Degradation of pyrimidine ribonucleosides by Pseudomonas aeruginosa

Pyrimidine ribonucleoside degradation in the human pathogen Pseudomonas aeruginosa ATCC 15692 was investigated. Either uracil, cytosine, 5-methylcytosine, thymine, uridine or cytidine supported P. aeruginosa growth as a nitrogen source when glucose served as the carbon source. Using thin-layer chromatographic analysis, the enzymes nucleoside hydrolase and cytosine deaninase were shown to be active in ATCC 15692. Compared to (NH₄)₂SO₄-grown cells, nucleoside hydrolase activity in ATCC 15692 approximately doubled after growth on 5-methylcytosine as a nitrogen source while its cytosine deaminase activity increased several-fold after growth on the pyrimidine bases and ribonucleosides examined as nitrogen sources. Regulation at the level of protein synthesis by 5-methylcytosine was indicated for nucleoside hydrolase and cytosine deaminase in P. aeruginosa.     

Utilization of 5-Bromouracil by Thymineless Bacteria

Several thymineless Escherichia coli strains have been examined for their ability to replicate their deoxyribonucleic acid when bromouracil is substituted for thymine. The procedure we describe was used to identify a thymineless strain with characteristics relatively favorable to its use in bromouracil labeling experiments. In addition, mutants with an “absolute” thymine requirement could be easily distinguished from one with a “leaky” thymine requirement.     

Kinetics of Thymine Photodimerization in DNA

The kinetics of thymine photodimerization in E. coli DNA have been measured at various wavelengths of ultraviolet light. The initial quantum yield is not strongly dependent on wavelength. The ratio of thymine dimer to thymine in the photostationary state is much more dependent on wavelength. At the 235 mμ photosteady state 1.7 per cent of the thymine is present as dimer. This shifts to 6.5 per cent at 254 mμ and to 20 per cent of 275 mμ. While the change in position of the photosteady state with wavelength fails to fit a simple model, the data do indicate that not all thymines are capable of participation in dimer formation.     

Mechanisms of targeted frameshift mutations: Insertions arising during error-prone or SOS synthesis of DNA containing cis-syn cyclobutane thymine dimers

It is still unclear how frameshift mutations arise at cyclobutane pyrimidine dimers. The polymerase model is commonly used to explain the mechanisms of various mutations. An alternative polymerase-tautomer model was developed for UV-induced mutagenesis. A mechanism was proposed for targeted insertions caused by cis-syn cyclobutane thymine dimers. Targeted insertions are frameshift mutations due to addition of one or more nucleotides in a DNA sequence opposite to a lesion capable of stopping DNA synthesis. Among other factors, cyclobutane pyrimidine dimers can cause targeted insertions. UV irradiation can change the tautomeric form of DNA bases. Five rare tautomeric forms are possible for thymine, and they are stable when the thymine is a component of a cyclobutane dimer. A structural analysis showed that none of the canonical nucleotides can be added opposite to a specific rare thymine tautomer so that hydrogen bonds form between the two bases. A single nucleotide gap is consequently left in the corresponding site of the nascent strand when a specialized or modified DNA polymerase drives SOS or error-prone DNA synthesis on a template containing cis-syn cyclobutane thymine dimers with a base occurring in the rare tautomeric form. If the DNA composition is homogenous within the region, the end of the growing DNA strand may slip to form a complementary pair with the nucleotide adjacent to the dimer according to the Streisinger model, thus producing a loop. A targeted insertion is thereby generated to make the daughter strand longer. Targeted insertions were for the first time assumed to result from the cis-syn cyclobutane thymine dimers wherein one or both of the bases occur in the specific tautomeric form that does not allow the addition and hydrogen bonding of any canonical nucleotide in the opposite position. A model was developed to explain how targeted insertions of one or more nucleotides are caused by cis-syn cyclobutane thymine dimers. Thus, the polymerase-tautomer model can explain the nature and formation of targeted frameshift mutations in addition to hot and cold spots or targeted or untargeted nucleotide substitutions.     

The biosynthesis of natural and unnatural polyoxins by Streptomyces cacaoi

The biosynthesis of the pyrimidine moiety and the uronic acid moiety of the polyoxins and the formation of unnatural polyoxins has been studied in Streptomyces cacaoi. Experimental evidence is provided for the biosynthesis of thymine via a pathway that is independent of thymidylate synthetase. This new thymine pathway is based on two experimental approaches. First, two known inhibitors of DNA synthesis (1-formylisoquinoline thiosemicarbazide and 5-fluoro-2′-deoxyuridine), when added to polyoxin-producing cultures of S. cacaoi, inhibit the synthesis of TMP from exogenously supplied uracil but do not inhibit the synthesis of the thymine or hydroxymethyluracil in the polyoxin complex. Second, exogenously supplied thymine and hydroxymethyluracil are taken up by S. cacaoi but are not incorporated into the thymine or hydroxymethyluracil of the polyoxin complex. The thymine is incorporated into the DNA. The uracil in polyoxin L could be the parent pyrimidine chromophore with C-1 additions occurring at carbon-5 to form thymine and hydroxymethyluracil. Carbon-3 of serine but not the methyl group of methionine is a one-carbon source for the formation of the thymine and hydroxymethyluracil in the polyoxin complex.S. cacaoi can synthesize unnatural polyoxins, as evidenced by the incorporation of 5-fluoro, 5-bromo, and 6-azauracil into the polyoxins; 5-iodo-, 2-thio-, or 4-thiouracil is not a substrate. Two new polyoxin analogs synthesized and characterized when 5-fluorouracil is added to the cultures are 5-fluoropolyoxin L and 5-fluoropolyoxin M. There is a marked change in the molar ratio of the uracil:thymine:hydroxymethyluracil chromophores in the polyoxin complex following the incorporation of 5-fluoro-, 5-bromo-, or 6-azauracil. Apparently, the unnatural polyoxins inhibit the addition of the C-1 unit to carbon-5 of uracil in the polyoxin complex. Polyoxin L and polyoxin C do not inhibit Escherichia coli and Streptococcus faecalis, but 5-fluoropolyoxin L and 5-fluoropolyoxin C inhibit both these organisms. There is little or no difference in the inhibition of the fluorinated and natural polyoxins against leukemia L-1210 cells. The fluoro group on carbon-5 of the uracil ring does not affect the enzyme-inhibition complex with chitin synthetase since the inhibition constant of fluoropolyoxins L is the same as has been reported for polyoxins A, D, and L.The ¹⁴C-labeling pattern in the 5′-amino-5′-deoxy-d-allofuranosyluronic acid moiety of the polyoxins from ¹⁴C-labeled glucose, allose, and glycerol suggests that the formation of this unique C-6 uronic acid in the polyoxins does not proceed via the direct oxidation of either d-glucose or d-allose to the -onic or -uronic acids. Glucose is converted to two three-carbon trioses, followed by either (i) the oxidation of one of the trioses to a threecarbon acid and subsequent condensation with another three-carbon sugar to form the C-6 uronic or (ii) an 80:20 equilibrium of the two trioses followed by condensation to a hexose which is then oxidized to the C-6 uronic acid.     

Thymine dimer excision in UV-irradiated and 5-fluorouracil-postincubatedEscherichia coli

The effect of post-irradiation cultivation with 5-fluorouracil on the excision of thymine dimers following UV irradiation was examined inEscherichia coli 15 T-U-his-. It was found that an increase of the number of surviving cells caused by 60-min post-incubation with 5-fluorouracil is not accompanied by any more rapid and complete removal of thymine dimers from the damaged molecule of DNA     

Synthesis of small polynucleotide chains in thymine-depleted bacteria

Bacteria that are depleted of intracellular thymidine nucleotide pools incorporate [³H]thymine at full specific activity, allowing the detection of early intermediates in DNA replication. A short pulse of [³H]thymine is incorporated almost exclusively into very small DNA chains which, during further incorporation of thymine, are converted into larger chains and high molecular weight DNA. The synthesis of these small DNA chains depends on the products of dna genes B, E and G. Analysis of the DNA by gel filtration on Sepharose 2B revealed an abundance of extremely short DNA chains while the frequency of larger chains decreased exponentially with increasing size. This size distribution of small DNA chains suggests a mechanism of DNA replication in which larger chains (Okazaki pieces, Okazaki et al., 1968a) arise through joining of extremely short polynucleotide chains in a process resembling crystallization rather than unidirectional chain elongation.     

NADH-dependent O-deethylation of p-nitrophenetole with rabbit liver microsomes.

A previous paper in this series (C. K. Mathews, (1972) J. Biol. Chem.247, 7430) showed that deoxynucleoside triphosphate pools expand manyfold when DNA synthesis is blocked genetically in infection by bacteriophage T4. This paper describes a more detailed analysis of this phenomenon. The key approach involves labeling with thymine or thymidine under conditions of infection where both phage and host bear mutations that inactivate thymidylate synthetase. Principal findings include the following: (1) Nucleotides in the expanded pools are derived in roughly equal measure from breakdown of host cell DNA and from nucleotide synthesis de novo after infection. (2) Thymidine diphosphate pool expansion is comparable, in rate and extent, to thymidine triphosphate pool expansion, but thymidine monophosphate pools accumulate much less. (3) The rate of expansion of the total thymine nucleotide pool following temperature upshift in infection by a temperature-sensitive gene 45 mutant is approximately equal to the rate of thymine incorporation into DNA immediately preceding the upshift. (4) Similarly, when DNA synthesis is restored by a downshift, the total thymine nucleotide pool drains at a rate commensurate with that of thymine incorporation into DNA. (5) Under these latter conditions the dTTP pool begins to drain earlier than the dTDP pool, suggesting that dTTP is the more proximal DNA precursor in this system.     

Sources of thymidine and analogs fueling futile damage-repair cycles and ss-gap accumulation during thymine starvation in Escherichia coli

Thymine deprivation in thyA mutant E. coli causes thymineless death (TLD) and is the mode of action of popular antibacterial and anticancer drugs, yet the mechanisms of TLD are still unclear. TLD comprises three defined phases: resistance, rapid exponential death (RED) and survival, with the nature of the resistance phase and of the transition to the RED phase holding key to TLD pathology. We propose that a limited source of endogenous thymine maintains replication forks through the resistance phase. When this source ends, forks undergo futile break-repair cycle during the RED phase, eventually rendering the chromosome non-functional. Two obvious sources of the endogenous thymine are degradation of broken chromosomal DNA and recruitment of thymine from stable RNA. However, mutants that cannot degrade broken chromosomal DNA or lack ribo-thymine, instead of shortening the resistance phase, deepen the RED phase, meaning that only a small fraction of T-starved cells tap into these sources. Interestingly, the substantial chromosomal DNA accumulation during the resistance phase is negated during the RED phase, suggesting futile cycle of incorporation and excision of wrong nucleotides. We tested incorporation of dU or rU, finding some evidence for both, but DNA-dU incorporation accelerates TLD only when intracellular [dUTP] is increased by the dut mutation. In the dut ung mutant, with increased DNA-dU incorporation and no DNA-dU excision, replication is in fact rescued even without dT, but TLD still occurs, suggesting different mechanisms. Finally, we found that continuous DNA synthesis during thymine starvation makes chromosomal DNA increasingly single-stranded, and even the dut ung defect does not completely block this ss-gap accumulation. We propose that instability of single-strand gaps underlies the pathology of thymine starvation.     

Isomérisation des 5-bromo-5,6-dihydro-6-hydroxythymidines. Préparation des formes anomères de la thymidine et de la 1-(2-désoxy-D-érythro-pentofuranosyl)thymine

The trans 5-(R), 6-(R) and 5-(S), 6-(S) diastereoisomeric forms of 5-bromo-5,6-dihydro-6-hydroxythymidine were obtained by the action of bromine upon thymidine in aqueous solution. Treatment of these compounds with warm M hydrobromic acid both rearranges the sugar moiety and cleaves the glycosylamine bond; the yields of both processes were determined. Reduction of the halohydrins gave three isomeric compounds derived from thymidine : 1-(2-deoxy-α-D-erythro-pentofuranosyl)thymine, 1-(2-deoxy-β-D-erythro-pentopyranosyl)thymine and 1-(2-deoxy-α-D-erythro-pentopyranosyl)thymine. These isomerisations were also shown in the treatment of thymidine with hydrobromic acid, but, in the latter case, the process is less productive than in the former one. A mechanism for these reactions is discussed.     

Parallel RNA-strand recognition by 2′-amino-β-l-LNA

A short synthetic route to the first β-l-ribo configured locked nucleic acid (LNA), that is, 2′-amino-β-l-LNA thymine phosphoramidite 6, has been developed from bicyclic nucleoside 1. Incorporation of 2′-amino-β-l-LNA thymine monomers into α-DNA strands results in probes forming stable duplexes with complementary RNA in parallel orientation.     

A thin-layer chromatographic method for separation of thymidine and deoxyuridine nucleotides

A thin-layer chromatographic method for the separation of thymidine and deoxyuridine nucleotides and nucleosides is described. This procedure involves the following sequence of steps: (i) Ion-exchange thin-layer chromatography to afford separation into fractions of increasing degree of phosphorylation, (ii) conversion of each fraction into an equivalent mixture of thymine and uracil through the combined actions of alkaline phosphatase and thymidine phosphorylase, and (iii) partition thin-layer chromatographic separation of thymine and uracil. A key feature of the method is the specificity afforded by the second step which converts only thymidine and deoxyuridine nucleotides and nucleosides to the corresponding pyrimidine bases. An application of the method to the study of [³H]deoxyuridine metabolism in L1210 cells, as well as the effect of methotrexate on this metabolism is also described.     

Thymineless Death in Escherichia coli: Strain Specificity

Thymineless death of various ultraviolet (UV)-sensitive strains of Escherichia coli B and K-12 was investigated. It was found that E. coli B, B_s−12, K-12 rec-21, and possibly K-12 Lon⁻, all sensitive to UV, were also sensitive to thymine starvation. However, other UV-sensitive strains of E. coli were found to display the typical resistant-type kinetics of thymineless death. The correlation of these results with various other cellular processes suggested that the filament-forming ability of the bacteria might be involved in the mechanism of thymineless death. It was apparent from the present results that capacity for host-cell reactivation, recombination ability, thymine dimer excision, and probably induction of a defective prophage had little to do with determining sensitivity to thymine deprivation.     

Deoxythymidine sugars are not direct precursors of DNA-thymine.

A theoretical model for the kinetics of uptake of a putative precursor molecule into nucleotide pools and into replicating DNA has been developed. The relationship between the accumulation of radioactively labeled precursors in the pool and the appearance of radioactivity in DNA is then derived. Experiments have been carried out in bacteria to compare the uptake of radioactive thymine into deoxythymidine triphosphate, deoxythymidine diphosphate sugars, and DNA to test the suitability of either compound as the direct precursor of thymine in DNA. New one-dimensional, thin-layer chromatographic procedures were used to determine the specific activity of deoxythymidine triphosphate and deoxythymidine triphosphate and deoxythymidine diphosphate sugars in growing cultures of 32PO4-labeled Escherichia coli during pulse labeling with [3H]-thymine. A comparison of the experimental data with our theoretical model supports the hypothesis that deoxythymidine triphosphate, but not deoxythymidine sugar, is the direct precursor of thymine in normally replicating DNA in vivo.     

Isocratic high-performance liquid chromatographic method for studying the metabolism of blood plasma pyrimidine nucleosides and bases: concentration and radioactivity measurements

A rapid and efficient isocratic high-performance liquid chromatographic method for studying the metabolism of blood plasma cytosine, uracil, thymine, cytidine, deoxycytidine and uridine has been elaborated. For each compound this method can measure concentrations in the range 0.5–200 μM and determine radioactivity. All the pyrimidine compounds can be eluted in less than 18 min, and the total time elapsed between collection of the blood and completion of the analysis need to exceed 3 h. All measurements can be performed on 0.25-ml blood samples. Blood plasma pyrimidine concentrations were determined for the rat, the rabbit, the guinea pig, the dog and the healthy human. This method could be well applied to experimentation on small animals using radiolabelled pyrimidine derivatives, in order to study the metabolic pathways of nucleotides and nucleic acids. It could also be used to characterize certain illnesses or cases of toxicity created by a chemotherapy affecting the plasma level of pyrimidine bases on nucleosides.     

Preferential formation of (5S,6R)-thymine glycol for oligodeoxyribonucleotide synthesis and analysis of drug binding to thymine glycol-containing DNA

We previously reported the chemical synthesis of oligonucleotides containing thymine glycol, a major form of oxidative DNA damage. In the preparation of the phosphoramidite building block, the predominant product of the osmium tetroxide oxidation of protected thymidine was (5R,6S)-thymidine glycol. To obtain the building block of the other isomer, (5S,6R)-thymidine glycol, in an amount sufficient for oligonucleotide synthesis, the Sharpless asymmetric dihydroxylation (AD) reaction was examined. Although the reaction was very slow, (5S,6R)-thymidine glycol was obtained in preference to the (5R,6S) isomer. The ratio of (5S,6R)- and (5R,6S)-thymidine glycols was 2:1, and a trans isomer was also formed. When an ionic liquid, 1-butyl-3-methylimidazolium hexafluorophosphate, was used as a co-solvent, the reaction became faster, and the yield was improved without changing the preference. The phosphoramidite building block of (5S,6R)-thymidine glycol was prepared, and oligonucleotides containing 5S-thymine glycol were synthesized. One of the oligonucleotides was used to analyze the binding of distamycin A to thymine glycol-containing DNA by Circular dichroism (CD) spectroscopy and surface plasmon resonance (SPR) measurements. Distamycin A bound to a duplex containing either isomer of thymine glycol within the AATT target site, and its binding was observed even when the thymine glycol was placed opposite cytosine.     

Thymine lesions produced by ionizing radiation in double-stranded DNA

A DNA glycosylase which catalyzes the release of thymine residues damaged by ring saturation, fragmentation, or ring contraction from double-stranded DNA has been used to characterize such base derivatives in gamma-irradiated DNA. It is shown by chromatographic analysis that irradiation of DNA in neutral solution generates the ring-saturated forms cis-thymine glycol, trans-thymine glycol, and a monohydroxydihydrothymine, probably 6-hydroxy-5,6-dihydrothymine. The latter compound is only observed after irradiation under hypoxic conditions. The ring-contracted thymine derivative 5-hydroxy-5-methylhydantoin is also formed, and it is the major lesion after irradiation of DNA under O2. Ring-fragmented products such as methyltartronylurea were only generated in small quantities. Isolation and analysis of the DNA from gamma-irradiated human cells also revealed the formation of ring-saturated thymine derivatives, but 5-hydroxy-5-methylhydantoin was not found in this case.