Abstract
The beta sliding clamp encircles DNA and tethers DNA polymerase III holoenzyme to the template for high processivity. The clamp loader, gamma complex (gamma 3 delta delta'chi psi), assembles beta around DNA in an ATP-fueled reaction. The delta subunit of the clamp loader opens the beta ring and is referred to as the wrench; ATP modulates contact between beta and delta among other functions. Crystal structures of delta.beta and the gamma 3 delta delta' minimal clamp loader make predictions of the clamp loader mechanism, which are tested in this report by mutagenesis. The delta wrench contacts beta at two sites. One site is at the beta dimer interface, where delta appears to distort the interface by via a steric clash between a helix on delta and a loop near the beta interface. The energy for this steric clash is thought to derive from the other site of interaction, in which delta binds to a hydrophobic pocket in beta. The current study demonstrates that rather than a simple steric clash with beta, delta specifically contacts beta at this site, but not through amino acid side chains, and thus is presumably mediated by peptide backbone atoms. The results also imply that the interaction of delta at the hydrophobic site on beta contributes to destabilization of the beta dimer interface rather than acting solely as a grip of delta on beta. Within the gamma complex, delta' is proposed to prevent delta from binding to beta in the absence of ATP. This report demonstrates that one or more gamma subunits also contribute to this role. The results also indicate that delta' acts as a backboard upon which the gamma subunits push to attain the ATP induced change needed for the delta wrench to bind and open the beta ring.
Highlights
Chromosomal replicases of both eukaryotes and prokaryotes derive their high processivity during synthesis from a ringshaped clamp that encircles DNA and binds to the chromosomal replicase [1,2,3,4,5]
The results indicate that ␦ acts as a backboard upon which the ␥ subunits push to attain the ATP induced change needed for the ␦ wrench to bind and open the  ring
Two other subunits, and , are associated with the clamp loader, but they are not required for clamp loading in vitro [9]. In order for this clamp loader to bind two molecules of DNA polymerase III core, two of the ␥ subunits are replaced by two subunits. and ␥ are encoded by the same gene; is the full-length product, and ␥ is a truncated version produced by a translational frameshift (10 –12)
Summary
Pol III, polymerase III; DTT, dithiothreitol; ssDNA, single-stranded DNA; RFC, replication factor C; PCNA, proliferating cell nuclear antigen. The interaction of ␦ with  at this site leads to an extensive conformational change of the  loop that is thought to be important for ring opening [20] This five-residue loop of  (residues 274 –278) is near the interface and connects to the interfacial ␣ helix that contains the two residues (Ile272 and Leu273) that form the hydrophobic core of the 2 dimer interface. It is proposed that as the ATP sites fill, conformation changes in ␥ are propagated around the pentamer to increase the gap between ␦ and ␦Ј, thereby allowing ␦ to bind to  for clamp opening [8, 17, 18, 22] In this state, with  and ATP bound to ␥ complex, a tight affinity for DNA is established [22, 24]. The studies contain surprises, but overall the results provide significant advancements in our understanding of how this complex machinery functions
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