SUMMARY
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 E. 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 strand (ss) DNA-binding region, or poly-Ala substitutions in place of residues 148-HQDVR-152 (β148–152), affecting the double strand (ds) DNA binding region, in order 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 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.
SummaryEscherichia coli strains expressing the mutant b159-sliding clamp protein (containing both a G66E and a G174A substitution) are temperature sensitive for growth and display altered DNA polymerase (pol) usage. We selected for suppressors of the dnaN159 allele able to grow at 42°C, and identified four intragenic suppressor alleles. One of these alleles (dnaN780) contained only the G66E substitution, while a second (dnaN781) contained only the G174A substitution. Genetic characterization of isogenic E. coli strains expressing these alleles indicated that certain phenotypes were dependent upon only the G174A substitution, while others required both the G66E and G174A substitutions. In order to understand the individual contributions of the G66E and the G174A substitution to the dnaN159 phenotypes, we utilized biochemical approaches to characterize the purified mutant b159 (G66E and G174A), b780 (G66E) and b781 (G174A) clamp proteins. The G66E substitution conferred a more pronounced effect on pol IV replication than it did pol II or pol III, while the G174A substitution conferred a greater effect on pol III and pol IV than it did pol II. Taken together, these findings indicate that pol II, pol III and pol IV interact with distinct, albeit overlapping surfaces of the b clamp.
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