With electrospray (ES), ions present in solution can be transferred to the gas phase. The method provides unique opportunities for studies of hitherto inaccessible ions. Collision-induced dissociation threshold measurements of gas phase ions produced by ES are described. The thresholds for the reactions M+L = M+ + L, where M+ is Na+ or K+ and L is acetone, dimethyl sulfoxide, acetamide, N-methylacetamide, N,N-dimethylacetamide, glycine, glycinamide, succinamide, and glycylglycine, were determined. Enthalpy changes for the reaction were derived from these data.

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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, and , which are not present in the crystal structures, were determined by measuring the activities of three forms of the clamp loader, ␥ 3 ␦␦, ␥ 3 ␦␦, and ␥ 3 ␦␦. The subunit is important for stabilizing an ATP-induced conformational state with high affinity for DNA, whereas the subunit does not contribute directly to clamp loading in our assays lacking singlestranded DNA-binding protein. The subunit also increases the affinity of the clamp loader for the clamp in assays in which ATP␥S is substituted for ATP. Interestingly, the affinity of the ␥ 3 ␦␦ complex for ␤ is no greater in the presence than in the absence of ATP␥S. A role for in stabilizing or promoting ATPand ATP␥S-induced conformational changes may explain why large conformational differences were not seen in ␥ 3 ␦␦ structures with and without bound ATP␥S. The ␤ clamp partially compensates for the activity of 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 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. 1-4). Sliding clamps are ring-shaped protein complexes that encircle DNA, and the Escherichia coli clamp consists of two identical arc-shaped ␤ subunits (5). 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 (6, 7). 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 subunit and one copy each of the ␥, ␦, ␦Ј, , and subunits (8 -11). The and ␥ subunits are products of the same gene, dnaX, and the ␥ subunit is a truncated form produced during translation (12-14). Clamp loaders containing three copies of the DnaX protein in any combination are fully active in clamp loading (9), however, the additional C-terminal extension present on mediates protein-protein interactions that are required to coordinate other activities at the replication fork (reviewed in Refs. 4,15,and 16).The functional core of clamp loaders is composed of five subunits that are members of the AAAϩ family of ATPases (ATPases associated with diverse cellular activities; for recent AAAϩ reviews see Refs. 17 and 18).2 Three copies of the ␥ subunit, and one copy each of the ␦ and ␦Ј subu...

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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).