Differential binding of <i>Escherichia coli</i> DNA polymerases to the β‐sliding clamp

Abstract

Summary Escherichia coli strains expressing the mutant β159‐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 β159 (G66E and G174A), β780 (G66E) and β781 (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 β clamp.