Specific amino acid residues in the β sliding clamp establish a DNA polymerase usage hierarchy in Escherichia coli

Escherichia coli dnaN159 strains encode a mutant form of the β sliding clamp (β159), causing them to display altered DNA polymerase (pol) usage. In order to better understand mechanisms of pol selection/switching in E. coli, we have further characterized pol usage in the dnaN159 strain. The dnaN159 allele contains two amino acid substitutions: G66E (glycine-66 to glutamic acid) and G174A (glycine-174 to alanine). Our results indicated that the G174A substitution impaired interaction of the β clamp with the α catalytic subunit of pol III. In light of this finding, we designed two additional dnaN alleles. One of these dnaN alleles contained a G174A substitution (β-G174A), while the other contained D173A, G174A and H175A substitutions (β-173-175). Examination of strains bearing these different dnaN alleles indicated that each conferred a distinct UV sensitive phenotype that was dependent upon a unique combination of ΔpolB (pol II), ΔdinB (pol IV) and/or ΔumuDC (pol V) alleles. Taken together, these findings indicate that mutations in the β clamp differentially affect the functions of these three pols, and suggest that pol II, pol IV and pol V are capable of influencing each others' abilities to gain access to the replication fork. These findings are discussed in terms of a model whereby amino acid residues in the vicinity of those mutated in β159 (G66 and G174) help to define a DNA polymerase usage hierarchy in E. coli following UV irradiation.