The clamp loader uses energy provided by ATP to load the sliding clamp onto a DNA template primer with a 5'-overhang. It exists as a subassembly of the DNA polymerase Ⅲ holoenzyme and also in a free form. The free form does not appear to arise from the dissociation of the holoenzyme because (1) the. Holoenzyme is stable when studied in vitro and can only be dissociated under harsh nonphysiological conditions and (2) the two forms have different subunit compositions: γ3δδ'ΧΨ in the free form and τ2γδδ'ΧΨ in the holoenzyme. Differences between the two forms are not as great as they may at first appear because both γandτsubunits are encoded by the same dnaX gene. Theτsubunit is the full-length product while theγsubunit is a truncated form that is missing about 1/3 of the residues at the C-terminus. The C-terminal region, which is present inτbut notγ, plays an important function in DNA polymerase Ⅲholoenzyme because it contains the site that binds the core polymerase and DnaB helicase.

    Mike O'Donnell and coworkers obtained considerable information about clamp loader structure and function by studying the structure and functions of aγ3δδ' complex they constructed by combining subunits in vitro. Thisγ3δδ' complex is called the minimal clamp loader because it is the smallest complex that can load sliding clamps onto DNA. Neither the Χ or Ψ subunit is required to load the clamp.

    The Ψ subunit stabilizes the clamp loader and the X subunit displaces primase from SSB. O'Donnell and coworkers obtained an important clue to the minimal clamp loader's mechanism of action by studying the effect that the δ subunit has on the sliding clamp. In one key experiment, they demonstrated that the δ subunit, working without the other subunits or ATP causes the β dimer to be released from a topologically-linked clamp · circular DNA complex. This release is not a catalytic or reversible process because the δ subunit remains tightly associated with the released clamp and the free δ subunit cannot load the clamp onto DNA. The δ subunit appears to work as a "wrench," forcing one of the two interfaces in the β dimer to open.

    O'Donnell and coworkers have also determined the high-resolution structure for the minimal clamp loader.The five subunits are arranged as a circular heteropentamer. The clamp loader behaves as a machine with moving parts when it loads the β clamp onto a DNA template-primer complex .Theγ(τ) subunits, which are the only polypeptide subunits that bind and hydrolyze ATP, act as the machine's motor. The δ' subunit, which appears to be stationary, regulates the δ subunit's ability to bind the β clamp. In the absence of ATP, δ' prevents δ (the wrench) from binding the-β ring and opening it. When ATP binds to the γ subunits, the δ subunit pulls away from δ' and thereby gains the ability to bind the β clamp. The clamp loader ·β clamp complex has a high affinity for a DNA template-primer complex with a 5'_overhang. After the clamp loader ·β clamp complex has interacted with the primed DNA, the bound ATP molecules are hydrolyzed, causing the clamp loader ·β clamp complex to dissociate and allowing the β clamp to close around the DNA.