzyme lacking the β subunit) were reconstituted from individual subunits and purified as described in Refs. 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 and 13Onrust R. Finkelstein J. Turner J. Naktinis V. O'Donnell M. J. Biol. Chem. 1995; 270: 13366-13377Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, respectively. Mutant β proteins were constructed using DNA oligonucleotide site-directed mutagenesis. Various N-terminal tagged versions of β (described below) were purified according to the previously described protocol for wild type β (7Kong X.P. Onrust R. O'Donnell M. Kuriyan J. Cell. 1992; 69: 425-437Abstract Full Text PDF PubMed Scopus (638) Google Scholar). Radiolabeling of tagged β with 32P was performed using [α-32P]ATP and cAMP-dependent protein kinase to a specific activity of ∼100 cpm/fmol as described (48Kelman Z. Naktinis V. O'Donnell M. Methods Enzymol. 1995; 262: 430-442Crossref PubMed Scopus (47) Google Scholar). The catalytic subunit of cAMP-dependent protein kinase produced in E. coli was a gift from Dr. Susan Taylor (University of California, San Diego). 3H-β was labeled by reductive methylation as described (48Kelman Z. Naktinis V. O'Donnell M. Methods Enzymol. 1995; 262: 430-442Crossref PubMed Scopus (47) Google Scholar). The β, L273A-β, and I272A/L273A-β proteins (3 μm as dimer) were sized by gel filtration (at 4 °C) on an FPLC HR 10/30 Superose 12 column (Amersham Pharmacia Biotech) equilibrated with Buffer A. The proteins were incubated in a final volume of 200 μl of Buffer A for 15 min at 15 °C and then applied to the column. After collecting 6-ml, 170-μl fractions were collected, and 25-μl aliquots of the indicated fractions were analyzed by SDS-polyacrylamide gel electrophoresis (15% gels); proteins were visualized by Coomassie Blue staining. For size standards, α (130 kDa), bovine serum albumin (66 kDa), and δ (39 kDa) were analyzed similarly. Interaction between δ and β was analyzed by incubating 9 μm δ with 12.5 μm wild type β (as dimer) or 25 μm I272A/L273A-β, (as monomer) for 15 min at 15 °C in a final volume of 200 μl of Buffer A, followed by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis analysis as described above. Singly primed M13mp18 ssDNA (20 fmol), 0.8 μg of SSB, 75 fmol of Pol III*, and 750 fmol of β (wild type and mutant concentrations are calculated as monomer) were incubated at 37 °C for 2 min in 25 μl (final volume) of DNA replication buffer (this buffer contains ATP, dCTP, and dGTP). DNA synthesis was initiated upon the addition of the remaining two deoxyribonucleoside triphosphates (60 μm dATP, 20 μm dTTP (final concentrations), and 1 μCi of [α-32P]dTTP). After 20 s, reactions were quenched with 25 μl of 40 mm EDTA and 1% SDS. Aliquots (20 μl) of the quenched reactions were analyzed by electrophoresis on a 1% TBE-agarose gel, and the radiolabeled DNA was visualized on a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA). Synthesis was quantitated by spotting 20-μl aliquots of the reaction on DE81 filters, followed by liquid scintillation counting as described (49Rowen L. Kornberg A. J. Biol. Chem. 1978; 253: 758-764Abstract Full Text PDF PubMed Google Scholar). The δ subunit (10 μl of 0.6 μm) was immobilized on a carboxymethylated dextran matrix-coated sensor chip (CM5; Biacore) by carbodiimide coupling in 10 mm sodium acetate (pH 5.5). SPR analysis was performed at 23 °C by injecting 15 μl of β or I272A/L273A−β (0.25 and 1.23 μm; concentrations for both are given as monomer) in SPR buffer, at a flow rate of 5 μl/s. After each analysis was complete, the chip surface was regenerated by injecting 10 μl of 0.1 m glycine (pH 9.5) over the chip, which releases bound β with no significant effect on the binding capacity of the immobilized δ protein. The kinetic constants for interaction between δ and β were determined by nonlinear curve fitting, using the BIAevaluation 2.1 software. The rate of dissociation (k off) was calculated by fitting the curves to a single exponential decay described by Equation 1,R=R0e−koff(t−t0)Equation 1 where R 0 represents the response andt 0 represents the time at the start of the dissociation phase. The association rate (k on) was calculated using the binding model A + B= AB and Equation 2,R=Req(1−e−konC+koff)(t−t0)Equation 2 where R eq is the response at steady state, C is the concentration of β, andt 0 is the time at the start of the association phase. The dissociation constant (K d) for interaction between β and δ was calculated ask off/k on. The two β mutants for this assay were constructed by placing the β gene into either the pHKEp vector or the pHKEpmut vector (50Kelman Z. Yao N. O'Donnell M. Gene (Amst.). 1995; 166: 177-178Crossref PubMed Scopus (43) Google Scholar). Both of these vectors place a 34-amino acid tag onto the N terminus of the protein. The tags contain a protein kinase site (to label the protein with 32P) and either a functional (pHKEp) or a nonfunctional (pHKEpmut) hemagglutinin (HA) epitope. The nonfunctional epitope was formed by replacing two amino acids; YPYDVPDYA was changed to YPYDVPAAA. After expression and purification, one β contains a functional HA epitope (haβ2) and the other β contains a nonfunctional HA epitope, which we use in this report in the phosphorylated form and refer to as32P-β2. The β with the mutated HA-epitope was labeled with 32P (32P-β) as described (48Kelman Z. Naktinis V. O'Donnell M. Methods Enzymol. 1995; 262: 430-442Crossref PubMed Scopus (47) Google Scholar). Titrations of these β variants showed that they were as active as wild type β in replication assays with Pol III* on SSB-coated M13mp18 ssDNA primed with a single oligonucleotide. Monoclonal antibody to the HA epitope was purchased from BabCo, and Protein A-Sepharose 4B was from Zymed Laboratories Inc. The HA antibody was conjugated to Protein A beads by incubation for 15 min at 25 °C in 400 μl of 20 mm Hepes (pH 7.4), 150 mm NaCl, 0.1% Triton X-100, 10% glycerol. Spontaneous protomer exchange was measured (i.e. no other proteins besides β) in 50-μl reactions containing 2 pmol of32P-β2 and 2 pmol ofhaβ2 in 20 mm Hepes (pH 7.5), 150 mm NaCl, and 10% glycerol. Reactions were incubated at 37 °C for 0, 1, 2, 4, 6, or 8 h before the addition of 50 μl of HA antibody-conjugated beads and placed at 4 °C for a further 30 min. Beads were pelleted, washed three times with 1 ml of 50 mm Tris-HCl (pH 8.0), 500 mm NaCl, 5 mm EDTA, 0.1% SDS, and 0.1% Triton X-100; resuspended in Eco-Lume (ICN); and counted in a scintillation counter. Control experiments were performed similarly except that either no antibody was conjugated to the beads or the haβ2 was not added to the reaction. To measure the effect of γ complex on β protomer exchange during clamp assembly onto DNA, 250 fmol each ofhaβ2 and 32P-β2were incubated for 5 min at 37 °C with 500 fmol of γ complex and 1.8 pmol of nicked pBS DNA in 70 μl of 20 nm Tris-HCl (pH. 7.5), 0.1 mm EDTA, 4% glycerol, and 8 mmMgCl2. The reaction was then applied to a 5-ml A15m gel filtration column equilibrated with the same buffer plus 0.15 m NaCl. Fractions of six drops each were collected, and those containing β on DNA were identified by scintillation counting and pooled (420 μl), and then the DNA was linearized upon treatment with 700 units of BamHI for 3 min at 37 °C to release β. To confirm that linearization was complete within this time, an aliquot (20 μl) was removed, quenched with 20 μl of 1% SDS, 40 mm EDTA, and then analyzed in a native agarose gel. Then 50 μl of HA antibody beads were added to the reaction, and incubation was continued for a further 30 min at 4 °C. The beads were pelleted; washed three times with 1 ml of 50 mm Tris-HCl (pH 8.0), 500 mm NaCl, 5 mm EDTA, 0.1% SDS, and 0.1% Triton X-100; resuspended in Eco-Lume (ICN); and counted in a scintillation counter. In a control experiment, the above procedure was repeated except that heterodimeric β was used in the assembly reaction by first preincubating 250 fmol of each β in one tube for 5 h at 37 °C before adding to the reaction containing γ complex and DNA. Reactions contained 67.5 pmol of3H-β2 (wild type β labeled by reductive methylation), 1.7 nmol of hisβ2, which contained a six-residue histidine tag on a 23-residue N-terminal leader (β was cloned into the pHK vector in Ref. 50Kelman Z. Yao N. O'Donnell M. Gene (Amst.). 1995; 166: 177-178Crossref PubMed Scopus (43) Google Scholar), and 6.6 nmol of δ (where present) in 200 μl of 20 mm Tris-HCl (pH 7.5), 10% glycerol, 8 mm MgCl2, and 100 mm NaCl. A control reaction utilized 1.7 nmol of unlabeled wild type β2 in placed of thehisβ2 derivative. Reactions were assembled on ice and then shifted to 37 °C, and aliquots of 20 μl were removed at 2 and 24 h of incubation. Upon removal of an aliquot, NaCl was added to a final concentration of 0.5 m, and the reaction was applied to a 1-ml nickel chelate column (HiTrap; Amersham Pharmacia Biotech) equilibrated in 20 mm Tris-HCl (pH 7.9), 5 mm imidazole, 8 mm MgCl2, and 10% glycerol. The column was washed with 5 ml of the same buffer and then eluted with 3 ml of 20 mm Tris-HCl (pH 7.9), 1m imidazole, 8 mm MgCl2, and 10% glycerol. Fractions of 1 ml were collected. The flow-through (wash) and bound (elution) fractions were analyzed by liquid scintillation counting and analyzed in a 10% SDS-polyacrylamide gel to confirm the presence of δ with β in the bound fractions. The typical yield of3H-β2 off the column was greater than 85%. We have shown previously that the β clamp is a tight dimer and remains a dimer even when diluted to a concentration of 50 nm (23Yao N. Turner J. Kelman Z. Stukenberg P.T. Dean F. Shechter D. Pan Z.Q. Hurwitz J. O'Donnell M. Genes Cells. 1996; 1: 10
Referência(s)