Mechanism of Loading the Escherichia coli DNA Polymerase III Sliding Clamp

Abstract

Sliding clamps tether DNA polymerases to DNA to increase the processivity of synthesis. The Escherichia coli ␥ complex loads the ␤ sliding clamp onto DNA in an ATP-dependent reaction in which ATP binding and hydrolysis modulate the affinity of the ␥ complex for ␤ and DNA. This is the second of two reports (Williams, C. R., Snyder, A. K., Kuzmič , P., O'Donnell, M., and Bloom, L. B. (2004) J. Biol. Chem. 279, 4376 -4385) addressing the question of how ATP binding and hydrolysis regulate specific interactions with DNA and ␤. Mutations were made to an Arg residue in a conserved SRC motif in the ␦ and ␥ subunits that interacts with the ATP site of the neighboring ␥ subunit. Mutation of the ␦ subunit reduced the ATP-dependent ␤ binding activity, whereas mutation of the ␥ subunits reduced the DNA binding activity of the ␥ complex. The ␥ complex containing the ␦ mutation gave a pre-steady-state burst of ATP hydrolysis, but at a reduced rate and amplitude relative to the wild-type ␥ complex. A pre-steady-state burst of ATP hydrolysis was not observed for the complex containing the ␥ mutations, consistent with the reduced DNA binding activity of this complex. The differential effects of these mutations suggest that ATP binding at the ␥ 1 site may be coupled to conformational changes that largely modulate interactions with ␤, whereas ATP binding at the ␥ 2 and/or ␥ 3 site may be coupled to conformational changes that have a major role in interactions with DNA. Additionally, these results show that the "arginine fingers" play a structural role in facilitating the formation of a conformation that has high affinity for ␤ and DNA.Sliding clamps tether DNA polymerases to their templates, enabling the polymerases to rapidly incorporate thousands of nucleotides into a growing polymer without dissociating from DNA. Crystal structures of sliding clamps from bacteria, bacteriophage, and humans have revealed a mechanism by which this is possible (1-4). Sliding clamps are composed of individual subunits that assemble into rings with a central opening large enough to encircle duplex DNA. These ring-shaped clamps must be assembled on DNA by the activity of a clamp loader that uses energy derived from ATP to power its mechanical task (reviewed in Ref. 5).In Escherichia coli, the clamp loader is composed of seven subunits, three copies of the dnaX gene product, ␦, ␦Ј, , and (6 -8). The DnaX proteins form the motor of the clamp loader, and each is capable of binding and hydrolyzing 1 molecule of ATP during the clamp loading process. In vivo, two forms of the DnaX protein are produced: a full-length gene product () and a truncated gene product (␥) with the same sequence, but only about two-thirds the length of (9 -11). The clamp loader at the replication fork most likely contains two copies of the subunit and a single copy of ␥ (7, 8). The unique C-terminal end present in interacts with other enzymes and coordinates the activities present at the replication fork, whereas the N-terminal domain shared by the and ␥ subunits provides the activ...

Multiple ATP Binding Is Required to Stabilize the “Activated” (Clamp Open) Clamp Loader of the T4 DNA Replication Complex

“…Thus three of the five clamp loader subunits of E. coli bind ATP (29), four or five subunits bind ATP in the yeast complex (19,26), and four bind ATP in the T4 system (14,22). It has been suggested for the E. coli complexes that ATP binding may expose the binding site for the clamp on the surface of the clamp loader complex, and that a particular ATP site may be centrally involved in modulating the interaction with the clamp and therefore presumably in driving clamp opening as well (31).…”

The Replication Factor C Clamp Loader Requires Arginine Finger Sensors to Drive DNA Binding and Proliferating Cell Nuclear Antigen Loading

“…Previous studies of prokaryotic and eukaryotic clamp loaders suggest that individual ATP sites play specific roles in clamp loading (29,44,49). Mutational analysis in yeast RFC has shown that ATP binding to sites C and D of RFC is essential for DNA binding (29), but it is not clear how ATP binding is coupled to DNA binding.…”

“…1C). In the E. coli ␥ complex clamp loader these arginine fingers have been shown to have two functions: they sense bound ATP and also play a catalytic role in ATP hydrolysis (43,44). The sensor functions of the arginine fingers in the ␥ complex clamp loader are thought to be coupled to conformational changes that promote binding to the clamp and DNA (44).…”

“…In the E. coli ␥ complex clamp loader these arginine fingers have been shown to have two functions: they sense bound ATP and also play a catalytic role in ATP hydrolysis (43,44). The sensor functions of the arginine fingers in the ␥ complex clamp loader are thought to be coupled to conformational changes that promote binding to the clamp and DNA (44). The arginine fingers are needed for ATP hydrolysis (43) and are presumed to act by stabilizing the growing negative charge as the ␥-phosphate is cleaved from ATP (21,42,45).…”

Discovery and Characterization of the Cryptic Ψ Subunit of the Pseudomonad DNA Replicase

“…A complex containing 3 ␦␦Ј (-complex), however, appears to be fully functional (23). Both and the shorter ␥ can hydrolyze ATP (in concert with ␦␦Ј), which opens the ring of the ␤ sliding clamp and positions ␤ on the primer terminus (23)(24)(25)(26)(27)(28)(29)(30)(31)(32). The clamp loader complex plays a role in elongation as well; interacts with and facilitates dimerization of the ␣ subunit (33,34).…”