Uracil Hydrate Research Articles

The comparative investigation of the dehydration methods of uracil hydrate derivatives

By following UV absorbance, UV absorption spectra and changes in high performance liquid chromatography hydrate peak elution areas during the reversion of uracil, uridine and 5′UMP hydrates induced by heating hydrated samples in boiling water for 10 min or by incubating acidified samples (pH 1) at room temperature for 24 h, it is shown that these methods do not lead to quantitative dehydration. The disappearance of hydrate peaks and the higher recovery of both the UV absorbance and UV absorption spectra obtained after heating acidified samples (pH 1) indicate that this method is best to achieve total dehydration. Catalysis of dehydration in phosphate buffer solution is also discussed.

Comparison of the Effects of UV Irradiation on 5-Methyl-Substituted and Unsubstituted Pyrimidines in Alternating Pyrimidine-Purine Sequences in DNA

We previously demonstrated the UV-induced formation of cytosine hydrate in DNA and its deamination product, uracil hydrate, via their release from the DNA backbone by the DNA glycosylase activity of Escherichia coli endonuclease III. Subsequently, endonuclease III-mediated release of thymine hydrate from UV-irradiated poly(dA-dT) was reported. Therefore, we asked whether 5-methylcytosine residues in DNA underwent photohydration and deamination to thymine hydrate in analogy to UV-induced deamination of cytosine. An alternating DNA copolymer containing 5-methylcytosine was irradiated with UVC and incubated with endonuclease III. No 5-methylcytosine hydrate was released. Instead, UV-induced nonenzymatic release of 5-methylcytosine occurred. Similarly, incubation of UV-irradiated poly(dA-dT) with endonuclease III did not release thymine hydrate; nonenzymatic release of thymine occurred. Nonenzymatic release of 5-methylpyrimidines was oxygen dependent, enhanced by ferric ion and inhibited by free radical scavengers. In contrast, photohydration of cytosine was oxygen independent, and only small amounts of cytosine were nonenzymatically released. Thus, 5-methylpyrimidine residues within alternating Pu-Py sequences in DNA do not undergo photohydration, but instead undergo cleavage of their N-glycosyl bonds yielding abasic (AP) sites. The inability to repair such AP sites may explain the UV sensitivity of E. coli xthnfo mutants, which lack AP endonuclease activity. We suggest that N-glycosyl bond cleavage is mediated by radical species formed via transfer of an electron from UV-excited triplet 5-methylpyrimidines to ground state oxygen and/or ferric ions.

Effect of pH and temperature on the stability of UV-induced repairable pyrimidine hydrates in DNA.

UV irradiation of cytosine yields 6-hydroxy-5,6-dihydrocytosine (cytosine hydrate) whether the cytosine is in solution as base, nucleoside, or nucleotide or on the DNA backbone. Cytosine hydrate decomposes by elimination of water, yielding cytosine, or by irreversible deamination, yielding uracil hydrate, which, in turn, decomposes by dehydration yielding uracil. To determine how pH and temperature affect these decomposition reactions, alternating poly(dG-[3H]dC) copolymer was irradiated at 254 nm and incubated under different conditions of pH and temperature. The cytosine hydrate and uracil hydrate content of the DNA was determined by the use of Escherichia coli endonuclease III, which releases pyrimidine hydrates from DNA by virtue of its DNA glycosylase activity. Uracil content was determined by using uracil-DNA glycosylase. The rate of decomposition of cytosine hydrate to cytosine was determined at 4 temperatures at pH 3.1, 5.4, and 7.4. The Ea was determined from the rates by using the Arrhenius equation and proved to be the same at pH 5.4 and 7.4, although the decomposition rate at pH 5.4 was faster at all temperatures. At pH 3.1, the Ea was reduced. These results suggest that the dehydration reaction is affected by two discrete protonations, most probably of the N-3 and the OH group of C-6 of cytosine hydrate. The deamination of cytosine hydrate to uracil hydrate was maximal at pH 3.1 at all temperatures. The doubly protonated cytosine hydrate probably is the common intermediate for both competing decomposition reactions, explaining why cytosine hydrate is prone to deamination at acid pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and function of Escherichia coli endonuclease III.

One of the most exciting areas of atomic structural studies on protein-DNA interactions has been the studies on on DNA repair enzymes. The atomic structures of endonuclease III and endonuclease V, a phage T4 enzyme that repairs pyrimidine dimers, are a significant advance, as they provide the first structural information concerning the mechanism by which enzymes can recognize altered bases in DNA. Endonuclease III from Escherichia coli is a DNA repair enzyme with two distinct activities: it is a DNA N-glycosylase that acts upon ring-saturated, ring-rearranged, and ring-contracted pyrimidines and is an AP lyase. The name endonuclease III was originally applied to an activity that nicked heavily UV-irradiated DNA. The enzyme was found to be identical with E. coli x-ray endonuclease, with an endonuclease that cleaves DNA damaged by UV irradiation, osmium tetroxide, x-rays, or acid, and with urea-DNA glycosylase. The identification of endonuclease III with these various other activities is due to the broad substrate specificity of the enzyme. It has been shown to release dihydrothymine, cis- and trans-thymine glycol, urea, methyltartronylurea and 5-hydroxy-5-methylhydantoin, 6-hydroxy-5,6-dihydrothymine, uracil hydrate, and cytosine hydrate from DNA. Endonuclease III has been shown to be an AP lyase rather than a true AP endonuclease; itmore » cleaves the phosphodiester bond adjacent to an AP site by a {beta}-elimination reaction rather than a hydrolytic reaction. The syn stereochemical course of the {beta}-elimination reaction has been defined: the 2{prime}-pro-S hydrogen is abstracted, and a trans {alpha}{beta}-unsaturated aldose is produced.« less

A computer simulation of the uracil hydrate mechanism for the photochemically induced deamination of free cytosine base in aqueous solution

The rate expression for the deamination of free cytosine base in unbuffered aqueous solution has been measured as In k = 8.301 - 75.230 kJ mol −1/ RT. Irradiation of similar solutions at 265 nm, followed by high performance liquid chromatography (HPLC) quantification of the uracil formed, allowed a quantum yield for uracil hydrate formation to be calculated as 4.9 × 10 −4. This result was used together with literature values of other rate constants to simulate a mechanism for the formation of uracil from UV-irradiated cytosine solutions. A value of 7.4 × 10 11 s −1 for the internal conversion of cytosine singlet to ground-state cytosine gave the best agreement between the experimental results and the simulation.

UV-induced pyrimidine hydrates in DNA are repaired by bacterial and mammalian DNA glycosylase activities.

Escherichia coli endonuclease III and mammalian repair enzymes cleave UV-irradiated DNA at AP sites formed by the removal of cytosine photoproducts by the DNA glycosylase activity of these enzymes. Poly(dG-[3H]dC) was UV irradiated and incubated with purified endonuclease III. 3H-Containing material was released in a fashion consistent with Michaelis-Menten kinetics. This 3H material was determined to be cytosine by chromatography in two independent systems and microderivatization. 3H-Containing material was not released from nonirradiated copolymer. When poly(dA-[3H]dU) was UV irradiated, endonuclease III released 3H-containing material that coeluted with uracil hydrate (6-hydroxy-5,6-dihydrouracil). Similar results are obtained by using extracts of HeLa cells. There results indicate that the modified cytosine residue recognized by endonuclease III and the mammalian enzyme is cytosine hydrate (6-hydroxy-5,6-dihydrocytosine). Once released from DNA through DNA-glycosylase action, the compound eliminates water, reverting to cytosine. This is consistent with the known instability of cytosine hydrate. The repairability of cytosine hydrate in DNA suggests that it is stable in DNA and potentially genotoxic.

Coding properties of ultraviolet photoproducts of uracil: I. Binding studies and polypeptide synthesis

Results of binding of aminoacyl tRNA to ribosomes in the presence of short well-defined nucleic acid sequences containing uracil photoproducts indicate that the uracil dimer can code as the sequences guanine-uracil and uracil-uracil, and the uracil hydrate can code as cytosine. These findings are confirmed in polypeptide synthesis in vitro in the presence of irradiated polyribonucleotides containing uracil. From the latter results it was calculated that the efficiency with which the hydrate codes as cytosine is about 100% in the first position of a codon but less than 20% in the second.

Coding properties of ultraviolet photoproducts of uracil: II. Phenotypic reversion of the amber mutation: Implication of the uracil hydrate

We have observed ultraviolet-induced phenotypic reversion of an amber mutation in vivo, a phenomenon predicted on the basis of studies in vitro of code changes brought about by ultraviolet irradiation of the RNA base uracil. Mathematical analysis indicated that the rate of production of reversion by ultraviolet light is of the same magnitude as the rate of production of uracil hydrate upon irradiation of poly U. Furthermore, the results lead to the conclusion that an amino acid with codon containing a uracil hydrate can be added to the growing polypeptide chain in vivo. Moreover, protein synthesis can proceed past this ultraviolet lesion.

Photochemical deamination of cytosine at 2537° A

The rate expression for the deamination of free cytosine base in unbuffered aqueous solution has been measured as In k = 8.301 - 75.230 kJ mol−1/RT. Irradiation of similar solutions at 265 nm, followed by high performance liquid chromatography (HPLC) quantification of the uracil formed, allowed a quantum yield for uracil hydrate formation to be calculated as 4.9 × 10−4. This result was used together with literature values of other rate constants to simulate a mechanism for the formation of uracil from UV-irradiated cytosine solutions. A value of 7.4 × 1011 s−1 for the internal conversion of cytosine singlet to ground-state cytosine gave the best agreement between the experimental results and the simulation.