Research Article

How a DNA Polymerase Clamp Loader Opens a Sliding Clamp

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Science  23 Dec 2011:
Vol. 334, Issue 6063, pp. 1675-1680
DOI: 10.1126/science.1211884

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  1. Fig. 1

    Clamp loaders and sliding clamps. (A) Clamp-loading reaction. The clamp loader has low affinity for both clamp and primer-template DNA in the absence of ATP. Upon binding ATP, the clamp loader can bind the clamp and open it. The binding of primer-template DNA activates ATP hydrolysis, leading to ejection of the clamp loader. (B) Three classes of clamp loaders. Bacterial clamp loaders are pentamers consisting of three proteins: δ (A position), γ (B, C, and D positions), and δ′ (E position). Eukaryotic clamp loaders (RFCs) consist of five different proteins, with the A subunit containing an A′ domain that bridges the gap between the A and E AAA+ modules. The T4 bacteriophage clamp loader consists of two proteins: gp44 (the B, C, D, and E subunits) and gp62 (the A subunit).

  2. Fig. 2

    Architecture of the T4 clamp loader–clamp–DNA complex (A) Structure of the T4 clamp loader bound to an open clamp. Ribbon and schematic diagrams of the complex between the T4 clamp loader (multicolored), the open T4 clamp (gray), which is broken between subunits I and III, and primer-template DNA. The gp62 protein (the A subunit; red) bridges the gap in the clamp with its A domain (a vestigial AAA+ module) on the lower part of the clamp and its A′ domain at the top. The duplex region of primer-template DNA (orange) is bound in the interior of the clamp loader (yellow ribbon in the schematic indicates contacts from the clamp loader) and the central pore of the open clamp, with the template overhang extruded through the gap between the A and A′ domains. (B) Interactions of the T4 clamp loader with the clamp. The six clamp interaction motifs of the clamp loader are displayed as surfaces with the remainder of the clamp loader shown as a thin ribbon. (C) Two orthogonal views of a closed clamp bound to DNA and the T4 clamp loader as in crystal form I. The clamp interacts with the DNA phosphate backbone through arginine residues from each clamp subunit (yellow). (D) Two orthogonal views of an open clamp bound to DNA and the T4 clamp loader. Diagram based on the open clamp complex in form II crystals. The side chains of Arg162 of subunit I and Arg87 of subunit III are represented as sticks without the surface displayed. (E) Two representations of distortions in the structure of the open clamp, relative to that of the closed clamp. Top: Displacement vectors between the two structures are shown, scaled up by a factor of 4. The magnitude of the displacement is also indicated by color (blue to red). Vectors are drawn in the direction of displacement from the planar to open conformation and are derived from local alignments of each of the six pseudo-symmetric domains in the clamp trimer. Bottom: Domain rotations derived from these local alignments are mapped onto a schematic diagram of the open clamp.

  3. Fig. 3

    Symmetric and cooperative recognition of DNA. (A) Spiral of AAA+ modules in the T4 clamp loader bound to an open clamp. The gp44 AAA+ modules, for which surfaces are displayed, form a spiral that tracks the minor groove of the DNA. The A subunit (gp62) is not shown. (B) Arginine fingers and ATP coordinate the AAA+ spiral and DNA binding. A top-down view with the arginine fingers and the ATP analog (ADP-BeF3) shown as spheres (collar domains not shown). (C) Cooperativity in ATP binding. The T4 clamp loader and the clamp, at concentrations of 2 and 5 μM, respectively, were incubated in the presence of 100 nM 5′TAMRA-labeled primer-template DNA. ADP-BeFx was titrated into the solution. As the concentration of the ATP analog increases, DNA binds to the clamp loader and the fluorescence anisotropy of the TAMRA probe increases in a highly sigmoidal fashion (nH,app = 3.3 ± 0.3).

  4. Fig. 4

    Hydrolysis-induced conformational changes. (A) The fully ATP-bound conformation of the open clamp–clamp loader–DNA complex from form II crystals. The clamp loader holds the clamp such that there is a gap between subunits I and III of the clamp. (B) Conformational changes in the clamp–clamp loader–DNA complex from ATP hydrolysis at the B subunit as seen in form III crystals. The structure of the complex is shown in the same orientation as in Fig. 4A (superposed using the AAA+ module of the C subunit). Clamp subunits I and III are sealed so that the clamp is now closed. The A and B subunits, as well as the entire collar region, undergo a large conformational change. (C) Schematic diagram describing ATP hydrolysis-induced changes in clamp interactions. The clamp and the AAA+ modules of the clamp loader are shown from the side. In the ATP-loaded state, all AAA+ modules are positioned perfectly to match the clamp binding sites. Upon ATP hydrolysis, the B subunit swings away from the C subunit, altering the spacing between the clamp binding loops. (D) ATP hydrolysis severs contacts between AAA+ modules. The AAA+ modules are shown as surfaces. The interface between the B and C AAA+ modules has been completely disrupted.

  5. Fig. 5

    A proposed DNA-dependent allosteric switch. (A) The switch residue of the T4 clamp loader (Lys80) is pointing into the interior chamber of the clamp loader to interact with DNA (DNA not shown). (B) In the absence of DNA, the calculated electrostatic potential is extremely positive in the DNA binding cleft (calculation performed without Mg2+ or ADP-BeF3 ligands).

  6. Fig. 6

    A detailed mechanism for the clamp loading reaction. The reaction cycle for the T4 clamp loader is shown as a schematic diagram. (1) In the absence of ATP, the clamp loader AAA+ modules cannot organize into a spiral shape. (2) Upon ATP binding, the AAA+ modules form a spiral that can bind and open the clamp. (3) Primer-template DNA must thread through the gaps between the clamp subunits I and III and the clamp loader A and A′ domains. (4) Upon DNA binding in the interior chamber of the clamp loader, ATP hydrolysis is activated, most likely through flipping of the switch residue and release of the Walker B glutamate. (5) ATP hydrolysis at the B subunit breaks the interface at the AAA+ modules of the B and C subunits and allows closure of the clamp around primer-template DNA. Further ATP hydrolyses at the C and D subunits dissolve the symmetric spiral of AAA+ modules, thus ejecting the clamp loader because the recognition of DNA and the clamp is broken. The clamp is now loaded onto primer-template DNA, and the clamp loader is free to recycle for another round of clamp loading.