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• W2052079265 abstract "Eukaryotic replication factor C is the heteropentameric complex that loads the replication clamp proliferating cell nuclear antigen (PCNA) onto primed DNA. In this study we used a derivative, designated RFC, with a N-terminal truncation of the Rfc1 subunit removing a DNA-binding domain not required for clamp loading. Interactions of yeast RFC with PCNA and DNA were studied by surface plasmon resonance. Binding of RFC to PCNA was stimulated by either adenosine (3-thiotriphosphate) (ATPγS) or ATP. RFC bound only to primer-template DNA coated with the single-stranded DNA-binding protein RPA if ATPγS was also present. Binding occurred without dissociation of RPA. ATP did not stimulate binding of RFC to DNA, suggesting that hydrolysis of ATP dissociated DNA-bound RFC. However, when RFC and PCNA together were flowed across the DNA chip in the presence of ATP, a signal was observed suggesting loading of PCNA by RFC. With ATPγS present instead of ATP, long-lived response signals were observed indicative of loading complexes arrested on the DNA. A primer with a 3′ single-stranded extension also allowed loading of PCNA; yet turnover of the reaction intermediates was dramatically slowed down. Filter binding experiments and analysis of proteins bound to DNA-magnetic beads confirmed the conclusions drawn from the surface plasmon resonance studies. Eukaryotic replication factor C is the heteropentameric complex that loads the replication clamp proliferating cell nuclear antigen (PCNA) onto primed DNA. In this study we used a derivative, designated RFC, with a N-terminal truncation of the Rfc1 subunit removing a DNA-binding domain not required for clamp loading. Interactions of yeast RFC with PCNA and DNA were studied by surface plasmon resonance. Binding of RFC to PCNA was stimulated by either adenosine (3-thiotriphosphate) (ATPγS) or ATP. RFC bound only to primer-template DNA coated with the single-stranded DNA-binding protein RPA if ATPγS was also present. Binding occurred without dissociation of RPA. ATP did not stimulate binding of RFC to DNA, suggesting that hydrolysis of ATP dissociated DNA-bound RFC. However, when RFC and PCNA together were flowed across the DNA chip in the presence of ATP, a signal was observed suggesting loading of PCNA by RFC. With ATPγS present instead of ATP, long-lived response signals were observed indicative of loading complexes arrested on the DNA. A primer with a 3′ single-stranded extension also allowed loading of PCNA; yet turnover of the reaction intermediates was dramatically slowed down. Filter binding experiments and analysis of proteins bound to DNA-magnetic beads confirmed the conclusions drawn from the surface plasmon resonance studies. replication factor C replication factor C with Rfc1-Δ(3–272) complex of Rfc2p, Rfc3p, Rfc4p, and Rfc5p replication protein A proliferating cell nuclear antigen single-stranded adenosine (3-thiotriphosphate) surface plasmon resonance resonance units The elongation apparatus for DNA replication is functionally conserved in all free living organisms, and a similar apparatus has been found in some bacteriophages, e.g. T4 (see Ref. 1Stillman B. Cell. 1994; 78: 725-728Abstract Full Text PDF PubMed Scopus (222) Google Scholar for a review). Processive DNA replication by the replicative DNA polymerase depends on its interaction with a toroidal shaped protein, the replication clamp. This clamp is loaded onto the template-primer junction by a protein complex, the clamp loader. Replication factor C (RF-C),1 the eukaryotic clamp loader, was first identified and purified as an essential component for SV40 DNA replication (2Tsurimoto T. Stillman B. Mol. Cell. Biol. 1989; 9: 609-619Crossref PubMed Scopus (164) Google Scholar). It is a multipolypeptide complex that loads the replication clamp proliferating cell nuclear antigen (PCNA) onto the template-primer junction in an ATP-dependent manner. PCNA is the processivity factor for several eukaryotic DNA polymerases including DNA polymerase δ. Yeast RF-C consists of a large subunit with a molecular mass of 95 kDa and four smaller subunits of 36–40 kDa. The genes encoding all five subunits are essential (3Li X. Burgers P.M. J. Biol. Chem. 1994; 269: 21880-21884Abstract Full Text PDF PubMed Google Scholar, 4Li X. Burgers P.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 868-872Crossref PubMed Scopus (56) Google Scholar, 5Noskov V. Maki S. Kawasaki Y. Leem S.H. Ono B. Araki H. Pavlov Y. Sugino A. Nucleic Acids Res. 1994; 22: 1527-1535Crossref PubMed Scopus (39) Google Scholar, 6Howell E.A. McAlear M.A. Rose D. Holm C. Mol. Cell. Biol. 1994; 14: 255-267Crossref PubMed Scopus (55) Google Scholar, 7Cullmann G. Fien K. Kobayashi R. Stillman B. Mol. Cell. Biol. 1995; 15: 4661-4671Crossref PubMed Scopus (213) Google Scholar, 8Gary S.L. Burgers P.M. Nucleic Acids Res. 1995; 23: 4986-4991Crossref PubMed Scopus (27) Google Scholar). All five subunits show sequence similarity to each other and to Rfc subunits from eukaryotes in general. This homology is localized in seven regions known as RF-C boxes II–VIII (reviewed in Ref. 7Cullmann G. Fien K. Kobayashi R. Stillman B. Mol. Cell. Biol. 1995; 15: 4661-4671Crossref PubMed Scopus (213) Google Scholar). RF-C boxes III and V contain sequences that show homology to nucleotide-binding proteins (9Walker J.E. Saraste M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4234) Google Scholar).RFC1 contains an additional box (I) in the N-terminal region that shows homology to prokaryotic DNA ligases and poly(ADP)-ribose polymerases (10Bunz F. Kobayashi R. Stillman B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11014-11018Crossref PubMed Scopus (76) Google Scholar). This box is not required for the clamp loading function of RF-C (11Uhlmann F. Cai J. Gibbs E. O'Donnell M. Hurwitz J. J. Biol. Chem. 1997; 272: 10058-10064Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 12Podust V.N. Tiwari N. Stephan S. Fanning E. J. Biol. Chem. 1998; 273: 31992-31999Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), and in fact, deletion of the N-terminal domain containing box I from yeast RFC1 shows no detectable replication phenotype and only a marginal repair phenotype (13Gomes X.V. Gary S.L. Burgers P.M. J. Biol. Chem. 2000; 275: 14541-14549Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The C termini of all five subunits are unique and are required for complex formation (11Uhlmann F. Cai J. Gibbs E. O'Donnell M. Hurwitz J. J. Biol. Chem. 1997; 272: 10058-10064Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 14Uhlmann F. Gibbs E. Cai J. O'Donnell M. Hurwitz J. J. Biol. Chem. 1997; 272: 10065-10071Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Biochemical studies of the eukaryotic clamp loader RF-C have established that the complex has a preferential binding affinity for template-primer junctions and has a single-stranded DNA-stimulated ATPase activity that is further activated by the presence of primer termini and PCNA (15Tsurimoto T. Stillman B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1023-1027Crossref PubMed Scopus (199) Google Scholar, 16Tsurimoto T. Stillman B. J. Biol. Chem. 1991; 266: 1950-1960Abstract Full Text PDF PubMed Google Scholar, 17Lee S.H. Kwong A.D. Pan Z.Q. Hurwitz J. J. Biol. Chem. 1991; 266: 594-602Abstract Full Text PDF PubMed Google Scholar, 18Yoder B.L. Burgers P.M.J. J. Biol. Chem. 1991; 266: 22689-22697Abstract Full Text PDF PubMed Google Scholar, 19Fien K. Stillman B. Mol. Cell. Biol. 1992; 12: 155-163Crossref PubMed Scopus (190) Google Scholar). In the presence of ATP or ATPγS a strong complex is formed between RF-C and PCNA, and in the presence of ATPγS a strong complex is formed between RF-C and DNA (16Tsurimoto T. Stillman B. J. Biol. Chem. 1991; 266: 1950-1960Abstract Full Text PDF PubMed Google Scholar, 20Gerik K.J. Gary S.L. Burgers P.M. J. Biol. Chem. 1997; 272: 1256-1262Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Hydrolysis of ATPγS is not observed, indicating that binding rather than hydrolysis of ATP drives formation of these two distinct complexes. Comprehensive mechanistic studies of the role of ATP in clamp loader interactions with the clamp and with DNA have been documented for the T4 and the Escherichia coli system (21Latham G.J. Pietroni P. Dong F. Young M.C. von Hippel P.H. J. Mol. Biol. 1996; 264: 426-439Crossref PubMed Scopus (30) Google Scholar, 22Sexton D.J. Kaboord B.F. Berdis A.J. Carver T.E. Benkovic S.J. Biochemistry. 1998; 37: 7749-7756Crossref PubMed Scopus (48) Google Scholar, 23Turner J. Hingorani M.M. Kelman Z. O'Donnell M. EMBO J. 1999; 18: 771-783Crossref PubMed Scopus (157) Google Scholar, 24Hingorani M.M. Bloom L.B. Goodman M.F. O'Donnell M. EMBO J. 1999; 18: 5131-5144Crossref PubMed Scopus (59) Google Scholar). Given the structural similarities of the three model systems, one might expect that the mechanism of clamp loading in eukaryotes would be similar in detail to T4 and E. coli. However, there are at least two reasons why there may be substantial differences. First, all subunits except RFC5 have a consensus ATP-binding domain, suggesting the possible involvement of four ATPs in the reaction pathway. However, unlike the T4 clamp loader, these putative four ATP molecules would be localized in unique rather than identical subunits, likely assigning an unique function to each subunit and the ATP bound to it. Second, there is accumulating evidence that the four small subunits constitute a core complex, designated Rfc2–5, that can associate with any subunit of a family of large subunits to form different complexes with distinct functions: with Rfc1p to form RF-C; with Rad24p to form Rad24Rfc2–5, which functions in checkpoint control; and with Chl12p to form Chl12Rfc2–5, which may function in a termination step in DNA replication (25Green C.M. Erdjument-Bromage H. Tempst P. Lowndes N.F. Curr. Biol. 2000; 10: 39-42Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 26Kouprina N. Kroll E. Kirillov A. Bannikov V. Zakharyev V. Larionov V. Genetics. 1994; 138: 1067-1079Crossref PubMed Google Scholar). Therefore, it is well possible that the eukaryotic clamp loader has evolved a modular binding and usage of ATP molecules that allow a more flexible adaptation for function in these different types of clamp loaders. In addition, specific functions may be associated with the Rfc2–5 core complex to guide its assembly into appropriate clamp loader complexes. In this series of papers we present studies detailing the mechanism of PCNA loading by RF-C and the requirement of ATP in this process. RF-C has at least two types of DNA-binding domains. DNA binding by a domain localized in the N-terminal third of Rfc1p is ATP-independent and unrelated to clamp loading. The DNA-binding sites in the C-terminal domain of Rfc1p and in other subunits of the complex may function as a coordinate unit that requires ATP for interaction with DNA. To facilitate our studies of the role of ATP in DNA binding and clamp loading, we have used a truncation derivative of RF-C in which the N-terminal domain carrying the ATP-independent DNA-binding domain has been deleted. Like the analogously truncated human RF-C, this derivative complex has an increased clamp loading activity that can be attributed to the loss of a competing DNA-binding domain for non-primer-template junctions (11Uhlmann F. Cai J. Gibbs E. O'Donnell M. Hurwitz J. J. Biol. Chem. 1997; 272: 10058-10064Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 12Podust V.N. Tiwari N. Stephan S. Fanning E. J. Biol. Chem. 1998; 273: 31992-31999Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 13Gomes X.V. Gary S.L. Burgers P.M. J. Biol. Chem. 2000; 275: 14541-14549Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The truncation derivative of RF-C containing Rfc1pΔ(3–272) has been used in all of our biochemical studies in these papers, and, for ease of reading, this complex has been simply designated as RFC. This paper details the ATP-dependent interactions of RFC and the Rfc2–5 core with DNA and PCNA and the effect of the single-stranded DNA-binding protein RPA and mismatched primer termini on binding and loading. These studies use surface plasmon resonance (SPR) for measuring interactions, but additional techniques have been presented to validate the SPR approach for studying clamp loading. The second paper details the quantitative aspects of ATP in the formation of complexes, clamp loading, and the order of the reaction pathway (27Gomes X.V. Gary Schmidt S.L. Burgers P.M. J. Biol. Chem. 2000; 276: 34776-34783Abstract Full Text Full Text PDF Scopus (86) Google Scholar). The third paper reports biochemical studies of mutant RFC complexes with mutations in the ATP-binding domains of four out of the five subunits (28Gary Schmidt S.L. Gomes X.V. Burgers P.M. J. Biol. Chem. 2000; 276: 34784-34791Abstract Full Text Full Text PDF Scopus (52) Google Scholar). A complementary genetic study of these mutants is reported in the fourth paper of this series (45Gary Schmidt S.L. Pautz A.L. Burgers P.M.J. J. Biol. Chem. 2001; 276: 34792-34800Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). PCNA, Rfc2–5, and replication protein A (RPA) were purified from E. coli overproduction strains as described (13Gomes X.V. Gary S.L. Burgers P.M. J. Biol. Chem. 2000; 275: 14541-14549Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 29Henricksen L.A. Umbricht C.B. Wold M.S. J. Biol. Chem. 1994; 269: 11121-11132Abstract Full Text PDF PubMed Google Scholar, 30Ayyagari R. Impellizzeri K.J. Yoder B.L. Gary S.L. Burgers P.M. Mol. Cell. Biol. 1995; 15: 4420-4429Crossref PubMed Scopus (186) Google Scholar). A truncated form of RF-C, in which residues 3–272 from Rfc1p was deleted, was used in this study (13Gomes X.V. Gary S.L. Burgers P.M. J. Biol. Chem. 2000; 275: 14541-14549Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The concentrations of RFC and PCNA were determined spectrophotometrically in 7 m guanidinium hydrochloride using the calculated extinction coefficient from the protein sequences. All other enzymes and oligonucleotides were obtained commercially. ATPγS was obtained from Roche Molecular Biochemicals. Buffer A contained 30 mm Hepes-NaOH, pH 7.5, 0.5 mmEDTA, 10% glycerol, 10 mm magnesium acetate, 125 mm sodium chloride, 0.1% ampholytes 3.5–9.8, and 0.01% Nonidet P40. Buffer B contained buffer A with 0.2 mg/ml bovine serum albumin. Whatman nitrocellulose filters (0.2 μm) were treated for 45 min with 0.4 m KOH and then equilibrated in buffer A. The V6 oligonucleotide (see Fig.1 A) was 5′-end labeled with 32P and hybridized to primer C12 in a V6:C12 molar ratio of 1:1.5. The DNA (0.65 nm) was incubated with 0.375–16 nm RFC in 30 μl of buffer B with 100 μm ATPγS for 5 min at 0 °C and then filtered through the nitrocellulose filter. The filter was washed with 0.5 ml of buffer A and dried, and the radioactivity was counted in a scintillation counter. 10-μl assays were performed in buffer B, except that the final NaCl concentration was 75 mm. The assays contained 0.1 μm Rfc2–5 or RFC, 50 μm [α-32P]ATP, 0.5 μm E. coli single-stranded binding protein, and when present 0.5 μm PCNA and 1 μm V6 or 1 μm V6/C12 DNA (see Fig. 1 A). After 6 min at 30 °C, the reaction was quenched with 3 μl of 50 mmEDTA, 1% SDS, 20 mm each of ADP and ATP. 3 μl was spotted on a polyethyleneimine cellulose sheet and dried. The sheet was washed in distilled water for 10 min, rinsed in ethanol, dried, and developed in 0.5 m LiCl, 1 m HCOOH. The sheets was dried and subjected to PhosphorImager analysis (Molecular Dynamics). SPR was performed in a BIAcore X apparatus. Buffer B was the running buffer used in the analysis. When a DNA chip was used, ∼2000 resonance units (RU) of streptavidin were immobilized on the surface of a dextran chip (pioneer F1) by carbodiimide coupling according to the manufacturer's instructions. A biotinylated 80-mer template (see Fig. 1 A), either alone or hybridized to an excess of primer C12 (or C12T, C12T3, or C12T10), was attached to the chip via the streptavidin-biotin linkage. Approximately 20–30 RU of template were immobilized. When a PCNA chip was used, ∼30–100 RU of PCNA were covalently immobilized on the surface of the dextran chip (CM5) by a carbodiimide-activated succinimide coupling method (amine coupling) according to the manufacturer's instructions. This mild coupling chemistry is analogous to one we previously used for the coupling of PCNA to agarose beads that proceeded largely with retention of RFC binding activity (20Gerik K.J. Gary S.L. Burgers P.M. J. Biol. Chem. 1997; 272: 1256-1262Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). The interaction between RFC with PCNA and with DNA was monitored at 20 °C by injecting 90 μl of the indicated concentrations of RFC over a PCNA chip or DNA chip at a flow rate of 30 μl/min. Higher flow rates did not significantly increase the rate of RFC binding to the chips, indicating that surface effects do not pose a serious problem with these low density chips. The dissociation constants (KD) were calculated using software provided by the manufacturer. Each KD value was obtained from in general 7–10 injections. Interaction measurements with the Rfc2–5 core were done similarly. The biotinylated 80-mer template V6 was hybridized to the 30-mer primer C12 (see Fig. 1 A) to generate the matched substrate or hybridized with C12T3 (5′-C12TTT) to generate the mismatched substrate. The primer-template substrate was immobilized onto streptavidin magnetic beads (Dynabeads) in 10 mmTris-HCl, pH 7.5, 1 mm EDTA, and 1 m NaCl by incubating at room temperature for 1–2 h. All washes were carried out using a Dynal magnet with a volume of buffer 100–200 times the bead volume for 1–2 min each at room temperature. The unbound substrate was washed off the beads twice with buffer B. The binding assay was performed in a 20-μl reaction in buffer B. About 500 fmol of DNA bead substrate (1 μl of beads) was coated with 5 pmol of yeast RPA for 1 min followed, where indicated, by the addition of 3 pmol of PCNA, 100 μm ATPγS, or 1 mm ATP and the indicated amounts of RFC. The reaction was incubated at 30 °C for 1 min. The beads were washed three times with wash buffer B. The same number of washes were carried out for each experiment regardless whether specific components were left out of the assay. Bead-bound proteins were boiled in sample loading buffer and separated on a SDS-10% polyacrylamide gel. The proteins were blotted onto a nitrocellulose membrane using a Mini Trans-Blot electrophoretic transfer cell from Bio-Rad. The blot was probed with a mixture of polyclonal antibodies raised in rabbit against PCNA and Rfc3p. Detection was carried out using an ECL chemiluminescence kit (Amersham Pharmacia Biotech) as recommended by the manufacturer. Different exposures of the blot were photographed with a CCD camera and digitized for quantitation. Previously, we had shown that RFC, i.e. RF-C lacking the ligase homology domain, is fully competent for clamp loading (13Gomes X.V. Gary S.L. Burgers P.M. J. Biol. Chem. 2000; 275: 14541-14549Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The ligase homology domain localizes to the N-terminal domain of Rfc1p and binds DNA regardless of the presence of ATP (31Fotedar R. Mossi R. Fitzgerald P. Rousselle T. Maga G. Brickner H. Messier H. Kasibhatla S. Hubscher U. Fotedar A. EMBO J. 1996; 15: 4423-4433Crossref PubMed Scopus (90) Google Scholar,32Allen B.L. Uhlmann F. Gaur L.K. Mulder B.A. Posey K.L. Jones L.B. Hardin S.H. Nucleic Acids Res. 1998; 26: 3877-3882Crossref PubMed Scopus (26) Google Scholar). Removal of the ligase homology domain revealed that binding of the remaining complex, i.e. RFC, to DNA was strongly stimulated by ATPγS, a nonhydrolyzable analog of ATP (13Gomes X.V. Gary S.L. Burgers P.M. J. Biol. Chem. 2000; 275: 14541-14549Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). These results are in agreement with earlier footprinting studies of human RF-C to DNA and recent binding studies with the E. coli γ-complex (16Tsurimoto T. Stillman B. J. Biol. Chem. 1991; 266: 1950-1960Abstract Full Text PDF PubMed Google Scholar,33Ason 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). To obtain a quantitative understanding of these interactions, we have used SPR. This technique, in which protein or DNA is attached to a dextran-coated surface and a second component is flowed across this chip to detect binding and dissociation in one experiment, may sometimes yield incorrect quantitative information because of surface effects (34Schuck P. Curr. Opin. Biotechnol. 1997; 8: 498-502Crossref PubMed Scopus (142) Google Scholar). In general these effects are minimized or eliminated if the surface of the chip is charged to a very low density with the immobilized component and if the flow rate across the chip is high enough so that rapid exchange between the surface-proximal and surface-distal fluid layers occurs. To determine whether SPR would be a useful technique providing accurate and meaningful quantitative information for interactions between DNA and RFC, we attached a 30/80-mer primer-template to the sensor chip via a biotin-streptavidin linkage (Fig. 1 A). The amount of DNA bound to the chip was kept very low at ∼20 RU to avoid problems inherent to high density chips (the maximum binding capacity of the chip is >1000 RU). Binding of RFC to the DNA chip in the presence of a saturating concentration of ATPγS (100 μm) was measured at increasing RFC concentrations (Fig. 1 C). Upon injection of RFC, rapid binding to the DNA chip was observed until an equilibrium, the steady state response, was reached. The observedkon values (3–4 × 107m−1 s−1) andkoff values (0.022–0.028 s−1) were largely independent on the RFC concentration between 1.2 and 75 nm, and a global fit of the data yielded aKD value of 6 ± 2 nm RFC. A plot of the steady state binding levels against RFC concentration gave aKD value of 5.5 ± 2 nm for the RFC-DNA interaction. In comparison, classical filter binding experiments under the exact same solution conditions with the same template-primer, but now labeled with 32P at the 5′-end of the 80-mer, gave a KD value of 2.1 ± 1 nm (Fig. 1 B). We consider theKD values obtained by these two techniques sufficiently close to conclude that SPR can provide reliable quantitative information about protein-protein and protein-DNA interactions. The DNA substrate used in this study is a partial duplex in which the A/G-rich primer strand is hybridized to a C/T-rich template strand and the SS regions of the template strand consist of oligo(dT) (Fig.1 A). The choice of this set eliminated the potential for secondary structure and allowed stable binding of RPA to the SS DNA (see below) (35Kim C. Snyder R.O. Wold M.S. Mol. Cell. Biol. 1992; 12: 3050-3059Crossref PubMed Scopus (241) Google Scholar). When a natural DNA template was used, derived from M13-mp18 sequences, the half-life of bound RPA was unacceptably low at 10–15 min. Furthermore, because of primer dimer and secondary structure formation, little or no dependence on a primer-template junction for binding by RFC was observed (data not shown). To evaluate whether SPR would permit detection of complexes containing both RPA and RFC, the DNA chip was first injected with RPA, followed by injection of RFC. Separate experiments showed that saturation binding of RPA was achieved with 40 nm RPA (data not shown). After injection of RPA stopped, an initial rapid dissociation of a minor fraction of weakly bound RPA was observed, followed by a very slow dissociation of the remaining RPA (t 12 = 50 min) (Fig.2). When during this slow dissociation phase 10 nm RFC in ATPγS buffer was injected, a sharp increase in signal was observed, followed by a subsequent decrease in signal when the injection of RFC was switched to that of buffer, suggesting that a binding of RFC was observed similar to that in Fig.1 C. Remarkably, after several minutes of dissociation, the response curve closely matched that of the control curve in which buffer was injected instead of RFC (Fig. 2, None). The same results were obtained with 25 nm RFC. This suggests that binding of RFC did not displace the bound RPA. Because an equilibrium was reached during the injection period when 10 or 25 nmRFC were injected, it is likely that the available primer-templates were multiple times sampled by RFC binding. In addition, because the site size of RPA is ∼30 nucleotides, only two RPA molecules would be expected to bind to the DNA, one on each side of the primer (35Kim C. Snyder R.O. Wold M.S. Mol. Cell. Biol. 1992; 12: 3050-3059Crossref PubMed Scopus (241) Google Scholar). Therefore, dissociation of one RPA molecule upon binding of RFC would result in a 50% decrease in the magnitude of the slow decay signal after RFC dissociation was complete, i.e. after ∼1000 s. This was not observed, indicating that RPA and RFC can bind the DNA substrate concurrently. Finally, when very high concentrations of RFC were injected on the RPA-coated DNA chip, some dissociation of the prebound RPA was observed. This was suggested by a decrease in the response signal during the binding phase after the initial maximum response had been reached (at 600–700 s) and, secondly, by the occurrence of a residual signal significantly lower than the control during the latter part of the dissociation phase (Fig. 2). Therefore, most of our DNA binding studies with RPA-coated DNA chips were carried out at 10–25 nm RFC. Although the 80-mer DNA template used in these studies did not contain any obvious sequences that could form secondary structures (Fig. 1 A), low but significant RFC binding was observed to a chip to which only the SS template oligonucleotide was attached (Fig.3 A). However, this binding was independent of a nucleotide cofactor, indicative of its nonspecific nature. Moreover, coating of the SS DNA with RPA prior to injection of RFC eliminated all binding of RFC (Fig. 3 B). In contrast, when primer-template was attached to the chip, robust binding of RFC was observed in the presence of ATPγS, and weak binding was observed when ATP or no nucleotide was present (Fig. 3 C). Interestingly, the level of binding in the presence of ATP was consistently and significantly lower than in its absence, suggesting that hydrolysis of ATP may actively promote dissociation of DNA-bound RFC. Coating of the primed DNA with RPA prior to injection of RFC virtually eliminated binding with or without ATP, whereas binding with ATPγS showed only a slight reduction (Fig. 3 D). The simplest explanation for these results is that binding of RFC to primed DNA requires binding of ATP but that hydrolysis of the bound ATP promotes complex dissociation. These studies assume that the observed differences between experiments with ATP and with ATPγS derive not from differences in binding affinities between these nucleotides to RFC but rather from the inability of ATPγS to undergo hydrolysis. Control studies established that ATPγS is not hydrolyzed by RFC under any binding condition, i.e. with or without PCNA and/or DNA (data not shown). The affinity of the Rfc2–5 core for primed DNA was extremely low and could not be reliably measured (data not shown and TableI). Furthermore, this low binding was unaffected by the presence of RPA or a nucleotide cofactor. When the NaCl concentration in the buffer used for SPR experiments was decreased from 125 to 75 mm, still no specific interaction between Rfc2–5 and DNA was observed, but nonspecific binding of Rfc2–5 to the chip matrix increased (data not shown). Despite these negative results, ATPase data presented below show indirectly that the core complex interacts with DNA albeit weakly.Table IInteraction of RFC and Rfc2–5 with PCNA and DNASensor ChipAnalytesKDkoffRFCPCNAATPATPγSnms−1PCNA+−−−210.035+−+−1.30.011+−−+1.50.011Rfc2–5−−−∼500Rfc2–5−+−∼500Rfc2–5−−+40Matched primer-template+−−−No Binding+−+−No Binding+−−+150.013++−−No Binding+++−BindingaKD cannot be calculated because of bi-modal dissociation.0.09, 0.007bkoff rates for dissociation of RFC and PCNA, respectively.++−+50.007−+−+No BindingRfc2–5−−+No BindingRfc2–5+−+No BindingForked primer template+−−+100.013+++−BindingaKD cannot be calculated because of bi-modal dissociation.0.02, 0.003bkoff rates for dissociation of RFC and PCNA, respectively.++−+1.30.003Increasing concentrations of RFC or Rfc2–5 were flowed across the sensor chip, and the KD values were determined from a plot of the steady state response against protein concentration (Fig.1 C). The Koff values were determined separately and for all experiments were independent of the concentration of RFC. All binding studies to the DNA chips were performed in the presence of RPA. No Binding indicates that the estimated KD is > 1000 nm.a KD cannot be calculated because of bi-modal dissociation.b koff rates for dissociation of RFC and PCNA, respectively. Open table in a new tab Increasing concentrations of RFC or Rfc2–5 were flowed across the sensor chip, and the KD values were determined from a plot of the steady state response against protein concentration (Fig.1 C). The Koff values were determined separately and for all experiments were independent of the concentration of RFC. All binding studies to the DNA chips were performed in the presence of RPA. No Binding indicates that the estimated KD is > 1000 nm. Previously, we have shown that ATP or ATPγS greatly enhances the binding of RFC to PCNA-agarose beads (20Gerik K.J. G" @default.
• W2052079265 created "2016-06-24" @default.
• W2052079265 creator A5065806062 @default.
• W2052079265 creator A5071638186 @default.
• W2052079265 date "2001-09-01" @default.
• W2052079265 modified "2023-10-07" @default.
• W2052079265 title "ATP Utilization by Yeast Replication Factor C" @default.
• W2052079265 cites W124504601 @default.
• W2052079265 cites W1517952165 @default.
• W2052079265 cites W1536976216 @default.
• W2052079265 cites W1540214411 @default.
• W2052079265 cites W1554226438 @default.
• W2052079265 cites W1580231709 @default.
• W2052079265 cites W1589132235 @default.
• W2052079265 cites W1605838085 @default.
• W2052079265 cites W1940379257 @default.
• W2052079265 cites W1965822554 @default.
• W2052079265 cites W1970030918 @default.
• W2052079265 cites W1975098427 @default.
• W2052079265 cites W1982016916 @default.
• W2052079265 cites W1988840026 @default.
• W2052079265 cites W1989821243 @default.
• W2052079265 cites W2018535900 @default.
• W2052079265 cites W2018705198 @default.
• W2052079265 cites W2021827554 @default.
• W2052079265 cites W2036743626 @default.
• W2052079265 cites W2036823751 @default.
• W2052079265 cites W2037431251 @default.
• W2052079265 cites W2052297952 @default.
• W2052079265 cites W2059060156 @default.
• W2052079265 cites W2060665340 @default.
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