title: 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.
abstract: The E. coli umuDC gene products participate in two temporally distinct roles: UmuD2C acts in a DNA damage checkpoint control, while UmuD'2C, 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 (βQ61K, βS107L, βD150N, βG157S, βV170M, βE202K, βM204K and βP363S) 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, βD150N and βP363S 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.
authors.affiliation: Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York, United States of America; Witebsky Center for Microbial Pathogenesis & Immunology, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York, United States of America.
authors.forename: Vignesh M P
authors.initials: VM
authors.lastname: Babu
authors.affiliation: Department of Biochemistry, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York, United States of America; Witebsky Center for Microbial Pathogenesis & Immunology, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York, United States of America; Genetics, Genomics and Bioinformatics Program, School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, New York, United States of America.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Sutton
mesh_terms: D000220:Adaptation, Biological / Q000235:genetics; D000483:Alleles; D003080:Cold Temperature; D004249:DNA Damage; D004258:DNA Polymerase III / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D023061:Gene Order; D050296:Microbial Viability / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D010957:Plasmids / Q000235:genetics*; D011487:Protein Conformation
chemical_list: D029968:Escherichia coli Proteins; C101139:beta subunit, DNA polymerase III; D004258:DNA Polymerase III