The effect of bisulfite on the dehalogenation of 5-chloro-, 5-bromo-, and 5-iodouracil

The 5-chloro-, bromo-, and iodo-analogs of uracil are dehalogenated in the presence of sodium bisulfite to yield 5,6 dihydrouracil-6-sulfonate as the final product. Under similar conditions, 5-fluorouracil adds bisulfite to yield 5-fluoro-5,6 dihydrouracil-6-sulfonate but is not dehalogenated. Ultraviolet absorption spectra of 5-bromouracil and 5-iodouracil reacting under pseudo first-order conditions with bisulfite indicate that dehalogenation proceeds via a pathway which has 5-halo-5,6-dihydrouracil-6-sulfonate and uracil as intermediates. In the case of 5-chlorouracil, the rate of bisulfite attack on the 6-position of the chlorouracil ring system is very slow relative to the rate of bisulfite addition to uracil. Hence, although dechlorination does occur, ultraviolet absorption spectra of reaction mixtures containing bisulfite and 5-chlorouracil do not reveal the uracil absorption peak observed with both 5-iodouracil and 5-bromouracil. Fluorine and proton nmr spectra indicate that bisulfite addition to 5-fluorouracil is stereoselective as is the case of bisulfite addition to uracil.     

The effect of thiols on the dehalogenation of 5-iodo and 5-bromouracil

The 5-iodo- and 5-bromo- analogs of uracil are dehalogenated in the presence of both cysteine and 2-mercaptoethanol to yield uracil. Presumably, the reaction involves the initial addition of the thiol group across the 5,6 double bond of the halopyrimidine to yield the corresponding 5-halo, 5,6-dihydrouracil-6-thioether which then dehalogenates to yield uracil. Under comparable conditions, cysteine causes more rapid dehalogenation of both halouracils than does 2-mercaptoethanol.Thiol containing compounds catalyze hydrogen-deuterium exchange at carbon five of uracil (1–3) and have been implicated as having a catalytic effect in the deamination of cytosine (4,5). Presumably, these reactions involve the reversible nucleophilic addition of the thiol group across the 5,6 double bond of the pyrimidine to yield the corresponding 5,6 dihydropyrimidine with a substituted thioether group on carbon six. This pathway is supported by comparable reactions involving the addition of bisulfite to the pyrimidine ring system (6–10). Different from the bisulfite addition compounds, the thioether containing dihydropyrimidine adducts have not been isolated and characterized; however, 5′-deoxy-5′,6-epithio-5,6-dihydro-2′,3′-0-isopropylideneuridine resulting from the intramolecular attack of the 5′ thiol group on carbon six of the uracil ring system of 5′-deoxy-5′-thio-2′,3′-0-isopropylideneuridine has been isolated and characterized (11).In a recent communication, we reported that bisulfite buffer systems catalyze the dehalogenation of 5-iodo-, 5-bromo-, and 5-chlorouracil (12). The object of this work is to demonstrate that cysteine and 2-mercaptoethanol, sulfur nucleophiles with more physiological importance than bisulfite, also cause halopyrimidine dehalogenation under nearly physiological conditions of temperature and pH.     

The dehalogenation of halocytosines by bisulfite buffers

Both 5-bromo- and 5-iodocytosine are rapidly dehalogenated in dilute bisulfite buffers to yield cytosine. With 5-bromocytosine, but not with 5-iodocytosine, extrapolation of semilogarithmic plots of extent reaction versus time indicates the bisulfite buffer concentration-dependent formation of an intermediate which subsequently reacts to control the rate of 5-bromocytosine dehalogenation. The disappearance of both halocytosines has a second-order dependence on bisulfite buffer concentration. Both imidazole and acetate buffers catalyze the reaction of 5-iodocytosine, but not that of 5-bromocytosine, with bisulfite. In the case of acetate buffer catalysis of the reaction of 5-iodocytosine with bisulfite, the dependence of the observed rate constants changes from first order to zero order as a function of increasing buffer concentration. The observed rate constants for 5-bromocytosine dehalogenation increase, reach a maximum at about 4.5, and then decrease as a function of pH. Iodometric titration of sulfite utilization coupled with spectrophotometric analysis of pyrimidine reactants and products indicates that 1 mole of sulfite is consumed per mole of halocytosine dehalogenated. The spectrophotometrically determined pK_a values for the conjugate acids of 5-bromo- and 5-iodocytosine at 25°C and ionic strength 1.0 M are 3.25 and 3.56, respectively. These results are discussed in terms of a multistep reaction pathway which is analogous to the bisulfite-catalyzed dehalogenation of the 5-halouracils.     

The addition of bisulfite to 5-fluorouracil. Evidence for a change in rate determining step

The kinetics of bisulfite addition to 5-fluorouracil were studied as a function of increasing concentrations of potential general acids. Values of $\text{k}{}_{\mathrm{obsd}}\text{[}\text{SO}{}_3{}^{\mathrm{=}}\text{]}$ measured at 25°C and ionic strength 1.0 M increased linearly and then became invariant with increasing concentrations of either HSO₃^? or (OHCH₂CH₂)₂N⁺C(CH₂OH)₃ HCl (BisTris⁺HCl). A small kinetic hydrogen-deuterium isotope effect ($\text{k}{}_{\mathrm{HS}}\text{k}{}_{\mathrm{DS}}\text{= 1.10}$) was observed for the general acid catalysed portion of the addition reaction. The kinetics of bisulfite elimination from 5-fluoro-5,6-dihydrouracil-6-sulfonate were studied in ethanolamine buffers. As previously observed with 1,3-dimethyl-5,6-dihydrouracil-6-sulfonate, this reaction is subject to general base catalysis and exhibits a large kinetic hydrogen-deuterium isotope effect (). The kinetic results for the addition reaction are consistent with a multistep reaction pathway involving the initial formation of an oxyanion sulfite addition intermediate (II) which subsequently adds a proton and undergoes tautomerization to yield the final 5-fluoro-5,6-dihydrouracil-6-sulfonate product. Thus the elimination of bisulfite from 5-fluoro-5,6-dihydrouracil-6-sulfonate probably proceeds by an ElcB mechanism which involves, at relatively low concentrations of general base, rate determining general base catalyzed proton abstraction from carbon 5 to yield intermediate II followed by the rapid elimination of sulfite to yield 5-fluorouracil. These results may be related to both the enzymatically catalyzed dehalogenation of bromoand iodouracil and the methylation of deoxyuridylate by thymidylate synthetase.     

The dehalogenation of halouracils by hydroxylamine buffers.

At room temperature, hydroxylamine dehalogenates 5-Br-and 5-I-uracil. 5-Cl-uracil reacts to a much less extent. Reaction with 5-F-uracil yields the 6-hydroxyamino-adduct as a product. Kinetics monitored spectrally indicate that dehalogenation involves the formation of a 5-halo-6-hydroxyamino-5, 6-dihydrouracil intermediate which then slowly dehalogenates. 5-Bromo-6-methoxy-5,6-dihydrothymine, a model for the above intermediate, also dehalogenates yielding thymine as a product.Hydroxylamine (NH₂OH), a mutagenic agent (1,2) reacts with pyrimidine rings promoting such reactions as the formation of 5,6-dihydro-N⁴-hydroxy-6-hydroxyaminocytosine from cytosine (3,4) and both urea and isoxazoles from uracil derivatives (2,5,6). It is believed to be unreactive toward 5-substituted uracil derivatives (2,5,6) but has been reported to cause the dehalogenation of 5-bromouracil derivatives yielding Br^? and uracil as products (2,7,8). The object of this report is to demonstrate the generality of NH₂OH addition to the 5-halouracils with the subsequent dehalogenation of both 5-Br-and 5-I-uracil; reactions which appear to proceed via pathways similar to bisulfite buffer mediated halouracil dehalogenation (9–13). A preliminary report of this work has appeared (14).     

The dehalogenation of the 5-halo-5,6-dihydrouracils: Uracil formation from 5-iodo- and 5-bromo-5,6-dihydrouracil

The elimination of halide ion from either 5-bromo- or 5-iodo-5,6-dihydrouracil to yield uracil is a slow reaction which, in the case of 5-iodo-5,6-dihydrouracil, is 400 times slower than the enzymatic release of ¹²⁵I^? from 5-[¹²⁵I]iodouracil. The elimination of HBr from 5-bromo-5,6-dihydrouracil is subject to general base catalysis by tris(hydroxymethyl)aminomethane (k₂^{Tris base} = 11 × 10^?4M^?1 min^?1, 37°C, ionic strength 1.0 M). At pH values near and above physiological, both the bromo- and iododihydropyrimidines are subject to hydrolysis of the dihydropyrimidine ring, a reaction which parallels halide elimination to yield uracil. The resulting 2-halo-3-ureidopropionate then cyclizes via intramolecular attack of the ureido oxygen atom to yield halide ion and 2-amino-2-oxazoline-5-carboxylic acid as final products. In dilute hydroxide ion, the kinetics of 5-bromo-5,6-dihydrouracil hydrolysis (25°C, ionic strength 1.0 M) show a change in rate-determining step as a function of increasing hydroxide ion concentration, a result which, as in the case of 5,6-dihydrouracil, can be explained in terms of the formation of a tetrahedral addition intermediate. The data are discussed relative to enzymatically catalyzed halopyrimidine dehalogenation.     

Reaction of 5-Carboxypyrimidine Derivatives with Sodium Bisulfite

5-Carboxyuracil derivatives were shown to react with aqueous sodium bisulfite in mild condition resulting in facile decarboxylation to give corresponding 5-decarboxy-5,6-dihydrouracil-6-sulfonates and uracils in good yield. The former compounds were quantitatively transformed to the latter in alkaline condition. Mechanistic feature of this reaction was discussed, which implied the initial nucleophilic addition of bisulfite across the 5,6-double bond. 5-Carboxycytosine was also shown to react similarly, however, accompanied by hydrolytic deamination.     

Transformation of Polyoxins D,E, and F with Sodium Bisulfite

Polyoxins D, E, and F which possess 5-carboxyuracil as the nucleobase were reacted selectively with sodium bisulfite at pH 4.0 resulting in facile decarboxylation to afford corresponding 5,6-dihydrouracil-6-sulfonates and uracil type polyoxins (polyoxins L, M, and K) in good yield. The former compounds were also converted to the latter almost quantitatively with mild alkali treatment. Biological activities of the transformed compounds were described.     

Co-operative mutagenic actions of bisulfite and nitrogen nucleophiles.

The combined effect of bisulfite and a nitrogen nucleophile, i.e. semicarbazide, methoxyamine or hydroxylamine, to chemically modify cytosine and to cause mutation and inactivation of bacteriophage lambda was investigated. A rapid transamination of cytidine with each of the amines took place in the presence of bisulfite, and the reaction product was solely the N(4)-transaminated 5,6-dihydrocytidine-6-sulfonate. Modifications of cytidine with bisulfite alone and with the nitrogen nucleophile alone were much slower reactions than those using a combination of bisulfite and the nucleophile. Whereas the product of the modification with the bisulfite/semicarbazide, 5,6-dihydro-4-semicarbazido-2-ketopyrimidine ribofuranoside-6-sulfonate, is convertible to 4-semicarbazido-2-ketopyrimidine ribofuranoside by treatment with a phosphate buffer, the products of the modification with the bisulfite/methoxyamine and with the bisulfite/hydroxylamine, i.e. 4-methoxy-5,6-dihydrocytidine-6-sulfonate and 4-hydroxy-5,6-dihydrocytidine-6-sulfonate, were stable in phosphate buffer.Inactivation and the “clear” mutation of bacteriophage lambda were observed when the phage was treated with sodium bisulfite in the presence of semicarbazide, methoxyamine or hydroxylamine. Under the conditions used, only very small increases in the mutation frequency were obtained by treatment of the phage with bisulfite alone or with the base alone. It was concluded that the residues, 5,6-dihydro-4-semicarbazido-2-ketopyrimidine-6-sulfonate, 4-methoxy-5, 6-dihydrocytosine-6-sulfonate and 4-hydroxy-5,6-dihydrocytosine-6-sulfonate in DNA are the causes of the mutation.When phage that had been inactivated by the semicarbazide/bisulfite reagent was subsequently treated with a phosphate buffer, a reactivation took place. The rate of the reactivation increased as the concentration of phosphate in the buffer increased. This reactivation was not accompanied by change in the mutation frequency. No reactivation was observed after a similar incubation when the prior inactivation had been induced by either methoxyamine/bisulfite or hydroxylamine/bisulfite. These results indicate that the 4-semicarbazido-2-ketopyrimidine residue is also mutagenic but is less lethal than the corresponding 5,6-dihydro-6-sulfonate structure.These results offer the first clear example of the co-operative mutagenic action of two different reagents.     

The eosinophil peroxidase-hydrogen peroxide-bromide system of human eosinophils generates 5-bromouracil, a mutagenic thymine analogue

Eosinophils use eosinophil peroxidase, hydrogen peroxide (H(2)O(2)), and bromide ion (Br(-)) to generate hypobromous acid (HOBr), a brominating intermediate. This potent oxidant may play a role in host defenses against invading parasites and eosinophil-mediated tissue damage. In this study, we explore the possibility that HOBr generated by eosinophil peroxidase might oxidize nucleic acids. When we exposed uracil, uridine, or deoxyuridine to reagent HOBr, each reaction mixture yielded a single major oxidation product that comigrated on reversed-phase HPLC with the corresponding authentic brominated pyrimidine. The eosinophil peroxidase-H(2)O(2)-Br(-) system also converted uracil into a single major oxidation product, and the yield was near-quantitative. Mass spectrometry, HPLC, UV--visible spectroscopy, and NMR spectroscopy identified the product as 5-bromouracil. Eosinophil peroxidase required H(2)O(2) and Br(-) to produce 5-bromouracil, implicating HOBr as an intermediate in the reaction. Primary and secondary bromamines also brominated uracil, suggesting that long-lived bromamines also might be physiologically relevant brominating intermediates. Human eosinophils used the eosinophil peroxidase-H(2)O(2)-Br(-) system to oxidize uracil. The product was identified as 5-bromouracil by mass spectrometry, HPLC, and UV--visible spectroscopy. Collectively, these results indicate that HOBr generated by eosinophil peroxidase oxidizes uracil to 5-bromouracil. Thymidine phosphorylase, a pyrimidine salvage enzyme, transforms 5-bromouracil to 5-bromodeoxyridine, a mutagenic analogue of thymidine. These findings raise the possibility that halogenated nucleobases generated by eosinophil peroxidase exert cytotoxic and mutagenic effects at eosinophil-rich sites of inflammation.     

Comparison of bisulfite modification of 5-methyldeoxycytidine and deoxycytidine residues.

Sodium bisulfite is a mutagen which can specifically deaminate more than 96% of the cytosine residues in single-stranded DNA via formation of a 5,6-dihydrocytosine-6-sulfonate intermediate. Under the same reaction conditions, only 2-3% of the 5-methylcytosine (m5Cyt) residues in single-stranded XP-12 DNA, which has 34 mole% m5Cyt, was converted to thymine (Thy) residues. In contrast, at the deoxynucleoside and free base levels, the same treatment with bisulfite and then alkali converted 51% and > 95%, respectively, of the m5Cyt to the corresponding Thy derivatives. However, the rate of reaction of m5Cyt and its deoxyribonucleoside was much slower than that of the analogous quantitative conversion of cytosine or deoxycytidine to uracil or deoxyuridine, respectively. The much lower reactivity of m5Cyt and its derivatives compared to that of the unmethylated analogs is primarily due to a decrease in the rate of formation of the sulfonate adduct.     

A polymerase engineered for bisulfite sequencing

Bisulfite sequencing is a key methodology in epigenetics. However, the standard workflow of bisulfite sequencing involves heat and strongly basic conditions to convert the intermediary product 5,6-dihydrouridine-6-sulfonate (dhU6S) (generated by reaction of bisulfite with deoxycytidine (dC)) to uracil (dU). These harsh conditions generally lead to sample loss and DNA damage while milder conditions may result in incomplete conversion of intermediates to uracil. Both can lead to poor recovery of bisulfite-treated DNA by the polymerase chain reaction (PCR) as either damaged DNA and/or intermediates of bisulfite treatment are poor substrate for standard DNA polymerases. Here we describe an engineered DNA polymerase (5D4) with an enhanced ability to replicate and PCR amplify bisulfite-treated DNA due to an ability to bypass both DNA lesions and bisulfite intermediates, allowing significantly milder conversion conditions and increased sensitivity in the PCR amplification of bisulfite-treated DNA. Incorporation of the 5D4 DNA polymerase into the bisulfite sequencing workflow thus promises significant sensitivity and efficiency gains.     

Reaction of uracil and thymine derivatives with sodium bisulfite. Studies on the mechanism and reduction of the adduct.

The rates and equilibria for the addition of sodium bisulfite to uracil, thymine, and their nucleosides have been studied for the pH range 3-9.5. The rate of addition for uracil is proportional to the concentration of sulfite ion and unionized uracil. The equilibrium constant (25 degrees C) for the reaction is (1.0 +/- 0.15) X 10(3) 1 - mol-1 for uracil and 0.62 +/- 0.03 1- mol-1 for thymine. A pH of 6-7, with a high bisulfite concentration is suggested for biochemical applications of the uracil reaction. The uracil reaction, which proceeds readily under physiological conditions and has a high equilibrium constant, may be a contributing cause of the biochemical effects of bisulfite and sulfur dioxide. Additional evidence on the structure of the thymine-bisulfite adduct has been obtained by nuclear magnetic resonance spectroscopy. This spectrum supports the assignment of structure as dihydrothymine-6-sulfonate. The uracil-bisulfite adduct is reduced by sodium borohydride to sodium 3-ureido-propanol-2-sulfonate. This reaction is suggested for the chemical modification of nucleic acids.     

Thymidylate synthetase catalyzed dehalogenation of 5-bromo-and 5-iodo-2′-deoxyuridylate

Thymidylate synthetase catalyzes a facile dehalogenation of 5-bromo-and 5-iodo-2′-deoxyuridylate in the presence of dithiothreitol. The chloro and fluoro nucleotides are not dehalogenated under similar conditions. A mechanism for this reaction is proposed which is in complete accord with previously ascertained features of the catalytic mechanism as well as model chemical reactions. This reaction is likely an important pathway in the biological dehalogenation of 5-halogenated uracil derivatives.     

Relationship between hydrogen consumption, dehalogenation, and the reduction of sulfur oxyanions by Desulfomonile tiedjei

Resting-cell suspensions of Desulfomonile tiedjei consumed H2 with 3-chloro-, 3-bromo-, and 3-iodobenzoate as electron acceptors with rates of 0.50, 0.44, and 0.04 mumol h-1 mg-1, respectively. However, benzoate and 3-fluorobenzoate were not metabolized by this bacterium. In addition, H2 uptake was at least fourfold faster when sulfate, sulfite, or thiosulfate was available as the electron acceptor instead of a haloaromatic substrate. When sulfite and 3-chlorobenzoate were both available for this purpose, the rate of H2 uptake by D. tiedjei was intermediate between that obtained with either electron acceptor alone. Hydrogen concentrations were reduced to comparably low levels when either 3-chlorobenzoate, sulfate, or sulfite was available as an electron acceptor, but significantly less H2 depletion was evident with benzoate or nitrate. Rates of 3-chlorobenzoate dechlorination increased from an endogenous rate of 14.5 to 17.1, 74.0, 81.1, and 82.3 nmol h-1 mg-1 with acetate, pyruvate, H2, and formate, respectively, as the electron donors. Sulfite and thiosulfate inhibited dehalogenation, but sulfate and NaCl had no effect. Dehalogenation and H2 metabolism were also inhibited by acetylene, molybdate, selenate, and metronidazole. Sulfite reduction and dehalogenation were inhibited by the same respiratory inhibitors. These results suggest that the reduction of sulfite and dehalogenation may share part of the same electron transport chain. The kinetics of H2 consumption and the direct inhibition of dehalogenation by sulfite and thiosulfate in D. tiedjei cells clearly indicate that the reduction of sulfur oxyanions is favored over aryl dehalogenation for the removal of reducing equivalents under anaerobic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship between hydrogen consumption, dehalogenation, and the reduction of sulfur oxyanions by Desulfomonile tiedjei.

Resting-cell suspensions of Desulfomonile tiedjei consumed H2 with 3-chloro-, 3-bromo-, and 3-iodobenzoate as electron acceptors with rates of 0.50, 0.44, and 0.04 mumol h-1 mg-1, respectively. However, benzoate and 3-fluorobenzoate were not metabolized by this bacterium. In addition, H2 uptake was at least fourfold faster when sulfate, sulfite, or thiosulfate was available as the electron acceptor instead of a haloaromatic substrate. When sulfite and 3-chlorobenzoate were both available for this purpose, the rate of H2 uptake by D. tiedjei was intermediate between that obtained with either electron acceptor alone. Hydrogen concentrations were reduced to comparably low levels when either 3-chlorobenzoate, sulfate, or sulfite was available as an electron acceptor, but significantly less H2 depletion was evident with benzoate or nitrate. Rates of 3-chlorobenzoate dechlorination increased from an endogenous rate of 14.5 to 17.1, 74.0, 81.1, and 82.3 nmol h-1 mg-1 with acetate, pyruvate, H2, and formate, respectively, as the electron donors. Sulfite and thiosulfate inhibited dehalogenation, but sulfate and NaCl had no effect. Dehalogenation and H2 metabolism were also inhibited by acetylene, molybdate, selenate, and metronidazole. Sulfite reduction and dehalogenation were inhibited by the same respiratory inhibitors. These results suggest that the reduction of sulfite and dehalogenation may share part of the same electron transport chain. The kinetics of H2 consumption and the direct inhibition of dehalogenation by sulfite and thiosulfate in D. tiedjei cells clearly indicate that the reduction of sulfur oxyanions is favored over aryl dehalogenation for the removal of reducing equivalents under anaerobic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reaction of cytidine with semicarbazide in the presence of bisulfite. A rapid modification specific for single-stranded polynucleotide.

Semicarbazide reacted rapidly with 5,6-dihydrocytidine-6-sulfonate, which was formed from cytidine by addition of bisulfite across the 5,6-double bond. The transaminated product, 5,6-dihydro-4-semicarbazido-2-ketotopyrimidine-6-sulfonate ribofuranoside, was identified by comparison with that formed by treatment of 4-semicarbazido-2-ketopyrimidine ribofuranoside with bisulfite. The progress of the transamination was monitored spectrophotometrically by use of a strong absorbance of the product in alkali. The reaction between cytidine and the semicarbazide-bisulfite mixture was optimal at pH 4.5. Complete transformation of cytidine into the product required only 5 min with the use of 3M semicarbazide-1M sodium bisulfite, pH 5.0, at the reaction temperature 37 degrees C. The product was stable in unbuffered solution but in phosphate buffers it underwent elimination of bisulfite to give 4-semicarbazido-2-ketopyrimidine ribofuranoside. The rate of the elimination at pH 7.0 and 37 degrees C increased proportionally with the increase of the phosphate concentration. Complete elimination was obtained by treatment with 1 M sodium phosphate for 2 h. When heat-denatured calf-thymus DNA was treated with 3 M semicarbazide-1 M bisulfite at 37 degrees C and pH 5.0 the transamination of reactive cytosine residues was completed by 10 min of incubation. At 20 degrees C, it required 85 min of incubation. Cytosine residues in native DNA did not react at all even by prolonged incubations. The modified DNA samples were further treated with a phosphate buffer at pH 7, producing 4-semicarbazido-2-ketopyrimidine residues in the DNA. Analysis of the base compositions of these samples by perchloric acid hydrolysis showed that the modification was selective to cytosine, which had been expected from studies with monomers. It also showed that the reactive cytosine residues in the denatured DNA, constitute about 80% of the total cytosine, which was consistent with the view that heat-denatured DNA still contains a considerable amount of secondary structure. The semicarbazide-bisulfite modification is expected to be a sensitive method to locate cytosine residues in single-stranded regions of polynucleotides.     

Thymidylate synthetase. Catalysis of dehalogenation of 5-bromo- and 5-iodo-2'-deoxyuridylate.

Tymidylate synthetase catalyzes the facile dehalogenation of 5-bromo-2'-deoxyuridylate (BrdUMP) and 5-iodo-2'-deoxyuridylate )IdUMP) to give 2'-deoxyuridylate (dUMP), the natural substrate of the enzyme. The reaction does not require folate cofactors and stoichiometrically consumes 2 equiv of thiol. In addition to dUMP, a minor product is formed during the debromination of BrdUMP which has been identified as a 5-alkylthio derivative formed by displacement of bromide ion by thiolate. The reaction has been found to proceed with a substantial alpha-secondary inverse tritium isotope effect (kT/kH = 1.212--1.258) with [2-14C,6-3H]-BrdUMP as the substrate. Similarly, an inverse tritiumisotope effect of 1.18 was observed in the nonenzymatic chemical counterpart of this reaction, the cysteine-promoted dehalogenation of [2-14C,6-3H]-5-bromo-2'-deoxyuridine. Previous evidence for the mechanism of action of this enzyme has rested largely on chemical model studies and on information obtained from its stoichiometric interaction with the inhibitor 5-fluoro-2'-deoxyuridylate. The magnitude of the secondary isotope effect during the enzymatic dehalogenation described here provides direct proof for nucleophilic catalysis and formation of 5,6-dihydroprimidine intermediates in a reaction catalyzed by thymidylate synthetase.     

Modification of E. coli ribosomes and coliphage MS2 RNA by bisulfite: effects on ribosomal binding and protein synthesis.

The reaction of E. coli 70s ribosomes with 0.2 M NaH-35 s03 (pH 7.1, 3.5hrs, 37 degree) led to the conversion of 4.5% of the uracil residues of the R, RNA into 5.6-dihydrouracil-6-sulfonate residues. The modified ribosomes exhibited a significant decrease in their ability to bind (14-C)-phenylalanyl-(RNA-phe and to incorporate (14-C)-phenylalanine into protein in the presence of polyuridylic acid. The ability of the modified ribosomes to form an initiation complex as measured by the A-U-G or coliphage MS2 RNA dependent binding of (14-C)-fmet-tRNA-fmet was also impaired, as was their ability to incorporate (14-C) lysine into protein with MS2 RNA as messenger. Treatment os MS RNA with 0.2 M sodium (35-S) bisulfite, pH 7.0 at 25 degrees C resulted in the substitution of 2.7% and 6.2% of the uracil residues by bisulfite after 1 and 3.5 hrs of reaction, respectively. Impairment of function of the MS2 RNA in both initiation complex formation and transplantation assays was observed. These reactions of uracil residues of mRNA and rRNA may be a cause of biological damage inflicted by sodium bisulfite and sulfur dioxide.     

Aufnahme Thymin- und Cytosin-analoger Verbindungen in die Bakterien-DesoxyribonukleinsÄure

Zusammenfassung Thymin wird vonE. coli im Vergleich zu anderen BakterienstÄmmen nur wenig aufgenommen, das gleiche gilt auch für 5-Chlor, 5-Brom- und 5-Joduracil. Die aufgenommenen radioaktiv [2-¹⁴C]-markierten Halogenpyrimidine werden von der Bakterienzelle dehalogeniert und in Uracil, Cytosin und Thymin umgewandelt. 5-Bromuracil wird zu etwa 50% dehalogeniert, 5-Chlor- und 5-Joduracil zu etwa 90%.5-Hydroxymethyluracil, 5-Hydroxymethylcytosin und 5-Methylcytosin werden von den Bakterien innerhalb der Fehlergrenze nicht aufgenommen. Die mesßbare Aufnahme von 5-Bromcytosin könnte mit dessen Umwandlung in 5-Bromuracil zusammenhÄngen.

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Summary In comparison with other bacterial strainsE. coli was found to incorporate thymine considerably in smaller amounts. Similar observations were also made for 5-chloro-, 5-bromo- and 5-iodouracil. We found that the halogenated pyrimidines labelled at 2-¹⁴C after incorporation into bacterial cells are dehalogenated and converted into uracil, cytosin and thymine; the extent of their dehalogenation varies, e. g. 5-bromouracil to 50%, and 5-chloroucil as well as 5-iodouracil to about 90%.Pyrimidine derivatives like 5-hydroxymethyluracil, 5-hydroxymethylcytosin and 5-methylcytosin were found not to be incorporated under the error limits into bacterial cells. The measurable amount of 5-bromocytosin incorporation could have been due to its conversion into 5-bromouracil.