Search

The ATP-bound form of the Escherichia coli DnaA replication initiator protein remodels the chromosomal origin of replication, oriC, to load the replicative helicase. The primary mechanism for regulating the activity of DnaA involves the Hda and β clamp proteins, which act together to dramatically stimulate the intrinsic DNA-dependent ATPase activity of DnaA via a process termed Regulatory Inactivation of DnaA. In addition to hyperinitiation, strains lacking hda function also exhibit cold sensitive growth at 30°C. Strains impaired for the other regulators of initiation (i.e., Δ seqA or Δ datA) fail to exhibit cold sensitivity. The goal of this study was to gain insight into why loss of hda function impedes growth. We used a genetic approach to isolate 9 suppressors of Δ hda cold sensitivity, and characterized the mechanistic basis by which these suppressors alleviated Δ hda cold sensitivity. Taken together, our results provide strong support for the view that the fundamental defect associated with Δ hda is diminished levels of DNA precursors, particularly dGTP and dATP. We discuss possible mechanisms by which the suppressors identified here may regulate dNTP pool size, as well as similarities in phenotypes between the Δ hda strain and hda+ strains exposed to the ribonucleotide reductase inhibitor hydroxyurea. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Expression of the Escherichia coli dnaN-encoded β clamp at ≥10-fold higher than chromosomally expressed levels impedes growth by interfering with DNA replication. We hypothesized that the excess β clamp sequesters the replicative DNA polymerase III (Pol III) to inhibit replication. As a test of this hypothesis, we obtained eight mutant clamps with an inability to impede growth and measured their ability to stimulate Pol III replication in vitro. Compared with the wild-type clamp, seven of the mutants were defective, consistent with their elevated cellular levels failing to sequester Pol III. However, the βE202K mutant that bears a glutamic acid-to-lysine substitution at residue 202 displayed an increased affinity for Pol IIIa and Pol III core (Pol IIIαεθ), suggesting that it could still sequester Pol III effectively. Of interest, βE202K supported in vitro DNA replication by Pol II and Pol IV but was defective with Pol III. Genetic experiments indicated that the dnaNE202K strain remained proficient in DNA damage-induced mutagenesis but was induced modestly for SOS and displayed sensitivity to UV light and methyl methanesulfonate. These results correlate an impaired ability of the mutant βE202K clamp to support Pol III replication in vivo with its in vitro defect in DNA replication. Taken together, our results (i) support the model that sequestration of Pol III contributes to growth inhibition, (ii) argue for the existence of an additional mechanism that contributes to lethality, and (iii) suggest that physical and functional interactions of the β clamp with Pol III are more extensive than appreciated currently. [ABSTRACT FROM AUTHOR]

Various lethal stresses, including bactericidal antibiotics, can trigger the production of reactive oxygen species (ROS) that contribute to killing. Incomplete base excision repair (BER) of oxidized nucleotides, especially 8-oxo-dG, has been identified as a major component of ROS-induced lethality. However, the relative contributions of this pathway to death vary widely between stresses, due in part to poorly understood complex differences in the physiological changes caused by these stresses. To identify new lethal stresses that kill cells through this pathway, we screened an essential protein degradation library and found that depletion of either DapB or Dxr leads to cell death through incomplete BER; the contribution of this pathway to overall cell death is greater for DapB than for Dxr. Depletion of either protein generates oxidative stress, which increases incorporation of 8-oxo-dG into the genome. This oxidative stress is causally related to cell death, as plating on an antioxidant provided a protective effect. Moreover, incomplete BER was central to this cell death, as mutants lacking the key BER DNA glycosylases MutM and MutY were less susceptible, while overexpression of the nucleotide sanitizer MutT, which degrades 8-oxo-dGTP to prevent its incorporation, was protective. RNA sequencing of cells depleted of these proteins revealed widely different transcriptional responses to these stresses. Our discovery that oxidative stress-induced incomplete BER is highly dependent on the exact physiological changes that the cell experiences helps explain the past confusion that arose concerning the role of ROS in antibiotic lethality. IMPORTANCE Bacterial cell death is a poorly understood process. The generation of reactive oxygen species (ROS) is an apparently common response to challenges by a wide variety of lethal stresses, including bactericidal antibiotics. Incomplete BER of nucleotides damaged by these ROS, especially 8-oxo-dG, is a significant contributing factor to this lethality, but the levels of its contribution vary widely between different lethal stresses. A better understanding of the conditions that cause cells to die because of incomplete BER may lead to improved strategies for targeting this mode of death as an adjunct to antimicrobial therapy.

The product of the human C21orf57 (huYBEY) gene is predicted to be a homologue of the highly conserved YbeY proteins found in nearly all bacteria. We show that, like its bacterial and chloroplast counterparts, the HuYbeY protein is an RNase and that it retains sufficient function in common with bacterial YbeY proteins to partially suppress numerous aspects of the complex phenotype of an Escherichia coli ΔybeY mutant. Expression of HuYbeY in Saccharomyces cerevisiae, which lacks a YbeY homologue, results in a severe growth phenotype. This observation suggests that the function of HuYbeY in human cells is likely regulated through specific interactions with partner proteins similarly to the way YbeY is regulated in bacteria., (Copyright © 2017 Elsevier Inc. All rights reserved.)