Loading clamps for DNA replication and repair

Abstract

Sliding clamps and clamp loaders were initially identified as DNA polymerase processivity factors. Sliding clamps are ring-shaped protein complexes that encircle and slide along duplex DNA, and clamp loaders are enzymes that load these clamps onto DNA. When bound to a sliding clamp, DNA polymerases remain tightly associated with the template being copied, but are able to translocate along DNA at rates limited by rates of nucleotide incorporation. Many different enzymes required for DNA replication and repair use sliding clamps. Clamps not only increase the processivity of these enzymes, but may also serve as an attachment point to coordinate the activities of enzymes required for a given process. Clamp loaders are members of the AAA+ family of ATPases and use energy from ATP binding and hydrolysis to catalyze the mechanical reaction of loading clamps onto DNA. Many structural and functional features of clamps and clamp loaders are conserved across all domains of life. Here, the mechanism of clamp loading is reviewed by comparing features of prokaryotic and eukaryotic clamps and clamp loaders.

Fig. 1. Structural features of sliding clamps

Ribbon diagrams of the E. coli β-clamp (left panel), human PCNA (center panel), and bacteriophage RB69 gp45 (right panel) are shown with each subunit in a different color. Beneath the ribbon diagrams are cartoons that illustrate key structural features of the clamps. Each clamp is composed of six globular domains that are arranged in a ring and interact mainly through noncovalent interactions. Peptide linkers on the outside of the rings covalently join globular domains into monomeric subunits. In β, three globular domains are covalently joined by peptide linkers to form a monomer, and in PCNA and gp45, two domains are covalently linked to form a monomer. Structures were generated from PDB files 2POL [1], 1AXC [26], and 1B77 [29], for β, PCNA, and gp45, respectively, using PyMol from DeLano Scientific LLC.

Fig. 2. Structural features of clamp loaders

A) Ribbon diagrams of the D-subunits from S. cerevisiae RFC (upper panel) and E. coli γ complex (lower panel) with bound ATPγS (yellow spheres) are shown. The subunits are oriented such that the C-terminal domain III is at the top. B) Ribbon diagrams of the five-subunit core clamp loaders, S. cerevisiae RFC (upper panel) and E. coli γ₃δδ ′ (lower panel) are shown. The view of RFC is looking down on the “collar” formed by the C-terminal domains (III). The view of the E. coli clamp loader is rotated 90° relative to that of RFC such that the collar formed by the C-terminal domains (III) is at the top of the structure. In this view, the tight packing of the C-terminal domains relative to the N-terminal domains and the gap between the A- and E-subunits can be seen. Bound ATPγS is shown as gray spheres. The A-subunit in the RFC structure was truncated for crystallography to residues 295 – 785 (of 861) and the other subunits are full-length. The γ subunits are truncated to residues 1 – 373 (of 431). C) Cartoon diagrams illustrating the arrangement of the five core subunits in eukaryotic and E. coli clamp loaders. The subunits are referred to as A – E to make comparisons between clamp loaders from different species. The subunit nomenclature is also given, and for RFC both the yeast (Rfc1 – 5) and human (e.g. p140) designations are given. All ribbon diagrams were generated from PDB files 1SXJ [33] and 1XXH [44] for the yeast and E. coli proteins, respectively, using PyMol from DeLano Scientific LLC.

Fig. 3. Clamp and DNA binding

A) A ribbon diagram of a S. cerevisiae RFC•PCNA complex is shown. The N-terminal domains (I) of subunits A, B, and C contact one face of the clamp. Contacts with the clamp are the greatest for subunit A and decrease around the ring to the point at which subunits D and E do not contact the clamp at all. The clamp is closed in this structure. In the open clamp loader•clamp complex, the cyan and magenta PCNA subunits are anticipated to swing up so that the N-terminal domain (I) of each subunit contacts the surface of the ring as illustrated in the cartoon in panel C. Arginine fingers in the conserved Ser-Arg-Cys motif were mutated to glutamine and ATPγS was substituted for ATP to prevent hydrolysis of ATP. The large A subunit was truncated to residues 295 – 785 (of 861), and is closer in size to the small RFC subunits in this structure. This diagram was generated from PDB file 1SXJ [33] using PyMol from DeLano Scientific LLC. B) A hypothetical model is shown that illustrates the footprint the clamp loader makes when binding the clamp to form an open clamp loader•clamp complex. The black circles represent contacts with the face of PCNA made by each RFC subunit (A – E). C) In the cartoon diagrams of RFC and PCNA, each subunit is colored as in the ribbon diagrams. Individual domains of each subunit are represented by spheres or ovals. The cartoon diagrams are based on structural and biochemical data discussed in the text. In general, the clamp loading reaction can be divided into two phases based on ATP requirements. In the first phase, formation of a ternary clamp loader•clamp•DNA complex is promoted by ATP binding. And in the second phase, DNA binding triggers hydrolysis of ATP and the release of the clamp on DNA.