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Recovery from vitamin B-12-induced unbalanced growth. The shortened cell cycle and the deoxyribonucleoside triphosphate pools.

ABSTRACT: The deoxyribonucleoside triphosphate pools are undetectable in vitamin B-12-deficient cells of Euglena gracillis, but appear rapidly after the replenishment with the vitamin. They reach a maximum size that is about 6 times that of normal exponentially growing cells, but decrease to almost zero as the cells divide. The pools expand again during the post-replenishment shortened cell cycle. However, the expansion takes place during rather than before the resumption of DNA synthesis. The maximum sizes reached are still larger than in normal cells. By using the protein-synthesis inhibitor cycloheximide and determining the pool size, we found that vitamin-deficient cells apparently accumulate a large amount of ribonucleoside triphosphate reductase apoenzyme, which lacks the vitamin B12 coenzyme. We showed that the production of the deoxyribonucleoside triphosphates is not closely coupled to DNA synthesis under our experimental conditions, and that the concentration of the deoxyribonucleoside triphosphate pools per unit of DNA synthesized is almost constant for all stages of growth examined.

SUBMITTER: Goetz GH

PROVIDER: S-EPMC1183941 | biostudies-other | 1978 Mar

REPOSITORIES: biostudies-other

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Deoxyribonucleoside triphosphate pools in vitamin B-12-deficient Euglenagracilis.

Project description:The size of the deoxyribonucleoside triphosphate pools of vitamin B-12-deficient cells of Euglena gracilis, and of vitamin B-12-deficient cells repleted with the vitamin, were measured. We found that the pools were very small, if they exist at all, in deficient cells but expand rapidly with the addition of the vitamin. The sizes of the pools decrease when DNA synthesis is completed, and are very small when the cells begin to divide.

| S-EPMC1163771 | biostudies-other

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Mechanisms of allosteric activation and inhibition of the deoxyribonucleoside triphosphate triphosphohydrolase from Enterococcus faecalis.

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Efficient Syntheses of Vitamin K Chain-Shortened Acid Metabolites.

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ITPA (inosine triphosphate pyrophosphatase): from surveillance of nucleotide pools to human disease and pharmacogenetics.

Project description:Cellular nucleotide pools are often contaminated by base analog nucleotides which interfere with a plethora of biological reactions, from DNA and RNA synthesis to cellular signaling. An evolutionarily conserved inosine triphosphate pyrophosphatase (ITPA) removes the non-canonical purine (d)NTPs inosine triphosphate and xanthosine triphosphate by hydrolyzing them into their monophosphate form and pyrophosphate. Mutations in the ITPA orthologs in model organisms lead to genetic instability and, in mice, to severe developmental anomalies. In humans there is genetic polymorphism in ITPA. One allele leads to a proline to threonine substitution at amino acid 32 and causes varying degrees of ITPA deficiency in tissues and plays a role in patients' response to drugs. Structural analysis of this mutant protein reveals that the protein is destabilized by the formation of a cavity in its hydrophobic core. The Pro32Thr allele is thought to cause the observed dominant negative effect because the resulting active enzyme monomer targets both homo- and heterodimers to degradation.

| S-EPMC3827912 | biostudies-literature

Phenotypic signatures arising from unbalanced bacterial growth.

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Synthesis of 4-Cyanoindole Nucleosides, 4-Cyanoindole-2'-Deoxyribonucleoside-5'-Triphosphate (4CIN-TP), and Enzymatic Incorporation of 4CIN-TP into DNA.

Project description:4-Cyanoindole-2'-deoxyribonucleoside (4CIN) is a fluorescent isomorphic nucleoside analogue with superior spectroscopic properties in terms of Stokes shift and quantum yield in comparison to the widely utilized isomorphic nucleoside analogue, 2-aminopurine-2'-deoxyribonucleoside (2APN). Notably, when inserted into single- or double-stranded DNA, 4CIN experiences substantially less in-strand fluorescence quenching compared to 2APN. Given the utility of these properties for a spectrum of research applications involving oligonucleotides and oligonucleotide-protein interactions (e.g., enzymatic processes, DNA hybridization, DNA damage), we envision that additional reagents based on 4-cyanoindole nucleosides may be widely utilized. This protocol expands on the previously published synthesis of 4CIN to include synthetic routes to both 4-cyanoindole-ribonucleoside (4CINr) and 4-cyanoindole-2'-deoxyribonucleoside-5'-triphosphate (4CIN-TP), as well as a method for the enzymatic incorporation of 4CIN-TP into DNA by a polymerase. These methods are anticipated to further enable the utilization of 4CIN in diverse applications involving DNA and RNA oligonucleotides. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Synthesis of 4-cyanoindole-2'-deoxyribonucleoside (4CIN) and 4CIN phosphoramidite 4 Basic Protocol 2: Synthesis of 4-cyanoindole-ribonucleoside (4CINr) Basic Protocol 3: Synthesis of 4-cyanoindole-2'-deoxyribonucleoside-5'-triphosphate (4CIN-TP) Basic Protocol 4: Steady state incorporation kinetics of 2AP-TP and 4CIN-TP by a DNA polymerase.

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