Cytosine Hydrate Research Articles

Experimental and Computational Hydrate Screening: Cytosine, 5-Flucytosine and Their Solid Solution.

The structural, temperature- and moisture dependent stability features of cytosine and 5-flucytosine monohydrates, two pharmaceutically important compounds, were rationalized using complementary experimental and computational approaches. Moisture sorption/desorption, water activity, thermal analysis and calorimetry were applied to determine the stability ranges of hydrate ↔ anhydrate systems, while X-ray diffraction, IR spectroscopy and crystal structure prediction provided the molecular level understanding. At 25 °C, the critical water activity for the cytosine hydrate ↔ anhydrate system is ~0.43 and for 5-flucytosine ~0.41. In 5-flucytosine the water molecules are arranged in open channels, therefore the kinetic desorption data, dehydration < 40% relative humidity (RH), conform with the thermodynamic data, whereas for the cytosine isolated site hydrate dehydration was observed at RH < 15%. Peritectic dissociation temperatures of the hydrates were measured to be 97 °C and 84.2 °C for cytosine and 5-flucytosine, respectively, and the monohydrate to anhydrate transition enthalpies to be around 10 kJ mol-1. Computed crystal energy landscapes not only revealed that the substitution of C5 (H or F) controls the packing and properties of cytosine/5-flucytosine solid forms, but also have enabled the finding of a monohydrate solid solution of the two substances which shows increased thermal- and moisture-dependent stability compared to 5-flucytosine monohydrate.

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

Vibronic structure of the lowest-energy UV absorption band in cytosine

Fine vibronic structure of the lowest-energy absorption band of cytosine was obtained by low temperature UV reflectivity measurement on cytosine hydrate single crystal. The absorption spectrum was then evaluated using advanced deconvolution procedure. It has been found that the spectrum consists of successions of Frank-Condon vibronic transitions with dominating role of the breathing vibrational mode. This fact suggests remarkable increasing of interatomic distances caused by electronic excitation of cytosine molecule.

Excision of cytosine hydrates from Z-DNA

Ultraviolet irradiation of DNA produces cytosine hydrate, released as a free base by E. coli endonuclease III. Cytosine hydrate excision was investigated by assaying photoproduct release from cytosine-radiolabeled, irradiated poly(dG-dC):poly(dG-dC). Conformational shifts between B-DNA and Z-DNA were affected by heating the polymer in either nickel chloride or cobaltous chloride, and were determined by circular dichroism. Rates of enzymic cytosine hydrate release did not differ between the different substrate conformations. Irradiation of left-handed poly(dG-dC):poly(dG-dC) resulted in cytosine hydrate formation. Therefore, neither formation nor enzymic excision of ultraviolet-induced cytosine hydrates are substantially affected by these DNA conformational states.

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.

Template properties of ultraviolet-irradiated poly(dC) replicated by E. coli DNA polymerase I: Indication for a role of apyrimidinic-sites in UV-induced mutagenesis

Ultraviolet irradiation alters the template properties of poly(dC) when replicated by Escherichia coli DNA polymerase I. These effects are due to base modifications. Some of them are identified as apurinic/apyrimidinic sites (AP-sites) by their sensitivity to AP-endonuclease B purified from Micrococcus luteus, and their template properties. The rate of formation of AP-sites in poly(dC) is estimated at 3 X 10(-7) site per nucleotide per J.m-2. Exposure of supercoiled or relaxed pBR322 DNA to UV light results also in the formation of sites sensitive to AP-endonuclease B. In this case, the rate of formation of AP-sites is the same in relaxed or supercoiled DNA: 0.3 X 10(-7) site per nucleotide per J.m-2. The apyrimidinic sites are generated through the processing of an ultraviolet induced primary lesion. We suggest that this lesion is cytosine hydrate by its rate of decay and preferential formation in single stranded DNA. Our results suggest that AP-sites might be a minor pathway leading to UV-induced mutagenesis.

Restricted rotation in crystalline cytosine and cytosine monohydrate

The n.m.r. parameters T1 and T1ρ, the spin-lattice relaxation times in the laboratory- and rotating-frames, respectively, have been utilized to study the molecular motions that occur in polycrystalline samples of cytosine and cytosine hydrate in the range 130–293 K. The deuterated analogues have also been studied. It is concluded that, in the monohydrate, the water molecule, but not the cytosine itself, may rotate (at a frequency of ca. 30 kHz at 200 K), the activation energy of rotation being 17.8±2.8 kJ mol–1. There is also evidence for the rotation of the amino group. Dehydration of cytosine hydrate commences around 328 K, and the enthalpy of dehydration is estimated to be 50 kJ mol–1.