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Structure of Escherichia coli dGTP triphosphohydrolase: a hexameric enzyme with DNA effector molecules.

ABSTRACT: The Escherichia coli dgt gene encodes a dGTP triphosphohydrolase whose detailed role still remains to be determined. Deletion of dgt creates a mutator phenotype, indicating that the dGTPase has a fidelity role, possibly by affecting the cellular dNTP pool. In the present study, we have investigated the structure of the Dgt protein at 3.1-Å resolution. One of the obtained structures revealed a protein hexamer that contained two molecules of single-stranded DNA. The presence of DNA caused significant conformational changes in the enzyme, including in the catalytic site of the enzyme. Dgt preparations lacking DNA were able to bind single-stranded DNA with high affinity (Kd ? 50 nM). DNA binding positively affected the activity of the enzyme: dGTPase activity displayed sigmoidal (cooperative) behavior without DNA but hyperbolic (Michaelis-Menten) kinetics in its presence, consistent with a specific lowering of the apparent Km for dGTP. A mutant Dgt enzyme was also created containing residue changes in the DNA binding cleft. This mutant enzyme, whereas still active, was incapable of DNA binding and could no longer be stimulated by addition of DNA. We also created an E. coli strain containing the mutant dgt gene on the chromosome replacing the wild-type gene. The mutant also displayed a mutator phenotype. Our results provide insight into the allosteric regulation of the enzyme and support a physiologically important role of DNA binding.

SUBMITTER: Singh D

PROVIDER: S-EPMC4400351 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Structure of Escherichia coli dGTP triphosphohydrolase: a hexameric enzyme with DNA effector molecules.

Singh Deepa D Gawel Damian D Itsko Mark M Hochkoeppler Alejandro A Krahn Juno M JM London Robert E RE Schaaper Roel M RM

The Journal of biological chemistry 20150218 16

The Escherichia coli dgt gene encodes a dGTP triphosphohydrolase whose detailed role still remains to be determined. Deletion of dgt creates a mutator phenotype, indicating that the dGTPase has a fidelity role, possibly by affecting the cellular dNTP pool. In the present study, we have investigated the structure of the Dgt protein at 3.1-Å resolution. One of the obtained structures revealed a protein hexamer that contained two molecules of single-stranded DNA. The presence of DNA caused signific ...[more]

PMID: 25694425

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Project description:dGTP starvation, a newly discovered phenomenon in which Escherichia coli cells are starved specifically for the DNA precursor dGTP, leads to impaired growth and, ultimately, cell death. Phenomenologically, it represents an example of nutritionally induced unbalanced growth: cell mass amplifies normally as dictated by the nutritional status of the medium, but DNA content growth is specifically impaired. The other known example of such a condition, thymineless death (TLD), involves starvation for the DNA precursor dTTP, which has been found to have important chemotherapeutic applications. Experimentally, dGTP starvation is induced by depriving an E. coligpt optA1 strain of its required purine source, hypoxanthine. In our studies of this phenomenon, we noted the emergence of a relatively high frequency of suppressor mutants that proved resistant to the treatment. To study such suppressors, we used next-generation sequencing on a collection of independently obtained mutants. A significant fraction was found to carry a defect in the PurR transcriptional repressor, controlling de novo purine biosynthesis, or in its downstream purEK operon. Thus, upregulation of de novo purine biosynthesis appears to be a major mode of overcoming the lethal effects of dGTP starvation. In addition, another large fraction of the suppressors contained a large tandem duplication of a 250- to 300-kb genomic region that included the purEK operon as well as the acrAB-encoded multidrug efflux system. Thus, the suppressive effects of the duplications could potentially involve beneficial effects of a number of genes/operons within the amplified regions.IMPORTANCE Concentrations of the four precursors for DNA synthesis (2'-deoxynucleoside-5'-triphosphates [dNTPs]) are critical for both the speed of DNA replication and its accuracy. Previously, we investigated consequences of dGTP starvation, where the DNA precursor dGTP was specifically reduced to a low level. Under this condition, E. coli cells continued cell growth but eventually developed a DNA replication defect, leading to cell death due to formation of unresolvable DNA structures. Nevertheless, dGTP-starved cultures eventually resumed growth due to the appearance of resistant mutants. Here, we used whole-genome DNA sequencing to identify the responsible suppressor mutations. We show that the majority of suppressors can circumvent death by upregulating purine de novo biosynthesis, leading to restoration of dGTP to acceptable levels.

Project description:UnlabelledOur laboratory recently discovered that Escherichia coli cells starved for the DNA precursor dGTP are killed efficiently (dGTP starvation) in a manner similar to that described for thymineless death (TLD). Conditions for specific dGTP starvation can be achieved by depriving an E. coli optA1 gpt strain of the purine nucleotide precursor hypoxanthine (Hx). To gain insight into the mechanisms underlying dGTP starvation, we conducted genome-wide gene expression analyses of actively growing optA1 gpt cells subjected to hypoxanthine deprivation for increasing periods. The data show that upon Hx withdrawal, the optA1 gpt strain displays a diminished ability to derepress the de novo purine biosynthesis genes, likely due to internal guanine accumulation. The impairment in fully inducing the purR regulon may be a contributing factor to the lethality of dGTP starvation. At later time points, and coinciding with cell lethality, strong induction of the SOS response was observed, supporting the concept of replication stress as a final cause of death. No evidence was observed in the starved cells for the participation of other stress responses, including the rpoS-mediated global stress response, reinforcing the lack of feedback of replication stress to the global metabolism of the cell. The genome-wide expression data also provide direct evidence for increased genome complexity during dGTP starvation, as a markedly increased gradient was observed for expression of genes located near the replication origin relative to those located toward the replication terminus.ImportanceControl of the supply of the building blocks (deoxynucleoside triphosphates [dNTPs]) for DNA replication is important for ensuring genome integrity and cell viability. When cells are starved specifically for one of the four dNTPs, dGTP, the process of DNA replication is disturbed in a manner that can lead to eventual death. In the present study, we investigated the transcriptional changes in the bacterium E. coli during dGTP starvation. The results show increasing DNA replication stress with an increased time of starvation, as evidenced by induction of the bacterial SOS system, as well as a notable lack of induction of other stress responses that could have saved the cells from cell death by slowing down cell growth.

Project description:Our laboratory has recently discovered that E. coli cells starved for the DNA precursor dGTP are killed efficiently (dGTP starvation) in a manner similar to that described for Thymineless Death (TLD). Conditions for specific dGTP starvation can be achieved by depriving an E. coli optA1 gpt strain of the purine nucleotide precursor hypoxanthine (Hx). To gain insight into the mechanisms underlying dGTP starvation, we conducted genome-wide gene expression analyses on actively growing optA1 gpt strains subjected to hypoxanthine deprivation for increasing periods of time. The data show that, upon Hx withdrawal, the optA1 gpt strain displays a diminished ability to de-repress the de novo purine biosynthesis genes, and this is likely due to internal guanine accumulation. The impairment to fully induce the purR regulon may be a contributing factor to the lethality of dGTP starvation. At later time points, and coinciding with cell lethality, strong induction of the SOS is observed, supporting the concept of replication stress as a final cause of death. No evidence was observed for the participation of other stress responses, including the rpoS-mediated global stress response in the starved cells, and reinforcing the lack of feedback of replication stress into the global metabolism of the cell. The genome-wide expression data also provide direct evidence for increased genome complexity during dGTP starvation, as a markedly increased gradient is observed for expression of genes located nearby the replication origin relative to those located towards the replication terminus. The present study was aimed at understanding the metabolic changes in dGTP-starved cells. To do so, we designed two experiments. The first experiment was aimed at investigating the transcriptional consequences of loss of the Hx purine source in each of the four strains (wt, optA1, gpt, and optA1 gpt). Here, hypoxanthine was withdrawn from actively growing cells starting at OD 0.1, and periodic 2-fold dilutions were applied to keep to OD of the growing cultures below or only slightly above 0.20 (low-dilution protocol). Samples were taken for microarray analysis at 15, 30, 45, 60, and 120 min. At each time point RNA samples were taken and analyzed for gene expression changes using the Affymetrix GeneChip E. coli Genome 2.0 Array. This experiment was conducted three times, independently. A second experiment was aimed at following the transcriptional changes in the optA1 gpt strain during the entire 6-hour time course including the filamentation and cell death stage. Here, we used a slightly different dilution protocol (high-dilution protocol), in which the strain was initially highly diluted (OD630 = 0.01) and further diluted at strategic points to prevent the OD to rise above 0.1. These conditions optimize the cell death phenomenon. This experiment was also conducted three times independently.

Dataset Information

Structure of Escherichia coli dGTP triphosphohydrolase: a hexameric enzyme with DNA effector molecules.

Publications

Structure of Escherichia coli dGTP triphosphohydrolase: a hexameric enzyme with DNA effector molecules.

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