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The  E. coli umuDC  gene products participate in two temporally distinct roles: UmuD 2 C acts in a DNA damage checkpoint control, while UmuD' 2 C, 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 β sliding clamp protein. Co-overexpression of the β clamp and Pol V severely blocked  E. coli  growth at 30°C. We previously used a genetic assay that was independent of the ability of β clamp to support  E. coli  viability to isolate 8 mutant clamp proteins (β Q61K , β S107L , β D150N , β G157S , β V170M , β E202K , β M204K  and β P363S ) that failed to block growth at 30°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, β D150N  and β P363S  were unable to support  E. coli  viability. The remaining 6 mutant clamps, each of which supported viability, were indistinguishable from β +  with respect to  umuDC  functions  in vivo . In light of these findings, we analyzed phenotypes of strains overexpressing either β clamp or Pol V alone. The strain overexpressing β + , but not those expressing mutant β clamps, displayed slowed growth irrespective of the incubation temperature. Moreover, growth of the Pol V-expressing strain was modestly slowed at 30°, but not 42°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°C.

plos one

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