Sliding Clamp–DNA Interactions Are Required for Viability and Contribute to DNA Polymerase Management in Escherichia coli

Sliding clamp proteins topologically encircle DNA and play vital roles in coordinating the actions of various DNA replication, repair, and damage tolerance proteins. At least three distinct surfaces of the Escherichia coli β clamp interact physically with the DNA that it topologically encircles. We utilized mutant β clamp proteins bearing G66E and G174A substitutions (β159), affecting the single-stranded DNA-binding region, or poly-Ala substitutions in place of residues 148-HQDVR-152 (β148–152), affecting the double-stranded DNA binding region, to determine the biological relevance of clamp–DNA interactions. As part of this work, we solved the X-ray crystal structure of β148–152, which verified that the poly-Ala substitutions failed to significantly alter the tertiary structure of the clamp. Based on functional assays, both β159 and β148–152 were impaired for loading and retention on a linear primed DNA in vitro. In the case of β148–152, this defect was not due to altered interactions with the DnaX clamp loader, but rather was the result of impaired β148–152–DNA interactions. Once loaded, β148–152 was proficient for DNA polymerase III (Pol III) replication in vitro. In contrast, β148–152 was severely impaired for Pol II and Pol IV replication and was similarly impaired for direct physical interactions with these Pols. Despite its ability to support Pol III replication in vitro, β148–152 was unable to support viability of E. coli. Nevertheless, physiological levels of β148–152 expressed from a plasmid efficiently complemented the temperature-sensitive growth phenotype of a strain expressing β159 (dnaN159), provided that Pol II and Pol IV were inactivated. Although this strain was impaired for Pol V-dependent mutagenesis, inactivation of Pol II and Pol IV restored the Pol V mutator phenotype. Taken together, these results support a model in which a sophisticated combination of competitive clamp–DNA, clamp–partner, and partner–DNA interactions serve to manage the actions of the different E. coli Pols in vivo.