A model for DNA polymerase switching involving a single cleft and the rim of the sliding clamp

Abstract

The actions of Escherichia coli DNA Polymerase IV (Pol IV) in mutagenesis are managed by its interaction with the ␤ sliding clamp. In the structure reported by Bunting et al. [EMBO J (2003) 22:5883-5892], the C-tail of Pol IV contacts a hydrophobic cleft on the clamp, while residues V303-P305 reach over the dimer interface to contact the rim of the adjacent clamp protomer. Using mutant forms of these proteins impaired for either the rim or the cleft contacts, we determined that the rim contact was dispensable for Pol IV replication in vitro, while the cleft contact was absolutely required. Using an in vitro assay to monitor Pol III*-Pol IV switching, we determined that a single cleft on the clamp was sufficient to support the switch, and that both the rim and cleft contacts were required. Results from genetic experiments support a role for the cleft and rim contacts in Pol IV function in vivo. Taken together, our findings challenge the toolbelt model and suggest instead that Pol IV contacts the rim of the clamp adjacent to the cleft that is bound by Pol III* before gaining control of the same cleft that is bound by Pol III*.mutagenesis ͉ toolbelt ͉ translesion DNA synthesis ͉ Pol IV ͉ DinB E ndogenous and exogenous agents continuously damage cellular DNA. As a result, faithful duplication of an organism's genetic information requires both high fidelity DNA polymerases (Pols), as well as a multitude of DNA repair and DNA damage tolerance mechanisms (reviewed in ref.

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

“…Previous efforts to reconstitute this model system for polymerase exchange have involved stalling Pol III on β at a primer terminus by nucleotide omission to synchronize a population of molecules and simulate a lesion-induced block (3,4). These studies were not able to resolve an exchange back to Pol III after Pol IV synthesis.…”

“…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).…”

“…Canonical clamps, such as the bacterial β and eukaryotic proliferating cell nuclear antigen (PCNA), are multimeric, with a binding cleft on each protomer. Biochemical experiments with bacterial (3,4) and eukaryotic (5) proteins have suggested that clamps can simultaneously bind multiple DNA polymerases during active DNA synthesis, serving as a molecular toolbelt (6). This multivalency may facilitate rapid polymerase exchange and lesion bypass.…”

“…Interactions of Pol IV with β and their implications for the toolbelt model have generated widespread interest (4,16). 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).…”

Essential roles for imuA ′- and imuB -encoded accessory factors in DnaE2-dependent mutagenesis in Mycobacterium tuberculosis

“…Notably, neither DnaE2 nor ImuA′ possesses an identifiable clamp-binding motif (1, 12). The β-clamp modulates the recruitment to the replication machinery of proteins involved in bacterial DNA replication and repair (13), binding replicative and specialist lesion bypass polymerases simultaneously to enable rapid interchange (14,15). The β-clamp-binding motif therefore suggested that ImuB might be crucial for DnaE2 function.…”