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PubMed1991

Kinetics of extension of O6-methylguanine paired with cytosine or thymine in defined oligonucleotide sequences.

The frequency of extending m6G.C or m6G.T pairs, when the 3' and 5' flanking neighbors of m6G are either cytosines or thymines, was investigated using primed 25-base-long oligonucleotides and the Klenow fragment of Escherichia coli DNA polymerase I (Kf). The efficiency, Vmax/Km, of extension to the following normal base pair was up to 40-fold greater than for the formation of the m6G.T or m6G.C pair. The frequencies of inserting either dCMP or dTMP opposite these m6G bases did not appear to be different in the two sequences, C-m6G-C and T-m6G-T, but extension was favored in the C-m6G-C sequence. The m6G.T pair extended to a C.G pair most efficiently, indicating that it was not a strong block to continued replication past the template lesion. Thus, m6G.T flanked by cytosines replicates more readily than when flanked by thymines, increasing G----A transitions. These data lend further support to the importance of sequence context in mutagenesis.

Base Composition↗

PubMed1992

Cytosine methylation can induce local distortions in the structure of duplex DNA.

Methyl groups at the C5 position of pyrimidines located within oligopurine-oligopyrimidine tracts in DNA have been shown previously to modulate curvature generated by those tracts. However, it was not known whether the influence of such methyl groups is consequent to the altered helical structure within the tracts themselves. In the current study, it is demonstrated that methylation of cytosines up to three base pairs away from a (dA)5.(dT)5 tract (A-tract) can still result in alterations of the net curvature of the A-tract-containing DNA, as measured by alterations in electrophoretic mobility. This latter effect depends strongly on both the sequence of the non-A-tract DNA and the positions of the methylated C residues. The current results lend further support to the notion that the biological consequences of cytosine methylation may be effected through local alterations in DNA structure as well as through direct protein-DNA interactions.

5-Methylcytosine↗

PubMed1994

Bisulfite induces tandem double CC-->TT mutations in double-stranded DNA. 2. Kinetics of cytosine deamination.

Deamination of cytosine to uracil in double-stranded DNA (ds DNA) by sodium bisulfite has been monitored with a sensitive genetic assay. In this system, reversion of a mutant in the lacZ alpha gene coding sequence of bacteriophage M13mp2 C141 was detected by employing an ung- bacterial strain defective in the enzyme uracil glycosylase. Within the 4-base target, it is possible to measure the rates of induction of C-->T, C-->A, C-->G, and CC-->TT mutations in DNA that has been incubated at physiological temperature and pH and then transfected into ung+ and ung- E. coli cells, respectively, for amplification and detection of the mutation. For concentrations of bisulfite from 1 to 50 mM, the reversion frequency in ung- cells increased linearly with time of incubation. The most interesting features of the bisulfite reaction were as follow: (1) Mutations were reduced 5-fold in ung+ cells, indicating ung is involved in repair of bisulfite-treated transforming DNA. (2) Sequencing of 157 revertants revealed that C-->T and tandem CC-->TT transition mutations comprised 100% of the mutations scored. (3) A unique finding was that, at the highest concentrations and longest incubation times, almost every mutant obtained in ds DNA exposed to bisulfite was found to be a CC-->TT tandem double mutation. (4) The high frequency of tandem double mutants is inconsistent with two random, independent mutational events and, coupled with the observed ung dependence, lends support to the concept of catalytic deamination, wherein bisulfite induces deamination in contiguous cytosines by a concerted mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage M13↗

PubMed1984

Sequence dependence of conformations of self-complementary duplex tetradeoxynucleotides containing cytosine and guanine.

The four self-complementary tetradeoxynucleotides which contain only cytosine and guanine are 5'-d-(CpGpCpG)-3', 5'-d(CpCpGpG)-3', 5'-d(GpCpGpC)-3', and 5'-d(GpGpCpC)-3'. The Raman spectra of aqueous solutions (about 0.05 M in monomer) of these tetranucleotides at pH 7 and 2 degrees C show clearly that these self-complementary tetranucleotides form double-stranded duplex structures of the canonical B type when the NaCl concentration is 0.5 M NaCl. If the temperature is raised to 50 degrees C, the Raman spectra show that in each case the double-helical B form melts in a non-cooperative way to a disordered single-chain form. On the other hand, if the salt concentration is raised to saturation, the Raman spectrum of only one of these four tetranucleotide solutions at 2 degrees C is changed in any substantial way. The Raman spectrum of the tetranucleotide 5'-d(CpGpCpG)-3' at 2.2 degrees C and at 4 M or higher salt concentration strongly resembles that of double-helical Z-form poly(dC-dG) taken under similar conditions. We conclude that the tetramer 5'-d(CpGpCpG)-3' is the only self-complementary double-helical tetranucleotide containing only cytosine and guanine in which the B-Z transition can be induced by increasing the salt concentration. This tetramer has several types of stacking interactions which differ markedly from stacking interactions in the other tetramers and may account for the enhanced stabilization of its Z conformation.

Base Sequence↗

PubMed1985

Model for interactions of amino acid side chains with Watson-Crick base pair of guanine and cytosine: crystal structure of 9-(2-carbamoylethyl)guanine and 1-methylcytosine complex.

As a model of interaction between the guanine-cytosine base pair and carbamoyl group, the crystal structure of 9-(2-carbamoylethyl)guanine-1-methylcytosine complex has been studied by X-ray method. The crystal data are a = 8.540 (1) A, b = 12.693 (3) A, c = 14.249 (2) A, beta = 94.02 (1) degrees, space group P21/c, Z = 4, dm = 1.50, dc = 1.49 g cm-3, and R = 0.10 for 1035 reflections. The bases form a Watson-Crick pair, and the carbamoyl group is hydrogen bonded with O(6) of guanine and N(4) of cytosine in the adjacent pairs. A structural correlation has been found between the hydrogen-bonding pattern and the secondary structural fitting of alpha-helical segment with B-form DNA.

Asparagine↗

PubMed1978

Interaction of aromatic residues of proteins with nucleic acids. Binding of oligopeptides to copolynucleotides of adenine and cytosine.

The binding of the peptide Lys-Trp-Lys to various single-stranded copolymers of adenine and cytosine has been investigated using circular dichroism (CD) and fluorescence measurements. Two types of complexes are formed, both involving electrostatic interactions between lysyl residues and phosphate groups. The fluorescence quantum yield of the first complex is identical with that of the free peptide. The other complex involves a stacking of the polynucleotide bases with the tryptophan residue whose fluorescence is quenched. The binding of the peptide leads to a conformational change of the copolymers as shown by the CD variations. The fluorescence and CD data have been analyzed according to the model involving the two types of complexes. The values of the binding constants have been studied as a function of the cytosine content of the copolymers. Analysis of the stacking process in term of nearest neighbor frequency demonstrates that this binding is sequence dependent and is favored in AA sequence as compared with AC, CA, and CC sequence. Thus this simple tripeptide is able to distinguish between various base sequences in a single-stranded nucleic acid.

Adenine↗

PubMed2002

Solution structure of a DNA duplex containing mispair-aligned N4C-ethyl-N4C interstrand cross-linked cytosines.

The solution structure of an interstrand cross-linked self-complementary oligodeoxynucleotide containing directly opposed alkylated N(4)C-ethyl-N(4)C cytosine bases was determined by molecular dynamics calculations guided by NMR-derived restraints. The undecamer d(CGAAACTTTCG)(2), where C represents directly opposed alkylated N(4)C-ethyl-N(4)C cytosine bases, serves as model for the cytotoxic cross-links formed by bifunctional alkylating agents used in cancer therapy. The structure of the duplex shows the cross-link protruding into the major groove. An increase in the diameter of the DNA at the pseudoplatform formed by the cross-linked residues creates an A-DNA characteristic hole in the central portion of the DNA. This results in a centrally underwound base step and a number of subsequent overwinding steps leading to an overall axis bend toward the major groove. The structure shows narrowing of both minor and major grooves in the proximity of the cross-link. The perturbation leads to preferential intrastrand base stacking, disruption of adjacent canonical (A.T) base pairing, and buckling of base pairs, the extent of which diminishes with progression away from the lesion site. Overall, the distortion induced by the cross-link spreads over three base pairs on the 5'- and 3'-sides of the cross-link.

Base Pair Mismatch↗

PubMed2005

Product release is rate-limiting in the activation of the prodrug 5-fluorocytosine by yeast cytosine deaminase.

Yeast cytosine deaminase (yCD), a zinc metalloenzyme, catalyzes the hydrolytic deamination of cytosine to uracil. The enzyme is of great biomedical interest because it also catalyzes the deamination of the prodrug 5-fluorocytosine (5FC) to form the anticancer drug 5-fluorouracil (5FU). yCD/5FC is one of the most widely used enzyme/prodrug combinations for gene-directed enzyme prodrug therapy for the treatment of cancers. A pH indicator assay has been developed for the measurement of the steady-state kinetic parameters for the deamination reaction. Transient kinetic studies have shown that the product release is a rate-limiting step in the activation of the prodrug 5FC by yCD. The rate constant of the chemical step for the forward reaction (250 s(-)(1)) is approximately 8 times that of the product release (31 s(-)(1)) and approximately 15 times k(cat) (17 s(-)(1)). The transient kinetic results are consistent with those of the steady-state kinetic analysis in the sense that the k(cat) and K(m) values calculated from the rate constants determined by the transient kinetic analysis are in close agreement with those measured by the steady-state kinetic analysis. NMR experiments have demonstrated that free 5FU is in slow exchange with its complex with yCD but has a low affinity for yCD. The transient kinetic and NMR results together suggest that the release of 5FU is rate-limiting in the activation of the prodrug 5FC by yCD and may involve multiple steps.

Antimetabolites, Antineoplastic↗

PubMed2005

Equilenin-derived DNA adducts to cytosine in DNA duplexes: structures and thermodynamics.

The drug Premarin is the most widely used formula for hormone replacement therapy. However, long-term exposure to estrogens from the Premarin drug increases the risk of breast cancer. Equilin and equilenin, major components of Premarin, are predominantly metabolized to 4-hydroxyequilenin (4-OHEN). The quinoids produced by 4-OHEN oxidation react with dG, dA, and dC to form unusual stable cyclic bulky adducts, with four stereoisomers identified for each base adduct. The 4-OHEN-dC adducts are most predominant. They are mutagenic in vitro and have been found in human tumor tissue. We have carried out molecular modeling and molecular dynamics simulations to investigate structures and thermodynamics of the four 4-OHEN-dC stereoisomeric adducts in DNA duplexes. Our results show that the structure of each stereoisomer adduct in duplex DNA is specifically governed by its unique stereochemistry. The bulky adducts, with an obstructed Watson-Crick edge and an equilenin ring system near perpendicular to the damaged cytosine, are located in the B-DNA major or minor groove, with the modified cytosine in the syn or anti conformation, respectively. The DNA duplex structures are distorted, in terms of Watson-Crick pairing at and near the lesion, stacking interactions, and groove dimensions. Stereochemistry determines the orientation of the equilenin rings with respect to the 5'- to 3'-direction of the modified strand, as well as the positioning of the equilenin moiety's methyl and hydroxyl groups for each stereoisomer. The unusual structures and the stereochemical effects underlie their biological processing as miscoding DNA lesions whose mutagenic properties may contribute to breast cancer.

Animals↗

PubMed1998

Murine DNA (cytosine-5-)-methyltransferase: steady-state and substrate trapping analyses of the kinetic mechanism.

DNA (cytosine-5-)-methyltransferase is essential for viable mammalian development and has a central function in the determination and maintenance of epigenetic methylation patterns. Steady-state and substrate trapping studies were performed to better understand how the enzyme functions. The catalytic efficiency was dependent on substrate DNA length. A 14-fold increase in KmDNA was observed as the length decreased from 5000 to 100 base pairs and kcat decreased by a third. Steady-state analyses were used to identify the order of substrate addition onto the enzyme and the order of product release. Double-reciprocal patterns of velocity versus substrate concentration intersected far from the origin and were nearly parallel. The kinetic mechanism does not appear to change when the DNA substrate is either 6250 or 100 base pairs in length. Isotope trapping studies showed that the initial enzyme-AdoMet complex was not catalytically competent; however, the initial enzyme-poly(dI.dC-dI.dC) complex was observed to be competent for catalysis. Product inhibition studies also support a sequential ordered bi-bi kinetic mechanism in which DNA binds to the enzyme first, followed by S-adenosyl-L-methionine, and then the products S-adenosyl-L-homocysteine and methylated DNA are released. The proposed mechanism is similar to the mechanism proposed for M. HhaI, a bacterial DNA (cytosine-5-)-methyltransferase. Evidence for an enzyme-DNA-DNA ternary complex is also presented.

Animals↗

PubMed2002

Theoretical studies of the cross-linking mechanisms between cytosine and tyrosine.

DNA-protein cross-linking is one of the many DNA lesions mediated by hydroxyl radicals, the most damaging among the reactive oxygen species in biological systems. Density functional theory methods are employed to investigate the complex reaction mechanisms of the formation of cytosine-tyrosine cross-links as observed in gamma-irradiated aqueous solutions of cytosine and tyrosine, as well as in gamma-irradiated nucleohistone. The majority of the radical addition mechanisms considered are found to have significant barriers and therefore to be thermodynamically unfavorable for the formation of the initial cross-linked product. Our calculated reaction potential energy surfaces suggest that a feasible complete mechanism consists of radical combination forming the initial cross-linked product, a hydrogen shuffle within the initial cross-linked product, and an acid-catalyzed dehydration reaction. Water and hydrogen-bonding interactions are suggested to play a key role in catalyzing the hydrogen-transfer step of the reaction.

Cytosine↗

PubMed2003

Ultrafast internal conversion of excited cytosine via the lowest pipi electronic singlet state.

Computational evidence at the CASPT2 level supports that the lowest excited state pipi* contributes to the S1/S0 crossing responsible for the ultrafast decay of singlet excited cytosine. The computed radiative lifetime, 33 ns, is consistent with the experimentally derived value, 40 ns. The nOpi* state does not play a direct role in the rapid repopulation of the ground state; it is involved in a S2/S1 crossing. Alternative mechanisms through excited states pisigma* or nNpi* are not competitive in cytosine.

Cytosine↗

PubMed2005

Triplet-state formation along the ultrafast decay of excited singlet cytosine.

We address the possibility of populating the lowest triplet state of cytosine by an "intrinsic"mechanism, namely, intersystem crossing (ISC) along the ultrafast internal conversion pathway of the electronically excited singlet species. For this purpose, we present a discussion of the ISC process and triplet-state reactivity based on theoretical analysis of the spin-orbit strength and the potential energy surfaces for the relevant singlet and triplet states of cytosine. High-level ab initio computations show that ISC is possible in wide regions of the singlet manifold along the reaction coordinate that controls the ultrafast internal conversion to the ground state. Thus, the ISC mechanism documented here provides a possibility to access the triplet state, which has a key role in the photochemistry of the nucleic acid bases.

Cytosine↗

PubMed2004

Catalytic mechanism of yeast cytosine deaminase: an ONIOM computational study.

The complete path for the deamination reaction catalyzed by yeast cytosine deaminase (yCD), a zinc metalloenzyme of significant biomedical interest, has been investigated using the ONIOM method. Cytosine deamination proceeds via a sequential mechanism involving the protonation of N(3), the nucleophilic attack of C(4) by the zinc-coordinated hydroxide, and the cleavage of the C(4)-N(4) bond. The last step is the rate determining step for the generation of the zinc bound uracil. Uracil is liberated from the Zn atom by an oxygen exchange mechanism that involves the formation of a gem-diol intermediate from the Zn bound uracil and a water molecule, the C(4)-O(Zn) cleavage, and the regeneration of the Zn-coordinated water. The rate determining step in the oxygen exchange is the formation of the gem-diol intermediate, which is also the rate determining step for the overall yCD-catalyzed deamination reaction.

Catalysis↗

PubMed2004

Triplet (pi,pi) reactivity of the guanine-cytosine DNA base pair: benign deactivation versus double tautomerization via intermolecular hydrogen transfer.

Ab initio computations (CASSCF/6-31G* supported by CAS-PT2 single-point calculations) are used to study the reactivity of the triplet excited state of the guanine-cytosine DNA base pair. When the triplet excitation is centered on cytosine there is a competition between benign deactivation to the ground state and a hydrogen transfer route that can trigger double tautomerization. The calculated barriers favor the benign deactivation, but this route goes through a singlet/triplet intersystem crossing with small spin-orbit coupling. Therefore, the potentially mutagenic, double tautomerization route cannot be ruled out completely, and the two paths are probably an alternative to the well-known cytidine photodimerization reaction.

Base Pairing↗

PubMed2005

Cytosine catalysis of nitrosative guanine deamination and interstrand cross-link formation.

Effects are discussed of the anisotropic DNA environment on nitrosative guanine deamination based on results of an ab initio study of the aggregate 3 formed by guaninediazonium ion 1 and cytosine 2. Within 3, the protonation of 2 by 1 is fast and exothermic and forms 6, an aggregate between betaine 4 (2-diazonium-9H-purin-6-olate) and cytosinium ion 5. Electronic structure analysis of 4 shows that this betaine is not mesoionic; only the negative charge is delocalized in the pi-system while the positive charge resides in the sigma-system. Potential energy surface exploration shows that both dediazoniation and ring-opening of betaine 4 in aggregate 6 are fast and exothermic and lead irreversibly to E-11, the aggregate between (E)-5-cyanoimino-4-oxomethylene-4,5-dihydroimidazole E-10 and 5. The computed pair binding energies for 3, 6, and E-11 greatly exceed the GC pair binding energy. While 1 can be a highly reactive intermediate in reactions of the "free nucleobase" (or its nucleoside and nucleotide), the cyanoimine 10 emerges as the key intermediate in nitrosative guanine deamination in ds-DNA and ds-oligonucleotides. In essence, the complementary nucleobase cytosine provides base catalysis and switches the sequence of deprotonation and dediazoniation. It is argued that this environment-induced switch causes entirely different reaction paths to products as compared to the respective "free nucleobase" chemistry, and the complete consistency is demonstrated of this mechanistic model with all known experimental results. Products might form directly from 10 by addition and ring closure, or their formation might involve water catalysis via 5-cyanoamino-4-imidazolecarboxylic acid 12 and/or 5-carbodiimidyl-4-imidazolecarboxylic acid 13. The pyrimidine ring-opened intermediates 10, 12, and 13 can account for the formations of xanthosine, the pH dependency and the environment dependency of oxanosine formation, the formation of the classical cross-link dG(N(2)())-to-dG(C2), including the known sequence specificity of its formation, and the formation of the structure-isomeric cross-link dG(N1)-to-dG(C2).

Carbodiimides↗

PubMed2005

Formation of intrastrand cross-link products between cytosine and adenine from UV irradiation of d((Br)CA) and duplex DNA containing a 5-bromocytosine.

Here, we showed that Pyrex-filtered UV light irradiation of d((Br)CA) gave rise to three types of intrastrand cross-link products, that is, d(C[5-N6]A), d(C[5-2]A), and d(C[5-8]A), where the C5 carbon atom of cytosine is covalently bonded to the N6 nitrogen atom, C2, and C8 carbon atoms of adenine, respectively. Furthermore, we demonstrated by LC-MS/MS that the UV irradiation of a 5-bromocytosine-containing duplex oligodeoxynucleotide (ODN) led to the formation of five cross-link products, that is, C[5-N6]A, C[5-2]A, C[5-8]A, A[2-5]C, and A[8-5]C, under both aerobic and anaerobic conditions. LC-MS/MS quantification results showed that the yields for the formation of these cross-link products are different. The presence of molecular oxygen reduces the yields for the formation of all cross-link products except A[2-5]C. To our knowledge, this is the first report about the formation of intrastrand cross-link products between cytosine and adenine in duplex DNA. The chemistry discovered here may facilitate the future preparations of oxidative cross-link lesion-bearing substrates for biochemical and biophysical studies.

Adenine↗

PubMed2004

Substituent effects on pyrid-2-yl ureas toward intramolecular hydrogen bonding and cytosine complexation.

Equilibria between two conformational isomers of pyrid-2-yl ureas, the (E,Z) and (Z,Z) forms, have been studied in DMF-d(7) at -70 degrees C. Most of them show a small preference for the (E,Z) form with an equilibrium constant K(i) around 1-2. However, the K(i) value for 1-methyl-2-(3-(pyrid-2-yl)ureido)pyridinium iodide (12) was found to be 14.2 +/- 1.2. That is 1 order of magnitude larger than those of the others, which indicates that the positively charged 1-methylpyridinium-2-yl substituent would facilitate the (E,Z) form formation. Pyrid-2-yl ureas bind cytosine in DMF-d(7) with binding constants K(B) ranging from 30 to 1700 M(-1). Electron withdrawing substituents, such as the 4-O(2)NC(6)H(4)- or 1-methylpyridinium-4-yl substituent, preferentially facilitate the intermolecular cytosine complexation with large binding constants.

Chemical Phenomena↗