A direct proofreader–clamp interaction stabilizes the Pol III replicase in the polymerization mode

Abstract

Processive DNA synthesis by the aeh core of the Escherichia coli Pol III replicase requires it to be bound to the b 2 clamp via a site in the a polymerase subunit. How the e proofreading exonuclease subunit influences DNA synthesis by a was not previously understood. In this work, bulk assays of DNA replication were used to uncover a non-proofreading activity of e. Combination of mutagenesis with biophysical studies and single-molecule leading-strand replication assays traced this activity to a novel b-binding site in e that, in conjunction with the site in a, maintains a closed state of the aeh-b 2 replicase in the polymerization mode of DNA synthesis. The e-b interaction, selected during evolution to be weak and thus suited for transient disruption to enable access of alternate polymerases and other clamp binding proteins, therefore makes an important contribution to the network of protein-protein interactions that finely tune stability of the replicase on the DNA template in its various conformational states.

Polymerase exchange on single DNA molecules reveals processivity clamp control of translesion synthesis

“…5B) (P < 0.01). Although the processivity of Pol III was reduced slightly with the single-cleft β + /β C , consistent with the model that the e subunit stabilizes it on the clamp (18,19), a high concentration of Pol IV with β + /β C reduced the Pol III processivity equivalently to the WT clamp condition (Fig. 5B), further supporting a role for the noncleft rim contact in Pol III displacement.…”

“…S2 A-C, time constant τ decreases from 19.7 to 12.4 s). Biophysical and structural data suggest that only one Pol III binds the clamp dimer (4,18,24,25), arguing that pauses observed during synthesis result from stochastic dissociation of Pol III from the clamp and the diffusionlimited recruitment of a new polymerase from solution (22).…”

“…The structure of the C-terminal little finger domain of Pol IV bound to β revealed that Pol IV can simultaneously interact with the cleft and the rim, a secondary site of β near its dimer interface, which positions the Pol IV catalytic domain well away from the DNA running through the center of the clamp (17). Although this potential inactive binding mode for Pol IV has been interpreted as evidence for the toolbelt model, Pol III was shown to contain a second weak CBM in its e exonuclease subunit (18) in addition to the CBM in its α catalytic subunit that has a strong affinity for the β clamp. A recent study proposed that Pol III would, therefore, occlude Pol IV from clamp binding during replication, only accommodating simultaneous binding after a lesion-induced stall (19).…”

Plasmid replication initiator interactions with origin 13-mers and polymerase subunits contribute to strand-specific replisome assembly

“…S8). Changing the QTSMAF motif into ATSMAF in e-subunit of Pol III core prevents interaction with the β-clamp (44). Experiments presented in this work demonstrate that the F138A substitution affects TrfA interaction with the β-clamp but also somehow affects helicase activity at oriV (Fig.…”

DNA Polymerase α Subunit Residues and Interactions Required for Efficient Initiation Complex Formation Identified by a Genetic Selection

“…Significant information is available regarding subunit interactions within the Pol III HE derived from quantification of physical interactions with subassemblies in solution and by determination of the structure of subassemblies (7)(8)(9)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33). More limited information is available regarding the dynamic interactions between Pol III HE subunits in the presence of all reaction components and the importance of these interactions at discrete reaction stages.…”