The Escherichia coli dnaN159 allele encodes a mutant form of the ␤-sliding clamp (␤159) that is impaired for interaction with the replicative DNA polymerase (Pol), Pol III. In addition, strains bearing the dnaN159 allele require functional Pol I for viability. We have utilized a combination of genetic and biochemical approaches to characterize the role(s) played by Pol I in the dnaN159 strain. Our findings indicate that elevated levels of Pol I partially suppress the temperature-sensitive growth phenotype of the dnaN159 strain.In addition, we demonstrate that the ␤ clamp stimulates the processivity of Pol I in vitro and that ␤159 is impaired for this activity. The reduced ability of ␤159 to stimulate Pol I in vitro correlates with our finding that single-stranded DNA (ssDNA) gap repair is impaired in the dnaN159 strain. Taken together, these results suggest that (i) the ␤ clamp-Pol I interaction may be important for proper Pol I function in vivo and (ii) in the absence of Pol I, ssDNA gaps may persist in the dnaN159 strain, leading to lethality of the dnaN159 ⌬polA strain.The Escherichia coli dnaN-encoded ␤-sliding clamp functions as a homodimer and is "loaded" onto primed DNA by the multisubunit DnaX clamp loader complex (5, 21). Once loaded, the ␤ clamp slides freely along duplex DNA and functions to tether a variety of different proteins involved in replication and repair to the DNA (4,6,7,9,19,23,24,26,28,(41)(42)(43). The dnaN159 allele encodes a mutant form of the ␤-sliding clamp (␤159) that bears two amino acid substitutions: G66E (glycine at position 66 replaced with glutamic acid) and G174A (14,35,38). The G174A substitution appears to impair the ability of a hydrophobic cleft in ␤ to interact with the eubacterial clamp-binding motif (QL[S/D]LF) that is conserved in most partner proteins reported to interact with the ␤ clamp (8, 38). E. coli strains bearing the dnaN159 allele display temperature-sensitive growth (14,35,38) and altered DNA polymerase (Pol) usage (29,(38)(39)(40). These phenotypes appear to result, at least in part, from impaired interactions of the mutant ␤159 clamp protein with the replicative DNA polymerase, Pol III (38). As a result of the impaired ␤159-Pol III interaction, E. coli strains bearing the dnaN159 allele display increased utilization of the three SOS-regulated DNA polymerases, Pol II (polB), Pol IV (dinB), and Pol V (umuDC) (29,(38)(39)(40). In contrast to these three Pols, which under certain conditions impede growth of the dnaN159 strain (29, 39, 40), presumably by impairing DNA replication, the catalytic DNA polymerase activity of Pol I (polA) is essential for viability of the dnaN159 strain (38). Importantly, Pol I function is not required by the isogenic dnaN ϩ strain (38), indicating that Pol I plays one or more essential roles in the dnaN159 strain. Since Pol I is a multifunctional protein that participates in DNA replication, as well as numerous DNA repair pathways, several possibilities exist, including Okazaki fragment maturation (21, 31) and single-stranded DNA ...