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PubMedDate not supplied

Adenovirus-mediated tissue-specific cytosine deaminase gene therapy for human hepatocellular carcinoma with different AFP expression levels.

To evaluate whether in vitro and in vivo transferring of Escherichia coli cytosine deaminase gene to a solid tumor will confer the sensitivity to the prodrug 5-fluorocytosine (5FC) on these cells, we constructed two replication-defective adenovirus vector in which the cytosine deaminase gene was driven by CAG promoter (Adex1CACD) and AFP gene 5'-flanking region (Adex1AFPCD), respectively. By transferring these two vectors to SMMC7721AFP(-) and HepG2 human hepatocellular carcinoma (HCC) cells in vitro, we found that Adex1CACD vector could effectively suppress SMMC7721AFP(-) and HepG2 cells growing in the presence of 5FC even if the infected cell is less to 20%, while Adex1AFPCD vector only conferred HepG2 cells sensitivity to 5FC. When Adex1CACD was directly injected into established subcutaneous SMMC7721AFP(-) tumors in nude mice receiving 5FC, the tumor growth was inhibited significantly, which was consistent with those in vitro results. Furthermore, the Adex1AFPCD plus 5FC suppressed SMMC7721AFP(+) tumor growth in vivo, but not SMMC7721AFP(-) tumor. The results suggested that the CAG promoter-controlled CD gene could effectively mediate the growth inhibition in different kinds of HCC combined with administration of 5FC, and the AFP promoter-controlled CD gene could only suppress the HCCs expressing high levels of AFP. Therefore, adenovirus-mediated tumor-specific gene transfer may be a potential strategy for local control of tumor growth.

Adenoviridae↗
PubMed1999

Evidence for a dynamic role for proline376 in the purine-cytosine permease of Saccharomyces cerevisiae.

The purine-cytosine permease (PCP), a carrier located in the plasma membrane of Saccharomyces cerevisiae, mediates the active transport of purine (adenine, guanine and hypoxanthine) and cytosine into the cell. Previous studies [Ferreira, T, Brèthes, D., Pinson, B., Napias, C. & Chevallier, J. et al. (1997) J. Biol. Chem. 272, 9697-9702] suggest that the hydrophilic segment 371-377 (-I-A-N-N-I-P-N-) of the polypeptide chain may play a key role in the correct three-dimensional structure of the active carrier. This paper describes the effects of mutations in this particular segment: a four-residue deletion, Delta374-377, and two substitutions, P376G and P376R. The Delta374-377 PCP was expressed in tiny amounts and was totally inactive. When compared with the wild-type, the P376G PCP showed slightly decreased amounts and was able to transport the bases with significantly increased affinity and decreased turnover. The P376R PCP was normally expressed and targeted to the plasma membrane; however, despite a normal number of base-binding sites [1000-1200 pmol.(mg protein)-1], this mutated carrier was completely unable to transport any of its ligands. In addition, the Kd(app) for hypoxanthine binding was completely independent of the pH (within the range 3.5-6.0), showing that the conformational change induced by ligand binding was no longer present. Our results show that the 374-377 segment is essential for the expression and activity of this carrier. They also show that the P376 residue is part of an unusual secondary structure, probably a beta-turn motif, which must play a crucial dynamic role in the translocation process.

Adenine↗
PubMed2005

Ab initio study of a biradical radiationless decay channel of the lowest excited electronic state of cytosine and its derivatives.

A theoretical model for the ultrafast S1-->S0 internal conversion of cytosine is presented, in which a state switch from the initially prepared 1pipi* state to the out-of-plane deformed excited state of biradical character controls the rate of the S1(1pipi*) decay. This mechanism successfully accounts for the dramatically longer S1 lifetimes of 5-fluorocytosine and N-acetylcytosine relative to cytosine. The replacement of the C5 hydrogen atom by a methyl group is predicted to lead to a substantial, but not dramatic, increase in the S1 lifetime, also consistent with experiment. It is this ability to correctly predict the substituent effects that distinguishes the present model from the previously proposed mechanisms.

Base Composition↗
PubMed2006

Formation of hydrogen-bridged cytosine dimers on Cu(110).

Cytosine was adsorbed onto a Cu(110) surface under UHV conditions. Annealing to 370 K resulted in the formation of a (6 x 6)gg low energy electron diffraction (LEED) pattern, even at submonolayer coverages. Examination of this structure with scanning tunneling microscopy (STM) revealed islands of zigzag chains at low coverages and large ordered domains at monolayer saturation. Further annealing to 480 K initiated a phase transition to a (6 x 2)gg structure observed both by LEED and STM. High resolution electron energy loss spectroscopy spectra for both overlayer structures exhibited mainly in-plane modes suggesting upright/tilted species on the surface. Based on the experimental data and supported by density functional theory calculations, a model is proposed for the (6 x 2)gg structure, which involves the formation of deprotonated hydrogen bridge-bonded cytosine dimers, adsorbed through the oxygen atoms.

Adsorption↗
PubMed2005

Investigating local conformations of double-stranded DNA by low-energy circular dichroism of pyrrolo-cytosine.

Local base stacking and conformational mobility play a major role in the structure and function of nucleic acids. We have recently shown that the low-energy CD spectrum of 2-aminopurine (2-AP), i.e., the CD spectral region above 300 nm, can be used to monitor conformational changes in polynucleotides at or near mono- and dinucleotide 2-AP residues that replace adenine residues in DNA and RNA. Here, we extend this technique to pyrrolo-cytosine (PC), a fluorescent analogue of cytosine. The low-energy CD spectrum of a PC dinucleotide in dsDNA exhibits an exciton couplet with two bands of opposite sign centered at 350 nm. This signal is characteristic of base stacking between adjacent PC residues in a right helical conformation. Isolated PC nucleotide residues inserted into polynucleotide chains also display chirality that reflects the asymmetric environment of their sequence context. Thus, we show that the low-energy CD spectra of C(PC)A and A(PC)C sequences in dsDNA have opposite signs. It appears that the measurement of the low-energy CD spectra of PC residues will usefully complement 2-AP measurements by serving to characterize the local conformations and dynamics of nucleic acids near C residues and G-C base pairs.

Circular Dichroism↗
PubMed2006

The deprotonated guanine-cytosine base pair.

Awareness of the harmful effects of radiation has increased interest in finding the mechanisms of DNA damage. Radical and anion formation among the DNA base pairs are thought to be important steps in such damage [Collins, G. P. (2003) Sci. Am. 289 (3), 26-27]. Energetic properties and optimized geometries of 10 radicals and their respective anions derived through hydrogen abstraction from the Watson-Crick guanine-cytosine (G-C) base pair have been studied using reliable theoretical methods. The most favorable deprotonated structure (dissociation energy 42 kcal x mol(-1), vertical detachment energy 3.79 eV) ejects the proton analogous to the cytosine glycosidic bond in DNA. This structure is a surprisingly large 12 kcal x mol(-1) lower in energy than any of the other nine deprotonated G-C structures. This system retains the qualitative G-C structure but with the H...O2 distance dramatically reduced from 1.88 to 1.58 A, an extremely short hydrogen bond. The most interesting deprotonated G-C structure is a "reverse wobble" incorporating two N-H...N hydrogen bonds. Three different types of relaxation energies (4.3-54 kcal x mol(-1)) are defined and reported to evaluate the energy released via different mechanisms for the preparation of the deprotonated species. Relative energies, adiabatic electron affinities (ranging from 1.93 to 3.65 eV), and pairing energies are determined to discern which radical will most alter the G-C properties. The most stable deprotonated base pair corresponds to the radical with the largest adiabatic electron affinity, 3.65 eV. This value is an enormous increase over the electron affinity (0.60 eV) of the closed-shell G-C base pair.

Base Pairing↗
PubMed2006

Active cytosine demethylation triggered by a nuclear receptor involves DNA strand breaks.

Cytosine methylation at CpG dinucleotides contributes to the epigenetic maintenance of gene silencing. Dynamic reprogramming of DNA methylation patterns is believed to play a key role during development and differentiation in vertebrates. The mechanisms of DNA demethylation remain unclear and controversial. Here, we present a detailed characterization of the demethylation of an endogenous gene in cultured cells. This demethylation is triggered in a regulatory region by a transcriptional activator, the glucocorticoid receptor. We show that DNA demethylation is an active process, occurring independently of DNA replication, and in a distributive manner without concerted demethylation of cytosines on both strands. We demonstrate that the DNA backbone is cleaved 3' to the methyl cytidine during demethylation, and we suggest that a DNA repair pathway may therefore be involved in this demethylation.

Animals↗
PubMed1972

Methylation of cytosine residues in DNA controlled by a drug resistance factor (host-induced modification-R factors-N 6 -methyladenine-5-methylcytosine).

The proportion of 5-methylcytosine (5MeCyt) and 6-methylaminopurine (N(6)-methyladenine, 6MeAde) in bacteriophage P22 DNA was analyzed as a function of the host-specificity the phage carried. In the DNA of P22 grown in strains harboring the modifying drug-resistance-transfer-factor N-3, the 5MeCyt content was at least twice that after growth in strains lacking the factor. In contrast, the 6MeAde level of P22 DNA was unaffected by the presence or absence of the factor. The 6MeAde and 5MeCyt levels were unaffected by factors 222 and N-1, which do not modify phage DNA. The 5MeCyt/6MeAde ratio was only slightly higher in the DNA of Salmonella strains that had received the N-3 factor. After transfer of the N-3 factor to Escherichia coli strain B, which normally lacks 5MeCyt, a high content of 5MeCyt is observed. We conclude that the N-3 factor controls a DNA methylase specific for cytosine residues. If the N-3 host specificity is imparted by cytosine methylation, this would be the first instance where a biological role for 5MeCyt has been elucidated.

Adenine↗
PubMed1974

An N-glycosidase from Escherichia coli that releases free uracil from DNA containing deaminated cytosine residues.

An enzyme that liberates uracil from single-stranded and double-stranded DNA containing deaminated cytosine residues and from deoxycytidylate-deoxyuridylate copolymers in the absence of Mg(++) has been purified 30-fold from cell extracts of E. coli. The enzyme does not release uracil from deoxyuridine, dUMP, uridine, or RNA, nor does it liberate the normally occurring pyrimidine bases, cytosine and thymine, from DNA. The enzymatic cleavage of N-glycosidic bonds in DNA occurs without concomitant cleavage of phosphodiester bonds, resulting in the formation of free uracil and DNA strands of unaltered chain length that contain apyrimidinic sites as reaction products. The enzyme may be active in DNA repair, converting deaminated dCMP residues to an easily repairable form.

Binding Sites↗
PubMed1984

Photoaffinity labeling and characterization of the cloned purine-cytosine transport system in Saccharomyces cerevisiae.

8-Azido[2-3H]adenine was used as a photoaffinity label for the purine-cytosine transport system. After irradiation in the presence of the photoaffinity label, the cells were converted into protoplasts, their plasma membranes were purified, and the membrane proteins were extracted and separated by NaDodSO4/PAGE. The radioactivity was specifically incorporated into a protein with a molecular weight of 120,000. Photoaffinity labeling of this protein could be blocked by irradiation in the presence of natural substrates for the transport system. The molecular weight as determined by NaDod-SO4/PAGE was found to be twice the value calculated from mRNA analysis of the cloned gene. Incubation of exponentially growing cells with tunicamycin, an antibiotic that inhibits glycosylation of proteins, resulted in a 40% decrease in the overall initial uptake rate, which correlates with the reduction of the labeled Mr 120,000 protein. Treatment of the extracted labeled plasma membrane proteins with glycosidic enzymes resulted in disappearance of the Mr 120,000 peak and the appearance of new peaks at Mr 60,000 and Mr 73,000. These findings indicate that the purine-cytosine transport protein is a glycoprotein.

Adenine↗
PubMed1986

Hemoglobin Long Island is caused by a single mutation (adenine to cytosine) resulting in a failure to cleave amino-terminal methionine.

Hemoglobin Long Island has two separate amino acid abnormalities of beta-globin structure: an extension of the NH2 terminus by a methionine residue and a histidine-to-proline substitution at the normal second position. The NH2-terminal methionine residue, the translation product of an AUG initiation codon, is present only transiently in nascent proteins. Because of the general biological implications of this abnormality, we investigated the nature of the genetic defect of this mutant. We determined the sequence of the relevant portion of the beta-globin mRNA by means of dideoxynucleotide chain termination of the complementary DNA (cDNA) in which an oligonucleotide complementary to codons 10-17 was used as a primer for reverse transcriptase. A histidine-to-proline substitution was confirmed in the mutant mRNA by identifying an adenine-to-cytosine transversion in the second codon. However, we were unable to find any other abnormality at either the AUG initiation codon or in the 56 bases upstream from the adenine-to-cytosine transversion (encompassing most of the 5' untranslated region of the mutant beta-globin mRNA). Thus, it appears that this single lesion probably interferes with the poorly understood methionine-cleaving mechanism that modulates most of prokaryotic and eukaryotic proteins.

Adenine↗
PubMed1986

Base pairing and mutagenesis: observation of a protonated base pair between 2-aminopurine and cytosine in an oligonucleotide by proton NMR.

2-Aminopurine (AP), a potent mutagenic base analogue, most frequently pairs with thymine. In the AP X T base pair, both bases adopt normal tautomeric forms. The mechanism for the mutagenic activity arises from its observed pairing with cytosine, which has been ascribed to an enhanced tendency to adopt the rare imino tautomeric form. NMR studies in H2O on all the exchangeable protons in an oligonucleotide duplex containing an AP X T base pair show Watson-Crick hydrogen bonding. When the thymine is replaced by cytosine in the duplex, we observe an AP X C base pair. Both amino protons of AP are seen excluding the rare tautomeric form. Although several alternative structures are possible, it is shown that the second hydrogen bond is formed by protonation of the AP X C base pair and that this is the dominant species under physiological conditions.

2-Aminopurine↗
PubMed1988

Cytosine methylation does not affect binding of transcription factor Sp1.

DNA methylation may be a component of a multilevel control mechanism that regulates eukaryotic gene expression. We used synthetic oligonucleotides to investigate the effect of cytosine methylation on the binding of the transcription factor Sp1 to its target sequence (a G + C-rich sequence known as a "GC box"). Concatemers of double-stranded 14-mers containing a GC box successfully competed with the human metallothionein IIA promoter for binding to Sp1 in DNase I protection experiments. The presence of 5-methylcytosine in the CpG sequence of the GC box did not influence Sp1 binding. The result was confirmed using double-stranded 20-mers containing 16 base pairs of complementary sequence. Electrophoretic gel retardation analysis of annealed 28-mers containing a GC box incubated with an Sp1-containing HeLa cell nuclear extract demonstrated the formation of DNA-protein complexes; formation of these complexes was not inhibited when an oligomer without a GC box was used as a competitor. Once again, the presence of a 5-methylcytosine residue in the GC box did not influence the binding of the protein to DNA. The results therefore preclude a direct effect of cytosine methylation on Sp1-DNA interactions.

5-Methylcytosine↗
PubMed1992

Mechanism of SOS mutagenesis of UV-irradiated DNA: mostly error-free processing of deaminated cytosine.

We measured the kinetics of growth and mutagenesis of UV-irradiated DNA of phages S13 and lambda that were undergoing SOS repair; the kinetics strongly suggest that most of SOS mutagenesis arises from the deamination of cytosine in cyclobutane pyrimidine dimers, producing C----T transitions. This occurs because the SOS mechanism bypasses T--T dimers promptly, while bypass of cytosine-containing dimers is delayed long enough for deamination to occur. The mutations are thus primarily the product of a faithful mechanism of lesion bypass by a DNA polymerase and are not, as had been generally thought, the product of an error-prone mechanism. All of these observations are explained by the A-rule, which is that adenine nucleotides are inserted noninstructionally opposite DNA lesions.

Cytosine↗
PubMed1996

Sequence-specific recognition of cytosine C5 and adenine N6 DNA methyltransferases requires different deformations of DNA.

DNA methyltransferases modify specific cytosines and adenines within 2-6 bp recognition sequences. We used scanning force microscopy and gel shift analysis to show that M.HhaI, a cytosine C-5 DNA methyltransferase, causes only a 2 degree bend upon binding its recognition site. Our results are consistent with prior crystallographic analysis showing that the enzyme stabilizes an extrahelical base while leaving the DNA duplex otherwise unperturbed. In contrast, similar analysis of M.EcoRI, an adenine N6 DNA methyltransferase, shows an average bend angle of approximately 52 degrees. This distortion of DNA conformation by M.EcoRI is shown to be important for sequence-specific binding.

Crystallography, X-Ray↗
PubMed1996

Inter-strand C-H...O hydrogen bonds stabilizing four-stranded intercalated molecules: stereoelectronic effects of O4' in cytosine-rich DNA.

DNA fragments with stretches of cytosine residues can fold into four-stranded structures in which two parallel duplexes, held together by hemiprotonated cytosine.cytosine+ (C.C+) base pairs, intercalate into each other with opposite polarity. The structural details of this intercalated DNA quadruplex have been assessed by solution NMR and single crystal x-ray diffraction studies of cytosine-rich sequences, including those present in metazoan telomeres. A conserved feature of these structures is the absence of stabilizing stacking interactions between the aromatic ring systems of adjacent C.C+ base pairs from intercalated duplexes. Effective stacking involves only the exocyclic keto groups and amino groups of the cytidine bases. The apparent absence of stability provided by stacking interactions between the bases in this intercalated DNA has prompted us to examine the available structures in detail, in particular with regard to unusual features that could compensate for the lack of base stacking. In addition to base-on-deoxyribose stacking and intra-cytidine C-H...O hydrogen bonds, this analysis reveals the presence of a hitherto unobserved, systematic intermolecular C-H...O hydrogen bonding network between the deoxyribose sugar moieties of antiparallel backbones in the four-stranded molecule.

Base Composition↗
PubMed1996

New 5' regions of the murine and human genes for DNA (cytosine-5)-methyltransferase.

DNA (cytosine-5)-methyltransferases (EC 2.1.1.37) maintain patterns of methylated cytosine residues in the mammalian genome; faithful maintenance of methylation patterns is required for normal development of mice, and aberrant methylation patterns are associated with certain human tumors and developmental abnormalities. The organization of coding sequences at the 5'-end of the murine and human DNA methyltransferase genes was investigated, and the DNA methyltransferase open reading frame was found to be longer than previously suspected. Expression of the complete open reading frame by in vitro transcription-translation and by transfection of expression constructs into COS7 cells resulted in the production of an active DNA methyltransferase of the same apparent mass as the endogenous protein, while translation from the second in-frame ATG codon produced a slightly smaller but fully active protein. Characterization of mRNA 5' sequences and the intron-exon structure of the 5' region of the murine and human genes indicated that a previously described promoter element (Rouleau, J., Tanigawa, G., and Szyf, M. (1992) J. Biol. Chem. 267, 7368-7377) actually lies in an intron that is more than 5 kilobases downstream of the transcription start sites.

Amino Acid Sequence↗
PubMed1998

Nitric oxide-induced deamination of cytosine and guanine in deoxynucleosides and oligonucleotides.

The autoxidation of nitric oxide (NO.) forms the nitrosating agent N2O3, which can directly damage DNA by deamination of DNA bases following nitrosation of their primary amine functionalities. Within the G:C base pair, deamination results in the formation of xanthine and uracil, respectively. To determine the effect of DNA structure on the deamination of guanine and cytosine, the NO.-induced deamination rate constants for deoxynucleosides, single- and double-stranded oligonucleotides, and a G-quartet oligonucleotide were measured. Deamination rate constants were determined relative to morpholine using a Silastic membrane to deliver NO. at a rate of approximately 10-20 nmol/ml/min for 60 min, yielding a final concentration of approximately 600-1200 microM NO2-. GC/MS analysis revealed formation of nanomolar levels of deamination products from millimolar concentrations of deoxynucleosides and oligomers. Deamination rate constants for cytosine and guanine in all types of DNA were lower than the morpholine nitrosation rate constant by a factor of approximately 10(3)-10(4). Xanthine was formed at twice the rate of uracil, and this may have important consequences for mechanisms of NO.-induced mutations. Single-stranded oligomers were 5 times more reactive than deoxynucleosides toward N2O3. Double-stranded oligomers were 10-fold less reactive than single-stranded oligomers, suggesting that Watson-Crick base pairing protects DNA from deamination. G-quartet structures were also protective, presumably because of hydrogen bonding. These results demonstrate that DNA structure is an important factor in determining the reactivity of DNA bases with NO.-derived species.

Cytosine↗