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PubMed1995

Function of Pro-185 in the ProCys of conserved motif IV in the EcoRII [cytosine-C5]-DNA methyltransferase.

ProCys in the conserved sequence motif IV of [cytosine-C5]-DNA methyltransferases is known to be part of the catalytic site. The Cys residue is directly involved in forming a covalent bond with the C6 of the target cytosine. We have found that substitution of Pro-185 with either Ala or Ser resulted in a reduced rate of methyl group transfer by the EcoRII DNA methyltransferase. In addition, we observed an increase in the Km for substrate S-adenosyl-L-methionine (AdoMet), but a decrease in the Km for substrate DNA. This is reflected in minor changes in kcat/Km for DNA, but in 10- to 100-fold reductions in kcat/Km for AdoMet. This suggests that Pro-185 is important to properly orient the activated cytosine and AdoMet for methyl group transfer by direct interaction with AdoMet and indirectly via the Cys interaction with cytosine.

Amino Acid Sequence↗
PubMed1982

A new Salmonella tester strain, TA97, for the detection of frameshift mutagens. A run of cytosines as a mutational hot-spot.

We have developed a new Salmonella tester strain, TA97, for use in the Salmonella/microsome mutagenicity test. DNA sequencing has shown that this strain contains an added cytosine, resulting in a run of six cytosines at the mutated site in the histidine D gene. Its mutagenic specificity is similar to that of the frameshift mutagen tester strain, TA1537, which also contains an added cytosine in a run of cytosines and is currently among the five standard tester strains used for general mutagen screening. We assessed the mutagenic potency of 21 frameshift mutagens for TA1537 and TA97. TA97 was considerably more sensitive than TA1537 to reversion by these frameshift mutagens. In addition, one agent, PR toxin (from Penicillium roqueforti), which was not detected by any of the previously existing standard tester strains, did revert TA97; and two substituted aryl-alkyl triazenes, which had not been reported previously to be frameshift mutagens, were mutagenic in this new tester strain. We suggest that TA1537 be replaced by TA97 for general screening of mutagenicity.

Antipsychotic Agents↗
PubMed1983

Regulation of viral and cellular oncogene expression by cytosine methylation.

Mink cells morphologically transformed by either Snyder-Theilen feline sarcoma virus (ST-FeSV) or Abelson murine leukemia virus (Abelson-MuLV) exhibit relatively high rates of reversion to the nontransformed phenotype. The proviral DNAs are conserved within the revertant lines and have not undergone changes in integration sites due to translocations or other genomic rearrangements. In contrast, expression of well-defined viral-encoded transforming proteins is blocked and elevated levels of phosphotyrosine characteristic of the parental transformed cells are reduced to control levels. Loss of the transformed phenotype is associated with increased cytosine methylation of proviral DNA sequences while levels of methylation resume control levels upon spontaneous retransformation of revertant clones. Following molecular cloning, and transfection to Rat-2 cells, ST-FeSV proviral DNAs from revertant and transformed cells induced similar numbers of transformed foci. Cytosine methylation sites involved in regulation of expression of the major ST-FeSV encoded transforming protein have been localized within the proviral DNA itself rather than in adjacent cellular flanking sequences. In contrast to the v-fes proviral DNA, c-fes, the cellular homolog of the ST-FeSV acquired transforming sequences, is highly methylated in cytosine residues in both transformed and revertant clones. These findings demonstrate regulation of viral oncogene-mediated transformation by cytosine methylation and suggest that expression of cellular homologs of viral oncogenes, such as c-fes, are also subject to regulation at this level.

Animals↗
PubMed1988

Photoexchange products of cytosine and 5-methylcytosine with N alpha-acetyl-L-lysine and L-lysine.

The photoinduced exchange reactions of cytosine (Ia) and 5-methylcytosine (IIa) with N alpha-acetyl-L-lysine, a derivative of the common amino acid L-lysine, were studied. These reactions of Ia and IIa at pH 7.5 produce, respectively, 2-N-acetylamino-6-(1-cytosinyl)hexanoic acid (Ib) and 2-N-acetylamino-6-(1-(5-methylcytosinyl]hexanoic acid (IIb) as major final products. In addition, small amounts of the corresponding deamination products were formed in the 5-methylcytosine-N alpha-acetyl-L-lysine and cytosine-N alpha-acetyl-L-lysine systems, namely 2-N-acetylamino-6-(1-thyminyl)-hexanoic acid and 2-N-acetylamino-6-(1-uracilyl)hexanoic acid. The compounds Ib and IIb were deacetylated by acid hydrolysis to yield the corresponding lysine products: 2-amino-6-(1-cytosinyl)hexanoic acid (Ic) and 2-amino-6-(1-(5-methylcytosinyl]hexanoic acid (IIc). The compound Ic was identified as a product in the photoreaction of cytosine with L-lysine at near neutral pH, while IIc is found as a product in the corresponding reaction of 5-methylcytosine. The occurrence of the above photoexchange reactions at pH values near those found in physiological systems could have biological implications; in particular, our observations suggest that cytosine and 5-methylcytosine residues, contained in DNA, might react with the epsilon-amino groups of lysine residues in proteins upon UV irradiation of nucleosomes and other DNA-protein complexes under physiological conditions.

5-Methylcytosine↗
PubMed2004

2-Ureidoquinoline: a useful molecular element for stabilizing single cytosine and thymine bulges.

We have demonstrated that aromatic heterocycles having hydrogen-bonding surfaces complementary to those of nucleotide bases are effective molecular elements for the binding to single nucleotide bulges and base mismatches. We here report that a new molecule, 2-ureidoquinoline having an alignment of hydrogen-bonding groups in the order of acceptor-donor-donor stabilizes single cytosine and thymine bulges in duplex DNAs. Furthermore, a dimeric form of 2-ureidoquinoline stabilizes cytosine-cytosine and cytosine-thymine mismatches.

Base Pair Mismatch↗
PubMed1999

Polymorphism for mutation of cytosine to thymine at location 677 in the methylenetetrahydrofolate reductase gene is associated with recurrent early fetal loss.

OBJECTIVE: This study was undertaken to determine whether a cytosine to thymine mutation at nucleotide 677 in the gene encoding for methylenetetrahydrofolate reductase is associated with particular subtypes of recurrent unexplained spontaneous abortion. STUDY DESIGN: The prevalences of cytosine to thymine mutation at nucleotide 677 in the gene encoding for methylenetetrahydrofolate reductase among 41 patients with recurrent unexplained spontaneous abortions and among 18 healthy control subjects were determined with polymerase chain reaction. RESULTS: Homozygosity and heterozygosity for the cytosine to thymine mutation at nucleotide 677 in the gene encoding for methylenetetrahydrofolate reductase were observed at nonsignificantly different rates among patients and control subjects (9% and 48% versus 22% and 38%, respectively, P <.95). Among patients with recurrent unexplained spontaneous abortions both homozygosity and heterozygosity were associated with significantly increased prevalence of recurrent early fetal loss rather than with repeated anembryonic gestations (P <.0001). CONCLUSION: The observation that polymorphism for the cytosine to thymine mutation at nucleotide 677 in the gene encoding for methylenetetrahydrofolate reductase is associated with repeated early fetal losses rather than with anembryonic gestations strengthens the argument for the role of hypercoagulability and abnormal uteroplacental vasculature in recurrent spontaneous abortion.

Abortion, Habitual↗
PubMed2003

In vivo deamination of cytosine-containing cyclobutane pyrimidine dimers in E. coli: a feasible part of UV-mutagenesis.

We have estimated in vivo deamination rates for cytosines in cyclobutane pyrimidine dimers (CPD or PyPy) in UV-irradiated E. coli deficient in uracil DNA glycosylase. The protocol consisted of UV-irradiation, holding in buffer to allow for deamination of cytosines in CPDs and photoreversal (PR) to establish uracils where cytosines in CPD deaminated. The deamination rate at TC photoproducts targeting glutamine tRNA suppressor mutations was estimated from the increase in the mutation frequency after PR (MF(PR)) that developed as UV-irradiated cells were held before PR. Evidence suggested that an earlier study with this protocol under-estimated the deamination rate at sites producing the same mutations in an E. coli B/r strain. With a K12 strain, where the targeting apparently is principally by CPD and not (6-4) photoproducts, a larger rate of k = 0.0091 min(-1) at 42 degrees C resulted. The dark assay for MF also increased significantly with time for deamination consistent with a model for efficient mutation by translesion synthesis at uracil-containing CPD. In addition, we used a strain constructed by Cupples and Miller in which beta-galactosidase was inactive because -GGG- was at codon 461 and would revert to Lac(+) only when replaced by -GAG- or -GAA- for glutamate. CC photoproducts at this target site in the opposite DNA strand could reveal effects of first and second deaminations in the same CPD. MF(PR) for Lac(+) mutations increased and then decreased as a function of deamination time (at six temperatures 36-48 degrees C). Fitting an approximate model equation that distinguished two different deamination rates to these data suggested a first deamination producing Lac(+) at a rate about eight-fold less than a second deamination restoring the Lac(-) phenotype. We conclude that deamination, changing a cytosine-containing CPD to a uracil-containing CPD, could be an integral part of UV-induced C-to-T mutations.

Cytosine↗
PubMed2001

Interactions of d(10) metal ions with hippuric acid and cytosine. X-ray structure of the first cadmium (II)-amino acid derivative-nucleobase ternary compound.

The interactions of Zn(II), Cd(II) and Hg(II) with hippuric acid (hipH) were studied and several novel compounds were synthesized and studied by NMR. Some new metal-hippuric-cytosine ternary compounds were formed and the structure of the [Cd(hip)(2)(cyt)(H(2)O)](2) ternary complex resolved. Each cadmium (II) atom has a distorted trigonal bipyramid coordination which is linked to a water molecule, a cytosine via N(3), a carboxylic oxygen atom of a hippurate moiety and two bridging dicoordinated hippurates bound through the carboxylic oxygen atoms. To these five main bonds, two longer ancillary interactions can be observed: the second oxygen of the monocoordinated hippurate group and the carboxylic oxygen of the cytosine ligand. The compound is stabilized by an intramolecular stacking between the benzene and cytosine rings and by the hydrogen bonds between the coordinated water molecules and the ligands. This is, to our knowledge, the first structure of a cadmium-amino acid derivative-natural nucleobase compound described so far.

Cadmium↗
PubMed1992

Recognition of a guanine-cytosine base pair by 8-oxoadenine.

Two oligodeoxyribonucleotides, d-CTTCTTTTTTATTTT, I(A), and d-ATTATTTTTTATTTT, II(A), where C is 5-methylcytosine and A is 8-oxoadenine, were prepared and their interactions with the duplex d-GAAGAAAAAAYAAAA/d-TTTTZTTTTTTCTTC, III.IV(Y.Z), were studied. Oligomers I(A) and II(A) each form triplexes with III.IV(G.C) at temperatures below 20 degrees C as shown by continuous variation experiments, melting experiments, and circular dichroism (CD) spectroscopy. The CD spectra of these triplexes are almost identical to those formed by I(C) and II(C), oligomers which contain cytosine in place of 8-oxoadenine. This suggests that the 8-oxoadenine-containing triplexes have conformations which are very similar to those of the cytosine-containing triplexes. The melting temperature (Tm) for dissociation of the third strand of triplex II.III.IV(A.G.C) is 22 degrees C at pH 7.0 and 8.0, whereas the Tm of the corresponding transition in triplex II.III.IV(C.G.C) decreases from 28 degrees C at pH 7.0 to 17 degrees C at pH 8.0. The pH dependence of the Tm in the latter triplex reflects the necessity of protonating the N-3 of cytosine in order for it to form two hydrogen bonds with G of the G.C base pair. It appears that the keto form of 8-oxoadenine can potentially form two hydrogen bonds with the N-7 and O-6 atoms of G of the G.C base pair, when the 8-oxoadenine is in the syn conformation and in contrast to cytosine does not require protonation of the base. Oligomer I(A) does not form triplexes with III.IV(Y.Z) when Y.Z is A.T or T.A.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine↗
PubMed1992

Template-directed interference footprinting of cytosine contacts in a protein-DNA complex: potent interference by 5-aza-2'-deoxycytidine.

In the template-directed interference (TDI) footprinting method (Hayashibara & Verdine, 1990), analogs of the naturally occurring DNA bases are incorporated into DNA enzymatically and assayed for interference of sequence-specific binding by a protein. Here we extend this method to include analysis of contacts of amino acid residues to the major groove surface of cytosine residues (TDI-C footprinting). The base analog 5-aza-2'-deoxycytidine, in which the hydrophobic 5-CH of cytosine is replaced by a hydrophilic aza nitrogen, was incorporated into DNA via the corresponding 5'-triphosphate. The analog was found to base pair with guanine during polymerization, resulting in substitution of 2'-deoxycytidine residues. TDI-C footprints of the lambda repressor-OL1 operator complex revealed apparent contacts to the cytosines at operator positions 7 and 8. Inspection of the high-resolution X-ray crystal structure of the lambda-OL1 complex (Clarke et al., 1992; Beamer & Pabo, 1992) revealed that C8 makes a hydrogen binding contact with the Lys3; C7, on the other hand, makes a previously unnoticed hydrophobic contact with the alkane side chain of Lys3. In only the consensus operator half-site was cytosine interference observed, suggesting that the nonconsensus arm binds DNA very differently if at all. The N-terminal arm represents the archetypal case of a sequence-specific peptide-DNA complex characterized at high resolution; thus, the present studies suggest strategies for design and screening of DNA binding peptides. The finding that 5-aza-2'-deoxycytidine inhibits sequence-specific DNA binding proteins may suggest an alternative rationale for the biological activities of this and related azapyrimidine nucleosides.

Antineoplastic Agents↗
PubMed1996

Murine DNA cytosine-C5 methyltransferase: pre-steady- and steady-state kinetic analysis with regulatory DNA sequences.

We present the first description of KmDNA, KdDNA, Kcat, and Kmethylation for a mammalian DNA methyltransferase. Homogeneous, 190 000 MTDNA (cytosine-5-)-methyltransferase isolated from mouse erythroleukemia cells has turnover constants of 0.15-0.59 h-1 with single-stranded and unmethylated double-stranded oligonucleotides containing a single CpG dinucleotide. These substrates were designed to mimic DNA transcriptional cis elements previously reported to have cytosine C-5-methylated regulation. The rate-limiting step for these substrates is the methylation step itself. In contrast, hemimethylated double-stranded substrates show burst kinetics, consistent with a rapid methylation event (3 h-1) followed by a slower step which determines steady-state Kcat. Hemimethylated and unmethylated double-stranded DNA shows similar binding affinities; these results reveal the molecular basis for the enzyme's preference for hemimethylated DNA to be the methyl transfer step. Substrates with multiple recognition sites do not show burst kinetics and have turnover rate constants of 6 h-1. Catalytic turnover for the mammalian enzyme is thus approximately 10-fold slower than that for the related bacterial enzymes. Our combined results show quantitatively that one enzyme is certainly capable of both maintenance and de novo methylation and that maintenance of the genomic methylation pattern is preferred over the de novo establishment of new patterns. Direct comparison of the mammalian enzyme with the bacterial DNA cytosine-C5 methyltransferase, M.SssI, indicates dramatic differences in preferences for single-stranded, double-stranded, and hemimethylated double-stranded substrates. Moreover, the specificity hierarchy shown for the M.SssI is derived from very different changes in K(m) and catalysis than those observed for the mammalian DCMTase. These results demonstrate that the M.SssI, and perhaps other DNA cytosine methyltransferases from bacteria, is functionally dissimilar to the mammalian enzyme.

Animals↗
PubMed1997

At physiological pH, d(CCG)15 forms a hairpin containing protonated cytosines and a distorted helix.

To investigate potential structures of d(CGG/CCG)n that might relate to their biological function and association with triplet repeat expansion diseases (TREDs), the structure of a single-stranded (ss) oligonucleotide containing d(CCG)15 [ss(CCG)15] was examined by studies of the pH and temperature dependence of electrophoretic mobility, UV absorbance, circular dichroism, chemical modification, and P1 nuclease digestion. ss(CCG)15 had an unusually high pKa (7.7 +/- 0.2). At pH 8.5, ss(CCG)15 formed a relatively unstable (Tm = 30 degrees C in 1 mM Na+) hairpin containing CpG base-pair steps. At pH 7.5, the hairpin contained protonated cytosines but no detectable C x +C base pairs, increased thermal stability (Tm = 37 degrees C), increased stacking of the CpG base-pair steps, and a single cytosine that was flipped away from the central portion of the helix. Examination of ss(CCG)18 and ss(CCG)20, which were designed to adopt hairpins containing alternative GpC base-pair steps, revealed hairpins containing CpG base-pair steps, pKas of approximately 8.2 and approximately 8.4, respectively, and distorted helices. The results suggest that DNA sequences containing (CCG)(n > or = 15) adopt hairpin conformations that contain CpG rather than GpC base-pair steps; the mismatched cytosines are protonated at physiological pH but are not H-bonded. We propose that protonation arises from the stacking of two cytosines in the minor groove of a distorted helix.

Base Sequence↗
PubMed2003

Theoretical and experimental investigations on the chemical reactions of positively charged phenyl radicals with cytosine and 1-methylcytosine.

The chemical behavior of positively charged phenyl radicals toward cytosine, 1-methylcytosine, and some pyrimidine analogues in the gas phase was investigated both theoretically by performing molecular orbital calculations and experimentally by using FT/ICR mass spectrometry. The phenyl radicals react with cytosine and 1-methylcytosine predominately by hydrogen abstraction and addition. For cytosine, the preferred site for hydrogen abstraction appears to be the amino group, and addition occurs preferentially at the N3 and N1 positions of the keto and enol tautomeric forms, respectively. For 1-methylcytosine, the methyl group is the thermodynamically favored site for hydrogen abstraction and N3 for addition. Possible structures and formation mechanisms are suggested for two unknown product ions formed upon the reaction of cytosine with the 3-dehydro-N-phenylpyridinium radical cation.

Benzene Derivatives↗
PubMed2005

Properties of a new fluorescent cytosine analogue, pyrrolocytosine.

Pyrrolocytosine is a novel, environment sensitive, fluorescent base that can be used in place of cytosine as a fluorescent marker in nucleic acids. In this work the results of a detailed computational investigation into the hybridization and photochemical properties of the base are reported. The interaction energy of the base pair formed between pyrrolocytosine and guanine, calculated at the MP2/6-31G(d,0.25)//HF/6-31G(d,p) level, was found to be -27.2 kcal mol(-1), comparing very favorably with the value calculated for the cytosine and guanine base pair, -25.8 kcal mol(-1). The wavelengths for the vertical transitions of pyrrolocytosine and cytosine were determined using both the configuration interaction technique, with singly excited configurations (CIS) and time-dependent density functional theory using the B3LYP functional (TDB3LYP). It was found that the spacing between the first pipi state and the first npi state was significantly larger in the case of pyrrolocytosine than cytosine, providing a rationale for the higher fluorescence quantum yield of the former. Hydrogen bonding of pyrrolocytosine to guanine did not affect the predicted fluorescence properties of pyrrolocytosine whereas stacking guanine above pyrrolocytosine, in a manner appropriate to B-form DNA, significantly reduced the predicted fluorescence. Calculations on the two base systems using the TDB3LYP method produced low-lying charge-transfer states which are not predicted when the CIS method is used and are not thought to be physically meaningful.

Cytosine↗
PubMed2001

Concentration by evaporation and the prebiotic synthesis of cytosine.

The efficient prebiotic synthesis of cytosine from urea and cyanoacetaldehyde (CA) has recently been claimed to be invalid on the basis of possible side reactions of the starting materials and the inapplicability of prebiotic syntheses using drying beach conditions. We therefore have investigated the synthesis of cytosine and uracil from urea and cyanoacetaldehyde at 100 degrees C under dry-down conditions, and in solution at 4 degrees C and -20 degrees C. We find that cytosine is produced from the low temperature experiments more efficiently than calculated from the Arrhenius extrapolation from higher temperatures, i.e., 60-120 degrees C. In addition, we find that CA dimer is as efficient as the monomer in cytosine synthesis. We also studied whether evaporating very dilute solutions of nonvolatile organic compounds will concentrate according to theory. Solutions as dilute as 10(-4) M concentrate from pure water approximately according to theory. Similar solutions in 0.5 M NaCl have less than theoretical concentrations due to absorption, but concentrations near dryness were very high.

Acetaldehyde↗
PubMed2002

Comments on 'concentration by evaporation and the prebiotic synthesis of cytosine'.

The claim by Nelson et al. (2001) that the reaction of cyanoacetaldehyde and urea provides 'an efficient prebiotic synthesis' of cytosine is disputed. The authors have not dealt with the important points presented in a criticism of this reaction (Shapiro, 1999): (1) The reactants undergo side reactions with common nucleophiles that appear to proceed more rapidly than cytosine formation, and (2) No reactions have been described thus far that would produce cytosine at a rate sufficient to compensate for its decomposition by deamination, and permit accumulation over extended periods of time. Instead, Nelson et al. have conducted 'drying-down' experiments, in an effort to simulate evaporations on the early Earth, but the design of these experiments is flawed. The initial reactant concentrations are much higher than might be expected in a natural setting, and potentially interfering substances such as glycine, cyanide and thiols have been excluded. 'Drying beaches and drying lagoons' have been invoked as sites for such a reaction but no effort has been made to describe the characteristics of such sites or to estimate their frequency with reference to the present Earth. In the absence of contradictory data, the conclusion put forward in Shapiro (1999) remains valid: 'It was quite unlikely that cytosine played a role in the origin of life'.

Cytosine↗
PubMed2001

Specific targeting of cytosine deaminase to solid tumors by engineered Clostridium acetobutylicum.

The presence of severe hypoxia and necrosis in solid tumors offers the potential to apply an anaerobic bacterial enzyme/prodrug approach in cancer treatment. In this context the apathogenic C. acetobutylicum was genetically engineered to express and secrete E. coli cytosine deaminase (CDase). Considerable levels of functional cytosine deaminase were detected in lysates and supernatants of recombinant C acetobutylicum cultures. After administration of the recombinant Clostridium to rhabdomyosarcoma bearing rats used as a model, cytosine deaminase could be detected at the tumor site. Moreover, following administration of the vascular targeting agent combretastatin A-4 phosphate significantly increased levels of cytosine deaminase were detected at the tumor site as a consequence of enlarged tumor necrosis and subsequently improved growth of C. acetobutylicum. The results provide evidence for the potential application of Clostrisdium-based therapeutic protein transfer to tumors in anticancer therapy.

Animals↗
PubMed2006

Low ionic strength reduces cytosine photoreactivity in UVC-irradiated isolated DNA.

Exposure of DNA to far-UV radiation leads to the formation of several types of dimeric lesions, including cyclobutane dimers and (6-4) photoproducts. In order to gain insights into the main parameters driving DNA photochemistry, the effect of ionic strength on the yield of formation of these photoproducts was investigated in UVC-irradiated samples of isolated genomic DNA. The main consequence of lowering the ionic strength was a decrease in the UVC-induced formation of thymine-cytosine and cytosine-cytosine photoproducts. The reactivity of thymine-thymine and cytosine-thymine doublets was hardly affected. Evidence was obtained against a major role played by duplex denaturation in these observations. A more likely explanation is a change in the DNA structure as the result of a larger extent of protonation at low counter-ions concentration.

Animals↗