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PubMed1997

Liquid chromatographic separation of hexopyranosylated cytosine nucleosides from their degradation products.

Development of a liquid chromatographic method which can separate each of a series of hexopyranosylated cytosine nucleosides from their degradation products formed at acid, neutral and basic pH is described. Both silica-based reverse-phase and polymer columns were examined. Influence of the mobile phase pH, ion-pairing agent, concentration of the buffer and type and concentration of organic modifier were systematically investigated. The concentration of the ion-pairing agent and the buffer were found to have a major effect on selectivity. Samples were finally analyzed on a poly(styrene-divinylbenzene), PLRP-S 100 A (8 microns) 250 x 4.6 mm I.D. column at 60 degrees C and with a mobile phase consisting of acetonitrile-sodium octanesulphonate (pH 2.5; 0.02 M)-potassium phosphate buffer (pH 2.5; 0.2 M)-water (X:25:50:25-X, v/v, where X is variable).

Antineoplastic Agents↗

PubMed2000

Guanine-cytosine rich regions of plasmid DNA can be the target in anti-plasmid effect of phenothiazines.

The antiplasmid effects of promethazine on E. coli is the consequence of special complex formed with a covalently closed circular (ccc) form of plasmid DNA. The exact target in this macromolecule, however, was not clarified until recently. Caffeine and the chemically similar guanosine-5'- monophosphate (GMP) could compete with the antiplasmid effect of promethazine, showing that promethazine or other phenothiazines preferentially bind to xanthine type molecules. Among the xanthines, GMP was more effective at complex-forming than adenosine-5'-monophosphate (AMP). In addition, the Z-DNA was more susceptible than B-DNA. Therefore, one could suppose that guanine-cytosine (G-C) rich regions have higher affinity than adenine-thymine (A-T) rich region on phenothiazines. Because the G-C rich regions have a special role in the DNA stability via three hydrogen bonds, we suppose that these regions could have a key role in some biological effects such as antiplasmid and anticancer activity.

Binding Sites↗

PubMed1998

Cytosine methylation of repeated sequences in eukaryotes: the role of DNA pairing.

In eukaryotic genomes, methylation of cytosine residues commonly occurs in repetitive sequences. This methylation correlates with reduced gene expression and suppression of recombination, and is thus thought to serve as a genome-defense mechanism that guards against the deleterious effects of multicopy transposable elements and aberrant gene duplications. Analysis of methylation in fungi and plants suggests that the ability of DNA repeats to pair with one another is a key to their selection for methylation. Recent data have outlined the substrate requirements for the establishment and maintenance of methylation in eukaryotic repeated sequences. Substrate-methylation patterns could help us to understand the way in which methyltransferase enzymes recognize their substrates.

Arabidopsis↗

PubMed2001

A possible prebiotic synthesis of purine, adenine, cytosine, and 4(3H)-pyrimidinone from formamide: implications for the origin of life.

The synthesis of prebiotic molecules is a major problem in chemical evolution as well as in any origin-of-life theory. We report here a plausible new prebiotic synthesis of naturally occurring purine and pyrimidine derivatives from formamide under catalytic conditions. In the presence of CaCO(3) and different inorganic oxides, namely silica, alumine, kaolin, and zeolite (Y type), neat formamide undergoes the formation of purine, adenine, cytosine, and 4(3H)-pyrimidinone, from acceptable to good yields. The role of catalysts showed to be not limited to the improvement of the yield but it is also relevant in providing a high selectivity in the products distribution.

Catalysis↗

PubMed2002

Absence of global genomic cytosine methylation pattern erasure during medaka (Oryzias latipes) early embryo development.

Two techniques were used to analyze global genomic 5-methyl cytosine methylation at CCGG sites of medaka embryo DNA. DNA was labeled by incorporation of microinjected radiolabeled deoxynucleotide into one-cell embryos. After Hpa II or Msp I digestion the radiolabeled DNA was fractionated in agarose gels and the distribution of label quantified throughout each sample lane to detect differences in fragment distribution. Alternately isolated DNA was digested with Hpa II or Msp I and the resulting generated termini end-labeled. The end-labeled digestion products were then analyzed for fragment distribution after gel fractionation. These techniques proved to be extremely sensitive, allowing comparison of genomic DNA methylation values from as few as 640 fish cells. The data suggest that in medaka embryos the vast majority (>90%) of genomic DNA is methylated at CCGG sites. Furthermore, these data support the conclusion that the extent of methylation at these sites does not change or changes very little during embryogenesis (from 16 cells to the hatchling). These data argue against active demethylation, or loss of methylation patterns by dilution, during the developmental stages between the one cell zygote and gastrulation. From a comparative viewpoint, these data may indicate that mammals and fishes methylate and demethylate their genomes in very different manners during development.

5-Methylcytosine↗

PubMed2001

Quantum chemistry predicted correlations between geometric isomerism (conformation) of -OH and =NH substituents and typical group frequencies of nucleic acid bases: cytosine.

Results of a search for correlations between typical group mode frequencies and conformation of -OH and =NH substituents is reported. The study is based on quantum chemical data (HF/6-31G (d, p) and MP2 (full)/6-31G (d, p) approximations) of 13 isomers of cytosine. It is closely related to investigations of stability and energetics of all isomers of the more common nucleic acid bases, which revealed correlations between conformation (geometric isomerism) of OH and =NH substituents on the one hand and conversion energies, relaxation of internal structural parameters, electric field gradients, etc. on the other hand. In the majority of cases alteration of conformation of these substituents is accompanied by systematic frequency shifts of stretching, in-plane and out-of-plane bending (torsional) modes conventionally assigned to such groups and by alteration of quantum chemically predicted estimates of harmonic valence force constants and structural parameters dominating the mode frequencies. For identification of group modes 'fractional' mass (other than 'natural' mass) isotope shifts of frequency and normal vectors proved useful. Likewise, estimation of effects of force constant and structural parameter alterations on frequency shifts by first order perturbation theory of the FG problem of partial structures contributed valuable insight into the origin of the shifts.

Adenine↗

PubMed2005

Association of cytosine-adenine repeat polymorphism of the estrogen receptor-beta gene with menopausal symptoms.

BACKGROUND: The nature and severity of menopausal symptoms are highly variable among women. Polymorphisms of the estrogen receptor-beta (ERbeta) gene, such as cytosine-adenine (CA) dinucleotide repeats in intron 5, have been implicated in various diseases. OBJECTIVE: We investigated the possible role of a CA dinucleotide repeat polymorphism in intron 5 of the ERbeta gene in the occurrence of menopausal and premenstrual symptoms. METHODS: Fifty-one postmenopausal Japanese women were interviewed about premenstrual symptoms, menopausal symptoms, and use of hormone replacement therapy. Menopausal symptoms were divided into vasomotor, psychological, and musculoskeletal symptoms, and summary scores were created on the basis of severity. CA repeat polymorphism of the ERbeta gene was examined using denaturing high-performance liquid chromatography with the WAVE DNA Fragment Analysis System (Transgenomic Inc., Omaha, Nebraska). RESULTS: The number of CA repeats of the ERP gene ranged from 14 to 25, and subjects could be divided into 3 groups: those with < or =17 repeats, or extremely short (E); 18 to 21 repeats, or short (S); and > or =22 repeats, or long (L). Four genotypes of CA repeats (EL, SS, SL, and LL) were found among the subjects, with SL being the most common. Relative to subjects with the SL genotype, women with SS had a 7.0-fold increased risk of vasomotor symptoms (odds ratio [OR] = 7.0; 95% CI, 1.25-39.15; P < 0.05), a 13.0-fold higher risk of psychological symptoms (OR = 13.0; 95% CI, 1.44-117.2; P < 0.01), and a 7.6-fold increased risk of premenstrual symptoms (OR = 7.6; 95% CI, 1.61-35.9; P = 0.01). The EL genotype was associated with an increased risk of vasomotor symptoms (P < 0.05), depression (P < 0.01), and premenstrual symptoms (P < 0.01). CONCLUSIONS: CA repeat polymorphism of the ERbeta gene may be associated with menopausal and premenstrual symptoms. Premenstrual symptom scores were significantly related to menopausal symptom scores.

Adenine↗

PubMed2003

Analysis of DNA (cytosine 5) methyltransferase mRNA sequence and expression in bovine preimplantation embryos, fetal and adult tissues.

Mammalian preimplantation development is a critical stage for establishment of the genomic methylation pattern and proper function of the enzymes responsible for this appear essential for normal development. To date, the vast majority of work concerning the developmental expression of the DNA cytosine 5-methyltansferases (Dnmts) has been conducted in mice. Here we report the sequence and expression of the Dnmt family during bovine preimplantation and fetal development. Bovine Dnmt mRNAs display strong sequence homology to those of human and mouse and similar to other species, exist as multiple isoforms. Two of these splice variants, which have been termed Dnmt2gamma and Dnmt3a4, represent previously unreported sequence combinations. Work presented here demonstrates early bovine embryos express mRNA coding for the somatic form of Dnmt1 and that this transcript fractionates with the ribosome. Unlike the murine model, mRNA encoding the de novo methyltransferases, Dnmt3a and 3b are present during preimplantation development and can also be found in the ribosomal subcellular fraction. Further, results of Real Time PCR analysis indicate significant differences in Dnmt mRNA expression levels exist among different tissue types as well as between fetal and adult stages. Recently, it has been postulated that the cause of abnormal methylation observed in cloned embryos may be due in part to misexpression of the Dnmt1o isoform during preimplantation development. Work presented here raises new and significant hypotheses that must be considered both regarding the cadre of DNA methyltranferases that direct epigenetic programming during normal development and regarding the implication of abnormal DNMT expression in cloned embryos.

Alternative Splicing↗

PubMed2026

Mapping Protein Occupancy on DNA with an Unnatural Cytosine Modification.

The epigenome provides a dynamic layer of gene regulatory control above the static genetic sequence. DNA base modifications are key epigenetic regulators, predominantly found within CpG contexts in mammalian genomes. Working in tandem with these DNA modifications, chromatin-associated proteins and transcription factors further control gene expression. Given the interplay of these factors, concurrent mapping of DNA base modifications with protein-DNA occupancy can greatly aid in interpreting the epigenome. Existing multimodal mapping methods include the use of DNA methyltransferases to mark accessible, protein-unbound DNA in non-CpG contexts. However, such approaches can either confound readouts with native DNA modifications or constrain users to third-generation sequencing approaches. To circumvent these limitations, we explored the possibility of introducing an unnatural DNA base modification, 5-carboxymethylcytosine, as an alternative label for protein occupancy. Here, we report our efforts to rationally engineer non-CpG-specific DNA methyltransferases to take on neomorphic DNA carboxymethyltransferase (CxMTase) activities. We find that DNA carboxymethylation of cytosines in GpC contexts shows broad compatibility with the most widely used epigenetic detection methods and can be used to reliably report on protein occupancy states. Using this approach, we reveal the single-molecule binding patterns of LexA, a master repressor in the bacterial DNA damage (SOS) response, at its self-regulated and endogenously methylated promoter. We thus show that unnatural DNA modifications can uncover novel biological insights and potentiate new approaches to multimodal epigenetic profiling.

DNA↗

PubMedDate not supplied

Interactions of cytosine derivatives with T.A interruptions in pyrimidine.purine.pyrimidine DNA triplexes.

The ability of triplex-forming oligopyrimidines to interact with duplex targets which contain a single pyr.pur interruption in their homopurine triplex binding site was studied. These oligonucleotides contain either N4-(3-carboxypropyl)deoxycytidine (3) or N4-(5-carboxytriazolyl)deoxycytidine (4) to target the pyr.pur interruption. The 3-carboxypropyl and the 5-carboxytriazolyl groups of these cytosine derivatives are designed to span the major groove of the duplex target. Molecular models suggest that the carboxyl group of 3 or 4 can serve as a hydrogen bond acceptor for the N6-amino hydrogen of A in a T.A base pair. Additional contacts are possible between the N4-amino hydrogen of 3 or 4 and the O4-carbonyl oxygen of T. UV melting experiments showed that a 15-mer, I(3), containing nucleoside 3 formed a stable triplex with duplex targets containing a single T.A interruption. The melting temperature of this triplex was 7 degrees C higher than that of a similar triplex containing a single G.T.A triad when a Tris buffer was employed. Oligomer I(3) also formed a triplex of lower stability with a target containing a G.C base pair but not with targets containing C.G, U.A, or A.T base pairs. A similar 15-mer, II, containing nucleoside 4 was found to be less selective than I(3) in its interaction with duplex targets. Thus, gel mobility shift experiments showed that in addition to interacting with T.A, oligomer II also formed triplexes with duplex targets containing U.A and C.G interruptions. These studies suggest that nucleoside derivatives which can potentially contact both bases of a pyr.pur interruption might provide a means to extend the range of sequences which can be recognized by triplex-forming oligonucleotides.

Base Sequence↗

PubMed1993

Kinetics of bisulfite-induced cytosine deamination in single-stranded DNA.

The rate of bisulfite-induced deamination of cytosine to uracil in single-stranded (ss) DNA at physiological temperature and pH was monitored by a sensitive genetic assay. The assay is based on reversion of a mutation in the lacZ alpha gene of bacteriophage M13mp2 and employs ung- (NR9404) and ung+ (MC1061) bacterial strains which are isogenic except for uracil glycosylase activity. For ss DNA incubated with 1-50 mM bisulfite and transfected into an ung- cell strain, the reversion frequency increased linearly with time of incubation and with concentration of bisulfite. Of 54 revertants sequenced, all were C-->T transitions. Reduction in reversion frequency upon transfecting ss DNA into ung+ cells indicated that the majority of mutations were occurring via a uracil intermediate. Assuming that all revertants arose via uracil, the pseudo-first-order rate constant for deamination in 10 mM sodium bisulfite and 10 mM Hepes-NaOH, pH 7.4, at 37 degrees C as measured by transfecting into an ung- cell strain was 3.5 x 10(-10) s-1, as compared to a spontaneous background rate constant of 0.6 x 10(-10) s-1 in buffer alone.

Bacteriophages↗

PubMed1993

Structural effects of the C2-methylhypoxanthine:cytosine base pair in B-DNA: A combined NMR and X-ray diffraction study of d(CGC[m2I]AATTCGCG).

C2-Methylhypoxanthine (m2I) is a synthetic analog of guanine with the N2-amino group replaced by a methyl group. We have studied the structural consequence of the m2I incorporation in DNA by a combination of X-ray crystallographic, NMR, and enzymatic analyses. The crystal structure of d(CGC[m2I]AATTCGCG) has been solved and refined to an R factor of 20.7% at 2.25-A resolution. In the DNA duplex, the two independent m2I:C base pairs maintain the Watson-Crick scheme. While the C2-methyl group of m2I is in van der Waals contact with the O2 of the base-paired cytosine, it only causes the base pair to have slightly higher propeller twist and buckle angles. Its solution structure was analyzed by the NMR refinement procedure SPEDREF [Robinson, H., & Wang, A. H.-J. (1992) Biochemistry 31, 3524-3533] using 2D nuclear Overhauser effect data. Two starting models, a relaxed fiber model and an X-ray model, were subjected to the NOE-constrained refinement using 1518 NOE cross-peak integrals to arrive at the final models with (NOE) R factors of 13.8% and 14.3%, respectively. The RMSD between the two refined models (all atoms included) is 1.23 A, which presently seems to be near the limit of convergence of NOE-based refinement. The local structures of the two models are in better agreement as measured by the RMSD of the dinucleotide steps, falling in the range 0.54-0.98 A. Both refined solution structures confirm that the m2I dodecamer structure is of the B-DNA type with a narrow minor groove at the AT region, as observed in the crystal. However, significant differences exist between the crystal and solution structures in parameters such as pseudorotation angles, propeller twist angles, etc. The solution structure tends to have a more uniform backbone conformation, an observation consistent with that concluded from the laser Raman study of d(CGCAAATTTGCG) [Benevides, J. M., Wang, A. H.-J., van der Marel, G. A., van Boom, J. H., & Thomas, G. J., J. (1988) Biochemistry 27, 931-938]. Three related dodecamers, d(CGCGAATTCGCG), d(CGC[m2I]AATTCGCG), and d(CGC[e6G]AATTCGCG), were tested as substrates for the restriction endonuclease EcoRI. The m2I dodecamer was active, but the e6G dodecamer was not. Our results illustrate the complementarity in terms of the structural information provided by the two methods, X-ray diffraction and NMR.

Base Composition↗

PubMed1991

Direct identification of the active-site nucleophile in a DNA (cytosine-5)-methyltransferase.

The overproduction, purification, and determination of the active-site catalytic nucleophile of the DNA (cytosine-5)-methyltransferase (DCMtase) enzyme M.HaeIII are reported. Incubation of purified M.HaeIII with an oligodeoxynucleotide specifically modified with the mechanism-based inhibitor 5-fluoro-2'-deoxycytidine [Osterman, D. G., et al. (1988) Biochemistry 27, 5204-5210], in the presence of the cofactor S-adenosyl-L-methionine (AdoMet), resulted in the formation of a covalent DNA-M.HaeIII complex, which was purified to homogeneity. Characterization of the intact complex showed it to consist of one molecule of the FdC-containing duplex oligonucleotide, one molecule of M.HaeIII, and one methyl group derived from AdoMet. Exhaustive proteolysis, reduction, and alkylation of the DNA-M.HaeIII complex led to the isolation of two DNA-bound peptides--one each from treatment with Pronase or trypsin--which were subjected to peptide sequencing in order to identify the DNA attachment site. Both peptides were derived from the region of M.HaeIII containing a Pro-Cys sequence that is conserved in all known DCMtases. At the position of this conserved Cys residue (Cys71), in the sequence of each peptide, was found an unidentified amino acid residue; all other amino acid residues were in accord with the known sequence. It is thus concluded that Cys71 of M.HaeIII forms a covalent bond to DNA during catalytic methyl transfer. This finding represents a direct experimental verification for the hypothesis that the conserved Cys residue of DCMtases is the catalytic nucleophile [Wu, J. C., & Santi, D. V. (1987) J. Biol. Chem. 262, 4778-4786].(ABSTRACT TRUNCATED AT 250 WORDS)

Amines↗

PubMed1992

Recognition of foldback DNA by the human DNA (cytosine-5-)-methyltransferase.

In order to specify the recognition requirements of the human DNA (cytosine-5-)-methyltransferase, two isomeric 48mers were synthesized so as to link a long block of DNA with a shorter complementary block of DNA through a tether consisting of five thymidine residues. These isomeric foldback molecules, differing only in the location of the 5-methyldeoxycytosine, were shown to be unimolecular, to contain a region of duplex DNA, and to contain a region of single-stranded DNA. When used as substrates for the DNA methyltransferase, only one of the isomers was methylated. A comparison of the structures of the two isomers allows us to begin to define the potential sites of interaction between the enzyme and the three nucleotides forming a structural motif consisting of 5-methyldeoxycytosine, its base-paired deoxyguanosine, and a deoxycytosine 5' to the paired deoxyguanosine.

Base Sequence↗

PubMed1993

Cytosine methylation enhances mitomycin C cross-linking.

Mitomycin C (MC) is a powerful antitumor agent that targets the DNA sequence CpG. Because it is likely that this dinucleotide will contain 5-methylcytosine in vivo, we have compared the cross-linking efficiency of MC for DNA containing either 5-methylcytosine or normal cytosine embedded in random-sequence DNA oligomers. We have found that mitomycin C displays a small but significant preference for methylated DNA. Recognition of an abnormal methylation pattern in the DNA of transformed cells may therefore be one mechanism by which MC exerts its chemotherapeutic effects.

5-Methylcytosine↗

PubMed1975

Calorimetric and potentiometric characterization of the ionization behavior of ribonuclease A and its complex with 3'-cytosine monophosphate.

The proton association behavior of ribonuclease A and its complex with 3'-cytosine monophosphate has been thermodynamically characterized in the pH range 4--8 at 25 degrees, mu = 0.05. Calorimetric and potentiometric titration data have been used to estimate the apparent pK values and enthalpy values for protonation of the four histidine residues of the protein, deltaHp. In the free enzyme the pK values were deduced to be 5.0, 5.8, 6.6, and 6.7 and deltaHp deduced to be -6.5, -6.5, -6.5, and -24 kcal/mol for residues 119, 12, 105, and 48, respectively. For the nucleotide-enzyme complex it was concluded that the apparent pK values of residues 119, 12, and 48 increased to an average value of about 7.2, the deltaHp values remaining constant for all histidine groups except 48. It was also concluded that only the dianionic phosphate form of the nucleotide inhibitor is bound to the enzyme in this pH range. These results are consistent with a thermodynamic model for the binding reaction in which inhibitor-enzyme association is coupled to the ionization of three imidazole residues (12, 119, and 48) and the interaction between the negative phosphate moiety of the inhibitor and the positively charged residues 12 and 119 is purely electrostatic. However, the "interaction" with residue 48 probably involves a conformational rearrangement of the macromolecule.

Binding Sites↗

PubMed2002

Nuclease resistance of oligonucleotides containing the tricyclic cytosine analogues phenoxazine and 9-(2-aminoethoxy)-phenoxazine ("G-clamp") and origins of their nuclease resistance properties.

The tricyclic cytosine analogues phenoxazine and 9-(2-aminoethoxy)-phenoxazine ("G-clamp") are known to significantly enhance the binding affinity of oligonucleotides to their complementary target DNA or RNA strands. To investigate their effect on the nuclease resistance, they were incorporated into model oligomers with a natural phosphodiester backbone, and enzymatic degradation was monitored in an in vitro assay with snake venom phosphodiesterase as the hydrolytic enzyme. In both cases, a single incorporation at the 3'-terminus completely protected the oligonucleotides against 3'-exonuclease attack. Further investigations indicate that the observed high nuclease resistance is not due to the lack of binding affinity to the enzyme's active site, since these modified oligonucleotides were able to inhibit degradation of a natural DNA fragment by bovine intestinal mucosal phosphodiesterase in a dose-dependent manner.

Animals↗

PubMed2002

DNA modification by 4-aza-3-ene-1,6-diynes: DNA cleavage, pH-dependent cytosine-specific interactions, and cancer cell cytotoxicity.

The (Z)-hex-1,5-diyne-3-ene reactive core common to the enediyne antitumor antibiotics undergoes a Bergman cyclization after proper activation to afford reactive diradical intermediates that are responsible for initiating DNA cleavage. Direct modification of the enediyne core has been proposed as a method to permit cancer cell-specific triggering of the diradical-generating cyclization. For example, 3-aza-3-ene-1,5-diynes undergo an aza-Bergman cyclization to afford the fleeting 2,5-didehydropyridine diradicals. While protonation of these aza-enediynes can afford products of diradical trapping, the hydrolytic instability of the 3-aza-3-ene-1,5-diyne moiety prevents its use in pH-triggered DNA cleaving anticancer agents. Recently, more hydrolytically stable systems incorporating the 4-aza-3-ene-1,6-diyne moiety were developed. We report here studies of the 4-aza-3-ene-1,6-diyne-containing benzimidazolium salt AZB002 [1-methyl-2-(phenylethynyl)-3-(3-phenylprop-2-ynyl)-3H-benzimidazolium tetrafluoroborate] and two structurally related heterocycles that lack the aza-enediyne functionality, AZB016 [1,3-dimethyl-2-(phenylethynyl)-3H-benzimidazolium triflate] and AZB004 [3-methyl-2-(phenylethynyl)benzothiazolium triflate]. The interaction of these compounds with supercoiled DNA, a double-stranded DNA fragment, and a short DNA duplex oligonucleotide was investigated. There are three distinct DNA interactions exhibited by AZB002: a frank strand scission leading to the relaxation of supercoiled DNA and formation of at least two different DNA adducts, one of which leads to cytosine-specific cleavage after piperidine/heat treatment. In contrast, analogues lacking the aza-enediyne functionality either fail to interact with DNA (AZB016) or cleave DNA at guanine residues, presumably through alkylation of the N-7 position (AZB004). We also investigated the cytotoxicity of AZB002 and the related heterocyclic compounds AZB004 and AZB016 and find that only the DNA interactive compounds AZB002 and AZB004 display significant cytotoxicity. In particular, AZB002 is cytotoxic against a wide range of cancer cell lines.

Alkynes↗