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1.
DNA Repair (Amst) ; 123: 103462, 2023 03.
Article in English | MEDLINE | ID: mdl-36738688

ABSTRACT

Mutation is a phenomenon inescapable for all life-forms, including bacteria. While bacterial mutation rates are generally low due to the operation of error-avoidance systems, sometimes they are elevated by many orders of magnitude. Such a state, known as a hypermutable state, can result from exposure to stress or to harmful environments. Studies of bacterial mutation frequencies and analysis of the precise types of mutations can provide insights into the mechanisms by which mutations occur and the possible involvement of error-avoidance pathways. Several approaches have been used for this, like reporter assays involving non-essential genes or mutation accumulation over multiple generations. However, these approaches give an indirect estimation, and a more direct approach for determining mutations is desirable. With the recent development of a DNA sequencing technique known as Duplex Sequencing, it is possible to detect rare variants in a population at a frequency of 1 in 107 base pairs or less. Here, we have applied Duplex Sequencing to study spontaneous mutations in E. coli. We also investigated the production of replication errors by using a mismatch-repair defective (mutL) strain as well as oxidative-stress associated mutations using a mutT-defective strain. For DNA from a wild-type strain we obtained mutant frequencies in the range of 10-7 to 10-8 depending on the specific base-pair substitution, but we argue that these mutants merely represent a background of the system, rather than mutations that occurred in vivo. In contrast, bona-fide in vivo mutations were identified for DNA from both the mutL and mutT strains, as indicated by specific increases in base substitutions that are fully consistent with their established in vivo roles. Notably, the data reproduce the specific context effects of in vivo mutations as well as the leading vs. lagging strand bias among DNA replication errors.


Subject(s)
Escherichia coli Proteins , Escherichia coli , Escherichia coli/genetics , Mutation , Sequence Analysis, DNA , DNA Replication , DNA Repair , DNA, Bacterial/genetics , Pyrophosphatases/genetics , Escherichia coli Proteins/genetics
2.
Proc Natl Acad Sci U S A ; 119(37): e2123092119, 2022 09 13.
Article in English | MEDLINE | ID: mdl-36067314

ABSTRACT

Levels of the cellular dNTPs, the direct precursors for DNA synthesis, are important for DNA replication fidelity, cell cycle control, and resistance against viruses. Escherichia coli encodes a dGTPase (2'-deoxyguanosine-5'-triphosphate [dGTP] triphosphohydrolase [dGTPase]; dgt gene, Dgt) that establishes the normal dGTP level required for accurate DNA replication but also plays a role in protecting E. coli against bacteriophage T7 infection by limiting the dGTP required for viral DNA replication. T7 counteracts Dgt using an inhibitor, the gene 1.2 product (Gp1.2). This interaction is a useful model system for studying the ongoing evolutionary virus/host "arms race." We determined the structure of Gp1.2 by NMR spectroscopy and solved high-resolution cryo-electron microscopy structures of the Dgt-Gp1.2 complex also including either dGTP substrate or GTP coinhibitor bound in the active site. These structures reveal the mechanism by which Gp1.2 inhibits Dgt and indicate that Gp1.2 preferentially binds the GTP-bound form of Dgt. Biochemical assays reveal that the two inhibitors use different modes of inhibition and bind to Dgt in combination to yield enhanced inhibition. We thus propose an in vivo inhibition model wherein the Dgt-Gp1.2 complex equilibrates with GTP to fully inactivate Dgt, limiting dGTP hydrolysis and preserving the dGTP pool for viral DNA replication.


Subject(s)
Bacteriophage T7 , Escherichia coli Proteins , Escherichia coli , GTP Phosphohydrolases , Guanosine Triphosphate , Viral Proteins , Bacteriophage T7/physiology , Cryoelectron Microscopy , DNA Replication , DNA, Viral/metabolism , Escherichia coli/enzymology , Escherichia coli/virology , Escherichia coli Proteins/chemistry , GTP Phosphohydrolases/metabolism , Guanosine Triphosphate/metabolism , Protein Conformation , Viral Proteins/chemistry , Virus Replication
3.
J Biol Chem ; 298(7): 102073, 2022 07.
Article in English | MEDLINE | ID: mdl-35643313

ABSTRACT

Deoxynucleoside triphosphate (dNTP) triphosphohydrolases (dNTPases) are important enzymes that may perform multiple functions in the cell, including regulating the dNTP pools and contributing to innate immunity against viruses. Among the homologs that are best studied are human sterile alpha motif and HD domain-containing protein 1 (SAMHD1), a tetrameric dNTPase, and the hexameric Escherichia coli dGTPase; however, it is unclear whether these are representative of all dNTPases given their wide distribution throughout life. Here, we investigated a hexameric homolog from the marine bacterium Leeuwenhoekiella blandensis, revealing that it is a dGTPase that is subject to allosteric activation by dATP, specifically. Allosteric regulation mediated solely by dATP represents a novel regulatory feature among dNTPases that may facilitate maintenance of cellular dNTP pools in L. blandensis. We present high-resolution X-ray crystallographic structures (1.80-2.26 Å) in catalytically important conformations as well as cryo-EM structures (2.1-2.7 Å) of the enzyme bound to dGTP and dATP ligands. The structures, the highest resolution cryo-EM structures of any SAMHD1-like dNTPase to date, reveal an intact metal-binding site with the dGTP substrate coordinated to three metal ions. These structural and biochemical data yield insights into the catalytic mechanism and support a conserved catalytic mechanism for the tetrameric and hexameric dNTPase homologs. We conclude that the allosteric activation by dATP appears to rely on structural connectivity between the allosteric and active sites, as opposed to the changes in oligomeric state upon ligand binding used by SAMHD1.


Subject(s)
Monomeric GTP-Binding Proteins , Allosteric Regulation/physiology , Escherichia coli/metabolism , Flavobacteriaceae , Humans , Models, Molecular , Monomeric GTP-Binding Proteins/metabolism , SAM Domain and HD Domain-Containing Protein 1/metabolism
4.
Nat Commun ; 12(1): 1957, 2021 03 30.
Article in English | MEDLINE | ID: mdl-33785757

ABSTRACT

Tomographic reconstruction of cryopreserved specimens imaged in an electron microscope followed by extraction and averaging of sub-volumes has been successfully used to derive atomic models of macromolecules in their biological environment. Eliminating biochemical isolation steps required by other techniques, this method opens up the cell to in-situ structural studies. However, the need to compensate for errors in targeting introduced during mechanical navigation of the specimen significantly slows down tomographic data collection thus limiting its practical value. Here, we introduce protocols for tilt-series acquisition and processing that accelerate data collection speed by up to an order of magnitude and improve map resolution compared to existing approaches. We achieve this by using beam-image shift to multiply the number of areas imaged at each stage position, by integrating geometrical constraints during imaging to achieve high precision targeting, and by performing per-tilt astigmatic CTF estimation and data-driven exposure weighting to improve final map resolution. We validated our beam image-shift electron cryo-tomography (BISECT) approach by determining the structure of a low molecular weight target (~300 kDa) at 3.6 Å resolution where density for individual side chains is clearly resolved.


Subject(s)
Cryoelectron Microscopy/methods , Electron Microscope Tomography/methods , Image Processing, Computer-Assisted/methods , Tomography, X-Ray Computed/methods , Algorithms , Imaging, Three-Dimensional/methods , Macromolecular Substances/chemistry , Macromolecular Substances/ultrastructure , Particle Size , Reproducibility of Results
5.
Microbiol Resour Announc ; 9(10)2020 Mar 05.
Article in English | MEDLINE | ID: mdl-32139577

ABSTRACT

Escherichia coli BL21-AI is a commercially available strain possessing a phage T7-based protein-expression system. A combination of tight regulation and high yield makes it widely used for high-level expression of toxic recombinant proteins. Here, we present the complete genome sequence of BL21-AI and provide insights on its genome.

6.
Sci Adv ; 5(9): eaaw3915, 2019 09.
Article in English | MEDLINE | ID: mdl-31535021

ABSTRACT

A recent article in Science Advances described the striking discovery that the commensal Staphylococcus epidermidis strain MO34 displays antimicrobial and antitumor activities by producing a small molecule, identified as the nucleobase analog 6-N-hydroxylaminopurine (6-HAP). However, in contradiction to the literature, the authors claimed that 6-HAP is nonmutagenic and proposed that the toxic effect of 6-HAP results from its ability to inhibit, in its base form, DNA synthesis. To resolve the discrepancy, we proved by genetic experiments with bacteria and yeast that extracts of MO34 do contain a mutagenic compound whose effects are identical to chemically synthesized 6-HAP. The MO34 extract induced the same mutation spectrum as authentic 6-HAP. Notably, the toxic and mutagenic effects of both synthetic and MO34-derived 6-HAP depended on conversion to the corresponding nucleotide. The nucleobase 6-HAP does not inhibit DNA synthesis in vitro, and we conclude that 6-HAP exerts its biological activity when incorporated into DNA.


Subject(s)
Neoplasms , Staphylococcus epidermidis , Adenine , Humans , Mutagenesis , Saccharomyces cerevisiae
7.
DNA Repair (Amst) ; 83: 102643, 2019 11.
Article in English | MEDLINE | ID: mdl-31324532

ABSTRACT

DNA Pol III holoenzyme (HE) is the major DNA replicase of Escherichia coli. It is a highly accurate enzyme responsible for simultaneously replicating the leading- and lagging DNA strands. Interestingly, the fidelity of replication for the two DNA strands is unequal, with a higher accuracy for lagging-strand replication. We have previously proposed this higher lagging-strand fidelity results from the more dissociative character of the lagging-strand polymerase. In support of this hypothesis, an E. coli mutant carrying a catalytic DNA polymerase subunit (DnaE915) characterized by decreased processivity yielded an antimutator phenotype (higher fidelity). The present work was undertaken to gain deeper insight into the factors that influence the fidelity of chromosomal DNA replication in E. coli. We used three different dnaE alleles (dnaE915, dnaE911, and dnaE941) that had previously been isolated as antimutators. We confirmed that each of the three dnaE alleles produced significant antimutator effects, but in addition showed that these antimutator effects proved largest for the normally less accurate leading strand. Additionally, in the presence of error-prone DNA polymerases, each of the three dnaE antimutator strains turned into mutators. The combined observations are fully supportive of our model in which the dissociative character of the DNA polymerase is an important determinant of in vivo replication fidelity. In this model, increased dissociation from terminal mismatches (i.e., potential mutations) leads to removal of the mismatches (antimutator effect), but in the presence of error-prone (or translesion) DNA polymerases the abandoned terminal mismatches become targets for error-prone extension (mutator effect). We also propose that these dnaE alleles are promising tools for studying polymerase exchanges at the replication fork.


Subject(s)
Alleles , DNA Polymerase III/genetics , DNA Replication , Escherichia coli/genetics , Mutation , DNA Polymerase beta/metabolism , Phenotype
8.
Proc Natl Acad Sci U S A ; 115(16): 4212-4217, 2018 04 17.
Article in English | MEDLINE | ID: mdl-29610333

ABSTRACT

The fidelity of DNA replication is a critical factor in the rate at which cells incur mutations. Due to the antiparallel orientation of the two chromosomal DNA strands, one strand (leading strand) is replicated in a mostly processive manner, while the other (lagging strand) is synthesized in short sections called Okazaki fragments. A fundamental question that remains to be answered is whether the two strands are copied with the same intrinsic fidelity. In most experimental systems, this question is difficult to answer, as the replication complex contains a different DNA polymerase for each strand, such as, for example, DNA polymerases δ and ε in eukaryotes. Here we have investigated this question in the bacterium Escherichia coli, in which the replicase (DNA polymerase III holoenzyme) contains two copies of the same polymerase (Pol III, the dnaE gene product), and hence the two strands are copied by the same polymerase. Our in vivo mutagenesis data indicate that the two DNA strands are not copied with the same accuracy, and that, remarkably, the lagging strand has the highest fidelity. We postulate that this effect results from the greater dissociative character of the lagging-strand polymerase, which provides additional options for error removal. Our conclusion is strongly supported by results with dnaE antimutator polymerases characterized by increased dissociation rates.


Subject(s)
DNA Polymerase III/metabolism , DNA Replication , Mutagenesis , Chromosomes, Bacterial/genetics , DNA/metabolism , DNA Polymerase III/genetics , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Escherichia coli Proteins/genetics , Lac Operon , Lac Repressors/genetics , Mutation Rate
9.
J Bacteriol ; 199(12)2017 06 15.
Article in English | MEDLINE | ID: mdl-28373271

ABSTRACT

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.


Subject(s)
Deoxyguanine Nucleotides/deficiency , Deoxyguanine Nucleotides/metabolism , Escherichia coli/growth & development , Escherichia coli/metabolism , Suppression, Genetic , Biosynthetic Pathways/genetics , Carrier Proteins/genetics , DNA Mutational Analysis , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Gene Duplication , High-Throughput Nucleotide Sequencing , Purines/biosynthesis , Repressor Proteins/genetics
10.
Mol Microbiol ; 104(3): 377-399, 2017 May.
Article in English | MEDLINE | ID: mdl-28130843

ABSTRACT

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 ß 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., ΔseqA or ΔdatA) 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 Δhda cold sensitivity, and characterized the mechanistic basis by which these suppressors alleviated Δhda cold sensitivity. Taken together, our results provide strong support for the view that the fundamental defect associated with Δhda 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 Δhda strain and hda+ strains exposed to the ribonucleotide reductase inhibitor hydroxyurea.


Subject(s)
Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Escherichia coli/genetics , Ribonucleoside Diphosphate Reductase/genetics , Ribonucleoside Diphosphate Reductase/metabolism , Adenosine Triphosphatases/metabolism , Alleles , Cold Temperature , DNA Helicases/genetics , DNA Helicases/metabolism , DNA Replication , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , DNA-Binding Proteins/metabolism , Deoxyadenine Nucleotides/genetics , Deoxyadenine Nucleotides/metabolism , Escherichia coli/enzymology , Escherichia coli/growth & development , Trans-Activators/genetics , Trans-Activators/metabolism
11.
J Bacteriol ; 198(11): 1631-44, 2016 06 01.
Article in English | MEDLINE | ID: mdl-27002130

ABSTRACT

UNLABELLED: Our 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. IMPORTANCE: Control 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.


Subject(s)
Deoxyguanine Nucleotides/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Transcriptome , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial/physiology , Protein Array Analysis
12.
Mutat Res ; 784-785: 16-24, 2016.
Article in English | MEDLINE | ID: mdl-26789486

ABSTRACT

Cells lacking deoxycytidine deaminase (DCD) have been shown to have imbalances in the normal dNTP pools that lead to multiple phenotypes, including increased mutagenesis, increased sensitivity to oxidizing agents, and to a number of antibiotics. In particular, there is an increased dCTP pool, often accompanied by a decreased dTTP pool. In the work presented here, we show that double mutants of Escherichia coli lacking both DCD and NDK (nucleoside diphosphate kinase) have even more extreme imbalances of dNTPs than mutants lacking only one or the other of these enzymes. In particular, the dCTP pool rises to very high levels, exceeding even the cellular ATP level by several-fold. This increased level of dCTP, coupled with more modest changes in other dNTPs, results in exceptionally high mutation levels. The high mutation levels are attenuated by the addition of thymidine. The results corroborate the critical importance of controlling DNA precursor levels for promoting genome stability. We also show that the addition of certain exogenous nucleosides can influence replication errors in DCD-proficient strains that are deficient in mismatch repair.


Subject(s)
Cytidine Deaminase/genetics , Escherichia coli/genetics , Mutation , Nucleoside-Diphosphate Kinase/genetics , Cytidine Deaminase/metabolism , DNA-Directed RNA Polymerases , Deoxyribonucleotides/genetics , Deoxyribonucleotides/metabolism , Escherichia coli/drug effects , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Mutation Rate , Nucleoside-Diphosphate Kinase/metabolism , Thymidine/pharmacology
13.
Anal Biochem ; 496: 43-9, 2016 Mar 01.
Article in English | MEDLINE | ID: mdl-26723493

ABSTRACT

We describe a continuous, spectrophotometric, enzyme-coupled assay useful to monitor reactions catalyzed by nucleoside triphosphohydrolases. In particular, using Escherichia coli deoxynucleoside triphosphohydrolase (Dgt), which hydrolyzes dGTP to deoxyguanosine and tripolyphosphate (PPPi) as the enzyme to be tested, we devised a procedure relying on purine nucleoside phosphorylase (PNPase) and xanthine oxidase (XOD) as the auxiliary enzymes. The deoxyguanosine released by Dgt can indeed be conveniently subjected to phosphorolysis by PNPase, yielding deoxyribose-1-phosphate and guanine, which in turn can be oxidized to 8-oxoguanine by XOD. By this means, it was possible to continuously detect Dgt activity at 297 nm, at which wavelength the difference between the molar extinction coefficients of 8-oxoguanine (8000 M(-1) cm(-1)) and guanine (1090 M(-1) cm(-1)) is maximal. The initial velocities of Dgt-catalyzed reactions were then determined in parallel with the enzyme-coupled assay and with a discontinuous high-performance liquid chromatography (HPLC) method able to selectively detect deoxyguanosine. Under appropriate conditions of excess auxiliary enzymes, the activities determined with our continuous enzyme-coupled assay were quantitatively comparable to those observed with the HPLC method. Moreover, the enzyme-coupled assay proved to be more sensitive than the chromatographic procedure, permitting reliable detection of Dgt activity at low dGTP substrate concentrations.


Subject(s)
Nucleoside-Triphosphatase/analysis , Spectrophotometry, Ultraviolet/methods , Alkaline Phosphatase/analysis , Chromatography, High Pressure Liquid , Escherichia coli/enzymology , Purine-Nucleoside Phosphorylase/analysis , Xanthine Oxidase/analysis
14.
Nucleic Acids Res ; 43(8): 4109-20, 2015 Apr 30.
Article in English | MEDLINE | ID: mdl-25824947

ABSTRACT

The Escherichia coli SOS system is a well-established model for the cellular response to DNA damage. Control of SOS depends largely on the RecA protein. When RecA is activated by single-stranded DNA in the presence of a nucleotide triphosphate cofactor, it mediates cleavage of the LexA repressor, leading to expression of the 30(+)-member SOS regulon. RecA activation generally requires the introduction of DNA damage. However, certain recA mutants, like recA730, bypass this requirement and display constitutive SOS expression as well as a spontaneous (SOS) mutator effect. Presently, we investigated the possible interaction between SOS and the cellular deoxynucleoside triphosphate (dNTP) pools. We found that dNTP pool changes caused by deficiencies in the ndk or dcd genes, encoding nucleoside diphosphate kinase and dCTP deaminase, respectively, had a strongly suppressive effect on constitutive SOS expression in recA730 strains. The suppression of the recA730 mutator effect was alleviated in a lexA-deficient background. Overall, the findings suggest a model in which the dNTP alterations in the ndk and dcd strains interfere with the activation of RecA, thereby preventing LexA cleavage and SOS induction.


Subject(s)
Deoxyribonucleotides/metabolism , Escherichia coli/drug effects , SOS Response, Genetics , Suppression, Genetic , Bacterial Proteins/genetics , DNA-Directed DNA Polymerase/metabolism , Drug Resistance, Bacterial/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Mutagenesis , Mutation , Nucleoside-Diphosphate Kinase/genetics , Nucleotide Deaminases/genetics , Rec A Recombinases/genetics , Regulon , Rifampin/pharmacology , Serine Endopeptidases/genetics
15.
J Biol Chem ; 290(16): 10418-29, 2015 Apr 17.
Article in English | MEDLINE | ID: mdl-25694425

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-Šresolution. 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.


Subject(s)
DNA, Bacterial/chemistry , Deoxyguanine Nucleotides/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/enzymology , Gene Expression Regulation, Bacterial , Phosphoric Monoester Hydrolases/chemistry , Allosteric Regulation , Catalytic Domain , Chromosomes, Bacterial/chemistry , Chromosomes, Bacterial/metabolism , Crystallography, X-Ray , DNA, Bacterial/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Deoxyguanine Nucleotides/metabolism , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Kinetics , Models, Molecular , Mutation , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/metabolism , Protein Multimerization , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
16.
PLoS Genet ; 10(5): e1004310, 2014 May.
Article in English | MEDLINE | ID: mdl-24810600

ABSTRACT

Starvation of cells for the DNA building block dTTP is strikingly lethal (thymineless death, TLD), and this effect is observed in all organisms. The phenomenon, discovered some 60 years ago, is widely used to kill cells in anticancer therapies, but many questions regarding the precise underlying mechanisms have remained. Here, we show for the first time that starvation for the DNA precursor dGTP can kill E. coli cells in a manner sharing many features with TLD. dGTP starvation is accomplished by combining up-regulation of a cellular dGTPase with a deficiency of the guanine salvage enzyme guanine-(hypoxanthine)-phosphoribosyltransferase. These cells, when grown in medium without an exogenous purine source like hypoxanthine or adenine, display a specific collapse of the dGTP pool, slow-down of chromosomal replication, the generation of multi-branched nucleoids, induction of the SOS system, and cell death. We conclude that starvation for a single DNA building block is sufficient to bring about cell death.


Subject(s)
Deoxyguanine Nucleotides/metabolism , Escherichia coli/metabolism , Thymine/metabolism , DNA, Bacterial/biosynthesis , Escherichia coli/genetics , Escherichia coli/growth & development , SOS Response, Genetics
17.
Mutat Res ; 759: 22-8, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24269257

ABSTRACT

The fidelity with which organisms replicate their chromosomal DNA is of considerable interest. Detailed studies in the bacterium Escherichia coli have indicated that the fidelity of leading- and lagging-strand DNA replication is not the same, based on experiments in which the orientation of certain mutational targets on the chromosome was inverted relative to the movement of the replication fork: different mutation rates for several base-pair substitutions were observed depending on this orientation. While these experiments are indicative of differential replication fidelity in the two strands, a conclusion whether leading or lagging strand is the more accurate depends on knowledge of the primary mispairing error responsible for the base substitutions in question. A broad analysis of in vitro base-pairing preferences of DNA polymerases led us to propose that lagging-strand is the more accurate strand. In the present work, we present more direct in vivo evidence in support of this proposal. We determine the orientation dependence of mutant frequencies in ndk and dcd strains, which carry defined dNTP pool alterations. As these pool alterations lead to predictable effects on the array of possible mispairing errors, they mark the strands in which the observed errors occur. The combined results support the proposed higher accuracy of lagging-strand replication in E. coli.


Subject(s)
DNA Replication , Deoxyribonucleotides/metabolism , Escherichia coli/genetics , Deoxyadenine Nucleotides/metabolism , Deoxycytosine Nucleotides/metabolism , Deoxyguanine Nucleotides/metabolism , Lac Operon , Thymine Nucleotides/metabolism
18.
Mutat Res ; 770: 79-84, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25771873

ABSTRACT

The lacI gene of Escherichia coli has been a highly useful target for studies of mutagenesis, particularly for analysis of the specificity (spectrum) of mutations generated under a variety of conditions and in various genetic backgrounds. The gene encodes the repressor of the lac operon, and lacI-defective mutants displaying constitutive expression of the operon are readily selected. DNA sequencing of the lacI mutants has often been confined to the N-terminal region of the protein, as it presents a conveniently short target with a high density of detectably mutable sites. Mutants in this region are easily selected due to their dominance in a genetic complementation test (lacI(d) mutants). A potential complication in these studies is that constitutive expression of lac may also arise due to mutations in the lac operator (lacO mutants). Under some conditions, for example when analyzing spontaneous mutations, lacO mutants can comprise a very high fraction of the constitutive mutants due to a strong base-substitution hotspot in the lac operator. Such mutational hot spots diminish the return of the sequencing effort and do not yield significant new information. For this reason, a procedure to eliminate the lacO mutants prior to DNA sequencing is desirable. Here, we report a simple method that allows screening out of lacO mutants. This method is based on the lack of resistance of lacO mutants to kanamycin under conditions when the kan gene is expressed from a plasmid under control of the lac promoter-operator (lacPO). We show data validating the new approach with sets of known lacI(d) and lacO mutants, and further apply it to the generation of a new collection of spontaneous mutations, where lacO mutants have historically been a significant contributor.


Subject(s)
Escherichia coli Proteins/genetics , Escherichia coli/genetics , Lac Operon/genetics , Lac Repressors/genetics , Mutagenesis , DNA Mutational Analysis , Genes, Bacterial , Genetic Complementation Test , Organisms, Genetically Modified , Plasmids/genetics , Transduction, Genetic
19.
Proc Natl Acad Sci U S A ; 110(46): 18596-601, 2013 Nov 12.
Article in English | MEDLINE | ID: mdl-24167285

ABSTRACT

The enzyme ribonucleotide reductase (RNR) plays a critical role in the production of deoxynucleoside-5'-triphosphates (dNTPs), the building blocks for DNA synthesis and replication. The levels of the cellular dNTPs are tightly controlled, in large part through allosteric control of RNR. One important reason for controlling the dNTPs relates to their ability to affect the fidelity of DNA replication and, hence, the cellular mutation rate. We have previously isolated a set of mutants of Escherichia coli RNR that are characterized by altered dNTP pools and increased mutation rates (mutator mutants). Here, we show that one particular set of RNR mutants, carrying alterations at the enzyme's allosteric specificity site, is characterized by relatively modest dNTP pool deviations but exceptionally strong mutator phenotypes, when measured in a mutational forward assay (>1,000-fold increases). We provide evidence indicating that this high mutability is due to a saturation of the DNA mismatch repair system, leading to hypermutability and error catastrophe. The results indicate that, surprisingly, even modest deviations of the cellular dNTP pools, particularly when the pool deviations promote particular types of replication errors, can have dramatic consequences for mutation rates.


Subject(s)
DNA Mismatch Repair/genetics , DNA Replication/genetics , Deoxyribonucleotides/metabolism , Escherichia coli/enzymology , Models, Molecular , Mutation Rate , Ribonucleotide Reductases/genetics , Base Sequence , Deoxyribonucleotides/genetics , Escherichia coli/genetics , Molecular Sequence Data , Mutation/genetics , Ribonucleotide Reductases/chemistry , Sequence Analysis, DNA
20.
mBio ; 4(6): e00661-13, 2013 Oct 29.
Article in English | MEDLINE | ID: mdl-24169576

ABSTRACT

UNLABELLED: The base analogs 6-N-hydroxylaminopurine (HAP) and 2-amino-HAP (AHAP) are potent mutagens in bacteria and eukaryotic organisms. Previously, we demonstrated that a defect in the Escherichia coli ycbX gene, encoding a molybdenum cofactor-dependent oxidoreductase, dramatically enhances sensitivity to the toxic and mutagenic action of these agents. In the present study, we describe the discovery and properties of a novel suppressor locus, yjcD, that strongly reduces the HAP sensitivity of the ycbX strain. Suppressor effects are also observed for other purine analogs, like AHAP, 6-mercaptopurine, 6-thioguanine, and 2-aminopurine. In contrast, the yjcD defect did not affect the sensitivity to the pyrimidine analog 5-fluorouracil. Homology searches have predicted that yjcD encodes a putative permease of the NCS2 family of nucleobase transporters. We further investigated the effects of inactivation of all other members of the NCS2 family, XanQ, XanP, PurP, UacT, UraA, RutG, YgfQ, YicO, and YbbY, and of the NCS1 family nucleobase permeases CodB and YbbW. None of these other defects significantly affected sensitivity to either HAP or AHAP. The combined data strongly suggest that YjcD is the primary importer for modified purine bases. We also present data showing that this protein may, in fact, also be a principal permease involved in transport of the normal purines guanine, hypoxanthine, and/or xanthine. IMPORTANCE: Nucleotide metabolism is a critical aspect of the overall metabolism of the cell, as it is central to the core processes of RNA and DNA synthesis. At the same time, nucleotide metabolism can be subverted by analogs of the normal DNA or RNA bases, leading to highly toxic and mutagenic effects. Thus, understanding how cells process both normal and modified bases is of fundamental importance. This work describes a novel suppressor of the toxicity of certain modified purine bases in the bacterium Escherichia coli. This suppressor encodes a putative high-affinity nucleobase transporter that mediates the import of the modified purine bases. It is also a likely candidate for the long-sought high-affinity importer for the normal purines, like guanine and hypoxanthine.


Subject(s)
Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/toxicity , Escherichia coli/drug effects , Escherichia coli/enzymology , Membrane Transport Proteins/metabolism , Purines/metabolism , Purines/toxicity , Escherichia coli/genetics , Membrane Transport Proteins/genetics , Mutation
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