A dnaN Plasmid Shuffle Strain for Rapid In Vivo Analysis of Mutant Escherichia coli β Clamps Provides Insight Into the Role of Clamp in umuDC-Mediated Cold Sensitivity

The E. coli umuDC gene products participate in two temporally distinct roles: UmuD(2)C acts in a DNA damage checkpoint control, while UmuD'C-2, also known as DNA polymerase V (Pol V), catalyzes replication past DNA lesions via a process termed translesion DNA synthesis. These different roles of the umuDC gene products are managed in part by the dnaN-encoded beta sliding clamp protein. Co-overexpression of the beta clamp and Pol V severely blocked E. coli growth at 30 degrees C. We previously used a genetic assay that was independent of the ability of beta clamp to support E. coli viability to isolate 8 mutant clamp proteins (beta(Q61K), beta(S107L), beta(D150N), beta(G157S), beta(V170M), beta(E202K), beta(M204K) and beta(P363S)) that failed to block growth at 30 degrees C when co-overexpressed with Pol V. It was unknown whether these mutant clamps were capable of supporting E. coli viability and normal umuDC functions in vivo. The goals of this study were to answer these questions. To this end, we developed a novel dnaN plasmid shuffle assay. Using this assay, beta(D150N) and beta(P363S) were unable to support E. coli viability. The remaining 6 mutant clamps, each of which supported viability, were indistinguishable from beta(+) with respect to umuDC functions in vivo. In light of these findings, we analyzed phenotypes of strains overexpressing either beta clamp or Pol V alone. The strain overexpressing beta(+), but not those expressing mutant beta clamps, displayed slowed growth irrespective of the incubation temperature. Moreover, growth of the Pol V-expressing strain was modestly slowed at 30 degrees, but not 42 degrees C. Taken together, these results suggest the mutant clamps were identified due to their inability to slow growth rather than an inability to interact with Pol V. They further suggest that cold sensitivity is due, at least in part, to the combination of their individual effects on growth at 30 degrees C.