Clamp loaders load ring-shaped sliding clamps onto DNA. Once loaded onto DNA, sliding clamps bind to DNA polymerases to increase the processivity of DNA synthesis. To load clamps onto DNA, an open clamp loader-clamp complex must form. An unresolved question is whether clamp loaders capture clamps that have transiently opened or whether clamp loaders bind closed clamps and actively open clamps. A simple fluorescence-based clamp opening assay was developed to address this question and to determine how ATP binding contributes to clamp opening. A direct comparison of real time binding and opening reactions revealed that the Escherichia coli γ complex binds β first and then opens the clamp. Mutation of conserved “arginine fingers” in the γ complex that interact with bound ATP decreased clamp opening activity showing that arginine fingers make an important contribution to the ATP-induced conformational changes that allow the clamp loader to pry open the clamp.

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Clamp loaders are multi-subunit complexes that use the energy derived from ATP binding and hydrolysis to assemble ring-shaped sliding clamps onto DNA. Sliding clamps in turn tether DNA polymerases to the templates being copied to increase the processivity of DNA synthesis. Here, the rate of clamp release during the clamp loading reaction was measured directly for the first time using a FRET-based assay in which the E. coli γ complex clamp loader (γ 3 δδ'χψ) was labeled with a fluorescent donor and the β-clamp was labeled with a nonfluorescent quencher. When a β·γ complex is added to DNA, there is a significant time lag before the clamp is released onto DNA. To establish what events take place during this time lag, the timing of clamp release was compared to the timing of DNA binding and ATP hydrolysis by measuring these reactions directly "side-by-side" in assays. DNA binding is relatively rapid and triggers hydrolysis of ATP. Both events occur prior to clamp release. Interestingly, the temporal correlation data and simple modeling studies indicate that the clamp loader releases DNA prior to the clamp and that DNA release may be coupled to clamp closing. Clamp release is relatively slow and likely to be the rate-limiting step in the overall clamp loading reaction cycle.Clamp loaders load sliding clamps, which serve as processivity factors for DNA polymerases, onto DNA. Sliding clamps are ring-shaped complexes of crescent-shaped monomers that encircle duplex DNA and bind DNA polymerases to tether them to the template being copied. Binding to sliding clamps increases the processivity of DNA polymerases from tens to thousands of nucleotides. Clamp loaders are molecular machines that ultimately open the ringshaped clamps and place the clamps around duplex DNA. Many structural and functional features of clamps and clamp loaders are conserved from bacteria to man, and the Escherichia coli sliding clamp and clamp loader have served as a useful model system for defining the mechanism of the clamp loading reaction (reviewed in (1-4)).The E. coli clamp loader contains seven polypeptides, three copies of the dnaX gene product, and one copy each of the δ, δ', χ and ψ subunits (5-7). The DnaX protein is present in two forms in the cell, a long form, τ, and a short form, γ. The short form is the result of a translational frameshift that truncates DnaX such that the γ subunit is about two-thirds the length of τ and of identical sequence except for the last amino acid residue (8-10).

In Escherichia coli, the ␥ complex clamp loader loads the ␤-sliding clamp onto DNA. The ␤ clamp tethers DNA polymerase III to DNA and enhances the efficiency of replication by increasing the processivity of DNA synthesis. In the presence of ATP, ␥ complex binds ␤ and DNA to form a ternary complex. Binding to primed template DNA triggers ␥ complex to hydrolyze ATP and release the clamp onto DNA. Here, we investigated the kinetics of forming a ternary complex by measuring rates of ␥ complex binding ␤ and DNA. A fluorescence intensity-based ␤ binding assay was developed in which the fluorescence of pyrene covalently attached to ␤ increases when bound by ␥ complex. Using this assay, an association rate constant of 2.3 ؋ 10 7 M ؊1 s ؊1 for ␥ complex binding ␤ was determined. The rate of ␤ binding was the same in experiments in which ␥ complex was preincubated with ATP before adding ␤ or added directly to ␤ and ATP. In contrast, when ␥ complex is preincubated with ATP, DNA binding is faster than when ␥ complex is added to DNA and ATP at the same time. Slow DNA binding in the absence of ATP preincubation is the result of a rate-limiting ATP-induced conformational change. Our results strongly suggest that the ATP-induced conformational changes that promote ␤ binding and DNA binding differ. The slow ATP-induced conformational change that precedes DNA binding may provide a kinetic preference for ␥ complex to bind ␤ before DNA during the clamp loading reaction cycle.Two accessory factors, a sliding clamp and a clamp loader, enhance the efficiency of DNA replication by increasing the processivity of DNA synthesis. The clamp loader assembles clamps onto DNA. The clamp binds the DNA polymerase to increase the processivity of DNA synthesis from tens to thousands of nucleotides in a single binding event. These processivity factors are conserved from bacteria to humans (reviewed in Refs. 1 and 2). The Escherichia coli ␤ clamp is composed of two identical protein monomers assembled into a ring-shaped dimer, which encircles duplex DNA (3, 4). The clamp slides freely along the DNA while tethering DNA polymerase III to the DNA template. The E. coli clamp loader is composed of seven subunits. At the replication fork, a complete clamp loader includes three copies of the dnaX gene product and one copy each of ␦, ␦Ј, , and (5-7). The dnaX gene produces two proteins, a full-length protein () and a truncated protein (␥), which is created by a translational frameshift (8 -10). Clamp loaders containing either three copies of the subunit ( complex, 3 ␦␦Ј) or three copies of the ␥ subunit (␥ complex, ␥ 3 ␦␦Ј) are fully active in clamp loading (6). The subunits have an additional C-terminal extension that coordinates the activities of the replisome (reviewed in Refs. 11 and 12). The clamp loader used in all experiments in this study is the ␥ complex.In the presence of ATP, ␥ complex binds with high affinity to ␤ and DNA to form a ternary complex. Binding to primed template DNA triggers the clamp loader to hydrolyze all three molecules of ATP (...

In the E. coli DNA replication system, the five‐subunit clamp loader, γ‐complex, loads the βsliding clamp onto DNA. The βclamp, a ring shaped dimer of identical subunits, tethers DNA polymerase III to DNA and enhances the efficiency of replication by increasing the processivity of DNA synthesis. In the presence of ATP, γ‐complex binds to βand DNA to form a ternary complex. Binding to primed template DNA triggers the clamp loader to hydrolyze ATP. Upon ATP hydrolysis, βis released onto DNA and γ‐complex dissociates from the complex. We have developed a βbinding fluorescence assay by covalently attaching a fluorophore, pyrene, onto a Cys residue (Q299C) ofβ. The fluorescence increases two‐fold when γ‐ complex binds β‐pyrene. Using this assay, the rates of association, kon of 3.5 x 107 M−1s−1 and dissociation, koff of 0.005 s−1 of γ‐complex binding to βwere determined in real time. In addition, the kinetics of γ‐ complex binding to DNA were measured in real time to compare to those for βbinding. When γ‐complex is pre‐incubated with ATP, the clamp loader can bind βor DNA with no preferred order. When there is no ATP pre‐incubation, γ‐complex must undergo a slow ATP‐induced conformational change before binding to DNA but does not have to undergo this change when binding toβ. Thus, there may be a kinetic preference for γ‐complex to bind βbefore DNA during the clamp loading reaction cycle.This work was supported by NIH grant GM055596.