Structure and Mechanism of the DNA Polymerase Processivity Clamp Loader

The replication of a genome is a fundamental biological process that every organism must perform in order to transmit its genetic makeup to its progeny. We have been studying the molecular machines that are used to rapidly and accurately replicate the DNA genome of bacteria. In addition to the polymerase (the actual copying machine), organisms have a set of assemblies that enable the polymerase to perform its jobs more rapidly. One of these assemblies is the processivity clamp, which is a ring shaped protein that encircles and slides along DNA and physically tethers the polymerase to the DNA that is being replicated. The main problem that we have been studying is how does a ring-shaped sliding clamp become assembled onto DNA, which is a long polymer. Organisms have solved this problem by using a clamp loader machine that physically opens the ring and loads it onto DNA. We have determined the structure of an intact and functional clamp loader machine. This structure, along with a second structure of the clamp, caught in the act of being opened, has allowed us to construct a model of the clamp loading reaction. Chromosomal replicases from prokaryotes to eukaryotes are organized around three functional components, a DNA polymerase, a ring-shaped processivity clamp, and a clamp-loading machine. DNA polymerases catalyze the addition of nucleotides to primed template DNA and, in isolation, are poorly processive enzymes (4,10,13). Presence of the processivity clamp, dramatically increase processivity. Processivity clamps are ring shaped proteins composed of 2 (eubacteria) or 3 (phage, archae, eukaryote) subunits (5). The clamps confer processivity by encircling DNA and physically tethering the polymerase subunit to the DNA as it tracks along the template (11). Assembly of sliding clamps on DNA is performed by the clamp loader assembly in a reaction fueled by ATP (12). In bacteria, the clamp loader is composed of 7 unique subunits (γ, δ, δ, χ, ψ) and the β 2 dimer is the processivity clamp. The clamp loader is an AAA+ ATPase and is the bacterial homologue of the eukaryotic replication factor C (RFC) (7, 8). We have determined the structure of the bacterial clamp loader γ 3 δδ’ and that of the δ wrench in complex with a clamp subunit (β:δ). These structures were solved using data measured at the National Synchrotron Light Source (X-25) at the Brookhaven National Laboratory, at the Structural Biology Center at the Advanced Photon Source (ID-19), the Stanford Synchrotron Radiation Laboratory (9.2), and the Advanced Light Source (5.02) at the Lawrence Berkeley Laboratory. The crystal structure of the β:δ assembly reveals that the δ subunit binds to the C-terminal side of the processivity clamp at a location near to, but not at the dimer interface (Figure 1) (3). We used a mutated (I272A, L273A) form of β (β 1 ), which exists as a stable monomer in solution and which binds to the wrench subunit (δ) with a 50 fold higher affinity than wild type (9). The δ subunit, which adopts the same fold as the other clamp loader subunits, places its N-terminal domain (β-interacting element) into a binding site composed of two domains (2 & 3) on β 1 . Two highly conserved hydrophobic δ residues (F73, L74) are wedged into a hydrophobic pocket on the clamp. Binding of the δ subunit requires a conformational change in β that renders the clamp interface incapable of closing. With respect to its structure in the dimeric clamp, β from the β:δ complex adopts a conformation of reduced curvature. This observation along with molecular dynamics simulations suggest a spring-loaded mechanism in which the β ring opens spontaneously once a dimer interface is perturbed by the β wrench. Figure 1. Structure of the β:δ complex.