Thymine Metabolism and Thymineless Death in Prokaryotes and Eukaryotes

Ahmad, Shamim; Kirk, Sandra H.; Eisenstark, A.

doi:10.1146/annurev.micro.52.1.591

Cited by 222 publications

(243 citation statements)

References 202 publications

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“…To make thymidine incorporation in bacterial cells linear during prolonged incubations, [ 3 H]thymidine labeling is usually done in thyA mutants growing in the presence of limited concentrations of thymine or thymidine (1, 6, 12). Unfortunately, thymine limitation exposes thyA mutant cells to the poorly understood phenomenon of "thymineless death," associated with fragmentation of the newly synthesized DNA (58). Indeed, thy-deficient B. subtilis cells are known to incorporate uracil in the newly replicated DNA, subsequent excision of which produces artifactual "LMW replication intermediates" (59).…”

Section: Resultsmentioning

confidence: 99%

The Replication Intermediates in Escherichia coli Are Not the Product of DNA Processing or Uracil Excision

Amado

Kuzminov

2006

Journal of Biological Chemistry

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The current model of DNA replication in Escherichia coli postulates continuous synthesis of the leading strand, based on in vitro experiments with purified enzymes. In contrast, in vivo experiments in E. coli and its bacteriophages, in which maturation of replication intermediates was blocked, report discontinuous DNA synthesis of both the lagging and the leading strands. To address this discrepancy, we analyzed nascent DNA species from ThyA ؉ E. coli cells replicating their DNA in ligase-deficient conditions to block maturation of replication intermediates. We report here that the bulk of the newly synthesized DNA isolated from ligase-deficient cells have a length between 0.3 and 3 kb, with a minor fraction being longer that 11 kb but shorter than the chromosome. The low molecular weight of the replication intermediates is unchanged by blocking linear DNA processing with a recBCD mutation or by blocking uracil excision with an ung mutation. These results are consistent with the previously proposed discontinuous replication of the leading strand in E. coli.Before the polarity of DNA strand extension was established, both DNA strands at the replication fork were thought to be synthesized continuously, being extended by two distinct DNA polymerases with opposite polarities (1, 2) (Fig. 1A). The finding that DNA polymerases extend DNA strands only in the 5Ј to 3Ј direction (reviewed in Ref.3) made it clear that at least the DNA strand synthesized in the direction opposite to the one of the replication fork movement has to be replicated in pieces and then assembled (maturated) into a full-length molecule (4 -6). Since this strand could not be synthesized continuously, its synthesis would lag behind the apparently continuous synthesis of the opposite DNA strand, which would thus lead the replication fork progress. The two strands were eventually called the "lagging" and the "leading" strands, respectively, and the hypothetical difference in their synthesis formed the basis of the semidiscontinuous model of DNA replication (Fig. 1B) (7). However, the semidiscontinuous paradigm is based on in vitro results and ignores a large body of in vivo data, which is reviewed below.Okazaki and colleagues (1, 8) used pulses of [ 3 H]thymidine to characterize the newly synthesized DNA of wild type Escherichia coli and Bacillus subtilis strains as well as E. coli bacteriophage T4. The cells were grown at 20°C to slow down the maturation of replication intermediates into the full-length DNA molecules, and short (10 -60-s) pulses of label were used (1, 8 -11). Using alkaline (denaturing) sucrose gradients to separate replication intermediates from their template strands, Okazaki and colleagues observed that the newly synthesized DNA after 2-10 s of labeling migrated mostly as low molecular weight (LMW) 2 species, with a mean length between 1 and 2 kb (1, 8). A chase with nonradioactive thymidine shifted the label into high molecular weight (HMW) DNA, proving that the LMW species are true replication intermediates (5, 8). These experi...

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Section: Resultsmentioning

confidence: 99%

The Replication Intermediates in Escherichia coli Are Not the Product of DNA Processing or Uracil Excision

Amado

Kuzminov

2006

Journal of Biological Chemistry

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“…coli cells are capable of producing de novo sufficient quantities of nucleoside precursors for DNA and RNA synthesis; however, externally supplied nucleosides can also be utilized via the salvage pathway and may even serve as a sole carbon or nitrogen source [51]. Interestingly, it was reported that wild type E. coli cells did not normally utilize exogenous thymine, presumably because they lacked an adequate 2′-deoxyribose-1-phosphate pool [52,53]. Exogenous thymidine was reported to be poorly incorporated into E. coli DNA because thymidine phosphorylase (deoA), which is inducible, degraded the thymidine to thymine [52].…”

Section: Effect Of Nucleoside Supplementation On Uracil Accumulationmentioning

confidence: 99%

Quantitative determination of uracil residues in Escherichia coli DNA: Contribution of ung, dug, and dut genes to uracil avoidance

et al. 2006

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The steady-state levels of uracil residues in DNA extracted from strains of Escherichia coli were measured and the influence of defects in the genes for uracil-DNA glycosylase (ung), doublestrand uracil-DNA glycosylase (dug), and dUTP pyrophosphatase (dut) on uracil accumulation was determined. A sensitive method, called the Ung-ARP assay, was developed that utilized E. coli Ung, T4pdg, and the Aldehyde Reactive Probe reagent to label a basic sites resulting from uracil excision with biotin. The limit of detection was one uracil residue per million DNA nucleotides (U/10 6 nt). Uracil levels in the genomic DNA of E. coli JM105 (ung + dug + ) were at the limit of detection, as were those of an isogenic dug mutant, regardless of growth phase. Inactivation of ung in JM105 resulted in 31 ± 2.6 U/10 6 nt during early log growth and 19 ± 1.7 U/ 10 6 nt in saturated phase. An ung dug double mutant (CY11) accumulated 33 ± 2.9 U/10 6 nt and 23 ± 1.8 U/10 6 nt during early log and saturated phase growth, respectively. When cultures of CY11 were supplemented with 20 ng/ml of 5-fluoro-2′-deoxyuridine, uracil levels in early log phase growth DNA rose to 125 ± 1.7 U/10 6 nt. Deoxyuridine supplementation reduced the amount of uracil in CY11 DNA, but uridine did not. Levels of uracil in DNA extracted from CJ236 (dut-1 ung-1) were determined to be 3000-8000 U/10 6 nt as measured by the Ung-ARP assay, twodimensional thin-layer chromatography of metabolically-labeled 32 P DNA, and LC/MS of uracil and thymine deoxynucleosides. DNA sequencing revealed that the sole molecular defect in the CJ236 dUTP pyrophosphatase gene was a C→T transition mutation that resulted in a Thr24Ile amino acid change.

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“…Mitomycin C and trimethoprim are both well-known inducers of the SOS response (Lewin & Amyes, 1991;Ahmad et al, 1998). Treatment of Ent.…”

Section: Morphological Effects Of Trimethoprimmentioning

confidence: 99%

Rapid antibiotic sensitivity testing and trimethoprim-mediated filamentation of clinical isolates of the Enterobacteriaceae assayed on a novel porous culture support

Ingham

Ende

Wever

et al. 2006

Journal of Medical Microbiology

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A porous inorganic material (Anopore) was employed as a microbial culture and microcolony imaging support. Rapid Anopore-based antibiotic sensitivity testing (AST) methods were developed to assess the growth of clinical isolates, with the primary focus on testing the response of the Enterobacteriaceae to trimethoprim, but with the method supporting a wider applicability in terms of strains and antibiotics. It was possible to detect the growth of Enterobacter aerogenes after 25 min culture and to distinguish a trimethoprim-sensitive from a trimethoprim-resistant strain with 40 min incubation. MIC 90 determinations were made on Anopore; these were in good agreement with the results from the Vitek 2 and E-test methods. The Anopore method correctly identified sensitive (40/40) and resistant (17/17) strains of the Enterobacteriaceae and other Gram-negative rods within only 2-3 h culture. Additionally, a trimethoprim-resistant subpopulation (10 % of population) could be detected by microcolony formation within 2 h, and a smaller subpopulation (1 %) after 3?5 h. These results suggest that this is a viable approach for the rapid AST of purified strains, and that it may be able to deal with mixed populations. The microscopic examination of microcolonies during AST is an advantage of this method which revealed additional information. Filamentation triggered by trimethoprim was discovered in many species of the Enterobacteriaceae for which this phenomenon has not previously been reported. Filamentation was characterized by heterogeneity in terms of cell length, and also uneven nucleic acid distribution and flattening of damaged cells. The development and application of Anopore-based AST within clinical diagnostics is discussed.

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Thymine Metabolism and Thymineless Death in Prokaryotes and Eukaryotes

Cited by 222 publications

References 202 publications

The Replication Intermediates in Escherichia coli Are Not the Product of DNA Processing or Uracil Excision

The Replication Intermediates in Escherichia coli Are Not the Product of DNA Processing or Uracil Excision

Quantitative determination of uracil residues in Escherichia coli DNA: Contribution of ung, dug, and dut genes to uracil avoidance

Rapid antibiotic sensitivity testing and trimethoprim-mediated filamentation of clinical isolates of the Enterobacteriaceae assayed on a novel porous culture support

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