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PubMed1986

Formation of cytosine glycol and 5,6-dihydroxycytosine in deoxyribonucleic acid on treatment with osmium tetroxide.

OsO4 selectively forms thymine glycol lesions in DNA. In the past, OsO4-treated DNA has been used as a substrate in studies of DNA repair utilizing base-excision repair enzymes such as DNA glycosylases. There is, however, no information available on the chemical identity of other OsO4-induced base lesions in DNA. A complete knowledge of such DNA lesions may be of importance for repair studies. Using a methodology developed recently for characterization of oxidative base damage in DNA, we provide evidence for the formation of cytosine glycol and 5,6-dihydroxycytosine moieties, in addition to thymine glycol, in DNA on treatment with OsO4. For this purpose, samples of OsO4-treated DNA were hydrolysed with formic acid, then trimethylsilylated and analysed by capillary gas chromatography-mass spectrometry. In addition to thymine glycol, 5-hydroxyuracil (isobarbituric acid), 5-hydroxycytosine and 5,6-dihydroxyuracil (isodialuric acid or dialuric acid) were identified in OsO4-treated DNA. It is suggested that 5-hydroxyuracil was formed by formic acid-induced deamination and dehydration of cytosine glycol, which was the actual oxidation product of the cytosine moiety in DNA. 5-Hydroxycytosine obviously resulted from dehydration of cytosine glycol, and 5,6-dihydroxyuracil from deamination of 5,6-dihydroxycytosine. This scheme was supported by the presence of 5-hydroxyuracil, uracil glycol and 5,6-dihydroxyuracil in OsO4-treated cytosine. Treatment of OsO4-treated cytosine with formic acid caused the complete conversion of uracil glycol into 5-hydroxyuracil. The implications of these findings relative to studies of DNA repair are discussed.

Barbiturates↗
PubMed2004

High-speed conversion of cytosine to uracil in bisulfite genomic sequencing analysis of DNA methylation.

Bisulfite genomic sequencing is a widely used technique for analyzing cytosine-methylation of DNA. By treating DNA with bisulfite, cytosine residues are deaminated to uracil, while leaving 5-methylcytosine largely intact. Subsequent PCR and nucleotide sequence analysis permit unequivocal determination of the methylation status at cytosine residues. A major caveat associated with the currently practiced procedure is that it takes 16-20 hr for completion of the conversion of cytosine to uracil. Here we report that a complete deamination of cytosine to uracil can be achieved in shorter periods by using a highly concentrated bisulfite solution at an elevated temperature. Time course experiments demonstrated that treating DNA with 9 M bisulfite for 20 min at 90 degrees C or 40 min at 70 degrees C all cytosine residues in the DNA were converted to uracil. Under these conditions, the majority of 5-methylcytosines remained intact. When a high molecular weight DNA derived from a cell line (containing a number of genes whose methylation status was known) was treated with bisulfite under the above conditions and amplified and sequenced, the results obtained were consistent with those reported in the literature. Although some degradation of DNA occurred during this process, the amount of treated DNA required for the amplification was nearly equal to that required for the conventional bisulfite genomic sequencing procedure. The increased speed of DNA methylation analysis with this novel procedure is expected to advance various aspects of DNA sciences.

Base Sequence↗
PubMed1990

Excision of cytosine hydrates from Z-DNA.

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

Circular Dichroism↗
PubMed1991

Cytosine methylated DNA synthesized by Taq polymerase used to assay methylation sensitivity of restriction endonuclease HinfI.

We have studied the resistance of cytosine methylated DNA to digestion by the restriction endonuclease HinfI, using a simple PCR procedure to synthesize DNA of known sequence in which every cytosine is methylated at the 5 position. We find that HinfI cannot digest cytosine methylated DNA at the concentrations normally used in restriction digests. Complete digestion is possible using a vast excess of enzyme; under these conditions, the rate of HinfI digestion for cytosine methylated DNA is at least 1440-fold slower than for unmethylated DNA. The presence of an additional methylated cytosine at the degenerate position internal to the recognition sequence does not appear to increase the resistance to HinfI digestion. We also tested HhaII, an isoschizomer of HinfI, and found that it is completely inactive on cytosine methylated DNA. The procedure we have used should be of general applicability in determination of the methylation sensitivities of other restiction enzymes, as well as studies of the effects of methylation on gene expression in direct DNA transfer experiments.

Base Sequence↗
PubMed1998

Intercalated cytosine motif and novel adenine clusters in the crystal structure of the Tetrahymena telomere.

The cytosine-rich strand of the Tetrahymena telomere consists of multiple repeats of sequence d(AACCCC). We have solved the crystal structure of the crystalline repeat sequence at 2.5 A resolution. The adenines form two different and previously unknown clusters (A clusters) in orthogonal directions with their counterparts from other strands, each containing a total of eight adenines. The clusters appear to be stable aggregates held together by base stacking and three different base-pairing modes. Two different types of cytosine tetraplexes are found in the crystal. Each four-stranded complex is composed of two intercalated parallel-stranded duplexes pointing in opposite directions, with hemiprotonated cytosine-cytosine (C.C+) base pairs. The outermost C.C+base pairs are from the 5'-end of each strand in one cytosine tetraplex and from the 3'-end of each strand in the other. The A clusters and the cytosine tetraplexes form two alternating stacking patterns, creating continuous base stacking in two perpendicular directions along the x - and z -axes. The adenine clusters could be organizational motifs for macromolecular RNA.

Adenine↗
PubMed2000

Examination of the DNA substrate selectivity of DNA cytosine methyltransferases using mass tagging.

The biological significance of cytosine methylation is as yet incompletely understood, but substantial and growing evidence strongly suggests that perturbation of methylation patterns, resulting from the infidelity of DNA cytosine methyltransferase, is an important component of the development of human cancer. We have developed a novel in vitro assay that allows us to quantitatively determine the DNA substrate preferences of cytosine methylases. This approach, which we call mass tagging, involves the labeling of target cytosine residues in synthetic DNA duplexes with stable isotopes, such as (15)N. Methylation is then measured by the formation of 5-methylcytosine (5mC) by gas chromatography/mass spectrometry. The DNA substrate selectivity is determined from the mass spectrum of the product 5mC. With the non-symmetrical duplex DNA substrate examined in this study we find that the bacterial methyltransferase HPA:II (duplex DNA recognition sequence CCGG) methylates the one methylatable cytosine of each strand similarly. Introduction of an A-C mispair at the methylation site shifts methylation exclusively to the mispaired cytosine residue. In direct competition assays with HPA:II methylase we observe that the mispaired substrate is methylated more extensively than the fully complementary, normal substrate, although both have one HPA:II methylation site. Through the use of this approach we will be able to learn more about the mechanisms by which methylation patterns can become altered.

Alkaloids↗
PubMed1995

Electrostatic properties of beta-cytidine and cytosine monohydrate from Bragg diffraction.

The charge-density distribution in the crystal structure of the nucleoside beta-cytidine at 123 K has been determined from X-ray diffraction data (Ag K alpha, lambda = 0.5608 A) using all 7233 reflections with sin theta/lambda < or = 1.14 A-1. Maps of electrostatic potential for the cytosine base in cytidine are similar to those derived from previous charge-density studies of cytosine monohydrate and 1-beta-D-arabinosyl-cytosine, after taking chemical differences into account. These results were obtained from pseudoatom multipole refinements including kappa as a variable to describe the expansion or contraction of the spherical valence shell for each atom type. A new structure refinement of this type has also been carried out for cytosine monohydrate. A survey of kappa values for hydrogen indicates that this is a variable which is not well determined experimentally. Variations in kappa H are relayed into the population parameters obtained for other pseudoatoms and can have a small but significant effect on molecular properties, such as the dipole moment. Assuming an average theoretical value of kappa H = 1.24, the molecular dipole moments calculated from the monopole and dipole pseudoatom population parameters are 17 (4) and 8.2 (15) Debye for cytidine nucleoside and cytosine in the monohydrate, respectively. Systems of atomic point charges are presented for cytidine, cytosine and water. These are generally satisfactory in reproducing the experimentally determined molecular electrostatic potentials and dipole moments.

Chemical Phenomena↗
PubMed1981

Bacteriophage T4 alc gene product: general inhibitor of transcription from cytosine-containing DNA.

The alc gene of bacteriophage T4 was originally defined on the basis of mutations which allow late protein synthesis directed by T4 DNA containing cytosine rather than hydroxymethylcytosine. The question remained whether the normal alc gene product (gpalc) also blocks the transcription of early genes from cytosine-containing DNA. Complementation experiments were performed between hydroxymethylcytosine-containing phage which direct gpalc synthesis but carry mutations in a given gene(s) and cytosine-containing phage carrying that gene(s). The required protein would then have to be directed by the cytosine-containing DNA: it is looked for directly on polyacrylamide gels or through its physiological effects or both. For all early proteins examined in this way, no synthesis was observed when 95 to 100% of the hydroxymethylcytosine was substituted by cytosine in the infecting DNA, whereas there was significant synthesis with 75% substitution or less. The results indicate that gpalc is carried in with the infecting DNA or is made very early to block transcription of all cytosine-containing DNA.

Cytosine↗
PubMed1996

Cytosine methylation: quantitation by automated genomic sequencing and GENESCAN analysis.

Bisulfite treatment and PCR amplification of genomic DNA permits the methylation analysis of any cytosine residue in a target sequence. By cloning and sequencing the PCR product, the methylation of individual molecules can be determined, whereas direct sequencing of the PCR product can provide an average of the methylation status in the population of molecules. Reliable quantitation of cytosine methylation by direct sequencing, however, has not been possible with current methods. In this paper we describe an accurate and innovative protocol to directly quantitate the methylation of any cytosine residue in the target sequence by fluorescence-based automated genomic sequencing. Only the cytosine and thymine residues of bisulfite-treated and amplified genomic DNA are sequenced. The degree of methylation is obtained by direct comparison of the cytosine and thymine signals, which have been labeled with the same fluorescent dyes. GENESCAN analysis is employed to achieve a fast and accurate estimate of methylation at every cytosine in the target sequence. Combining direct bisulfite genomic sequencing and GENESCAN analysis permits the rapid survey of detailed DNA methylation profiles. Using this approach we have found the unexpected result that multicopy plasmid DNA grown in a Dcm host is not always fully methylated as suggested by restriction enzyme data.

Autoanalysis↗
PubMed2006

N-terminal and C-terminal cytosine deaminase domain of APOBEC3G inhibit hepatitis B virus replication.

AIM: To investigate the effect of human apolipoprotein B mRNA-editing enzyme catalytic-polypeptide 3G (APOBEC3G) and its N-terminal or C-terminal cytosine deaminase domain-mediated antiviral activity against hepatitis B virus (HBV) in vitro and in vivo. METHODS: The mammalian hepatoma cells HepG2 and HuH7 were cotransfected with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vector and 1.3-fold-overlength HBV DNA as well as the linear monomeric HBV of genotype B and C. For in vivo study, an HBV vector-based mouse model was used in which APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vectors were co-delivered with 1.3-fold-overlength HBV DNA via high-volume tail vein injection. Levels of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) in the media of the transfected cells and in the sera of mice were determined by ELISA. The expression of hepatitis B virus core antigen (HBcAg) in the transfected cells was determined by Western blot analysis. Core-associated HBV DNA was examined by Southern blot analysis. Levels of HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by quantitative PCR and quantitative RT-PCR analysis, respectively. RESULTS: Human APOBEC3G exerted an anti-HBV activity in a dose-dependent manner in HepG2 cells, and comparable suppressive effects were observed on genotype B and C as that of genotype A. Interestingly, the N-terminal or C-terminal cytosine deaminase domain alone could also inhibit HBV replication in HepG2 cells as well as Huh7 cells. Consistent with in vitro results, the levels of HBsAg in the sera of mice were dramatically decreased, with more than 50 times decrease in the levels of serum HBV DNA and core-associated RNA in the liver of mice treated with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain as compared to the controls. CONCLUSION: Our findings provide probably the first evidence showing that APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain could suppress HBV replication in vitro and in vivo.

APOBEC-3G Deaminase↗
PubMed1990

Variation and inheritance of cytosine methylation patterns in wheat at the high molecular weight glutenin and ribosomal RNA gene loci.

Chromosome marking by cytosine methylation has been examined in two gene systems in wheat--at the loci encoding high molecular weight (HMW) glutenin subunits (seed proteins) and ribosomal RNA. Variation in cytosine methylation occurs between progeny in highly inbred lines around the HMW glutenin locus. The variation is inherited through meiosis to F1 and F2 generations but occasionally a new variant arises. Specific cytosine residues lose their methyl group in the seed, the organ where the genes are expressed. Within the multigene family of ribosomal RNA genes, several subsets of genes can be defined based upon the cytosine methylation patterns. High activity of a ribosomal RNA gene locus is correlated with loss of methylation at specific cytosine residues, especially in the promoter and upstream regulatory regions. A model is described in which the subset of genes selected to be used are those to which specific regulatory proteins and transcription complexes bind most favourably. Binding of such proteins inhibits cytosine methylation and so marks the subset of genes for expression in subsequent cell generations. Examples are described where new types of RNA genes are introduced via sexual crosses that result in changes to the methylation patterns of the ribosomal RNA genes. The processes determining the changes begin, it is believed, in the fertilised egg.

Cytosine↗
PubMed1984

On the molecular basis of transition mutations. Frequency of forming 2-aminopurine-cytosine base mispairs in the G X C----A X T mutational pathway by T4 DNA polymerase in vitro.

An in vitro model system including wild-type T4 DNA polymerase, the mutagenic nucleotide analogue 2-aminopurine deoxyribonucleoside triphosphate, and poly[d(A,C)] X oligo(dT) poly(dC) X oligo(dG) template-primers is used to measure the frequency of 2-aminopurine X cytosine base mispairs formed in the G X C----A X T mutational pathway. Incorporation and turnover of the analogue into DNA is dependent on the presence of cytosine on the template strand and is reduced significantly in the presence of dGTP. 2-Aminopurine X cytosine mispairs are observed to occur at a 2-3 order of magnitude greater frequency than adenine X cytosine mispairs. The frequency of inserting 2-aminopurine deoxyribonucleoside monophosphate in place of dGMP opposite template cytosine sites is about 3-6% when either strong or weak base-stacking partners are present on the primer strand. However, enzymatic proofreading of the mispair strongly depends on base-stacking partners. Greater than 85% of misinserted 2-aminopurine deoxynucleotides are excised whenever the mispairs are formed next to 5'-primer thymine sites. A 5-fold reduction in proofreading frequency occurs when the mispair is formed with 2-aminopurine deoxynucleoside monophosphate stacked adjacent to a 5'-primer guanine. The frequency of 2-aminopurine X cytosine base mispair formation in the G X C----A X T pathway is similar to that found previously in the A X T----G X C pathway (Watanabe, S. M., and Goodman, M.F. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 2864-2868). We propose a criterion for base selection by DNA polymerase to account for the unexpected similarity in base mispairing rates in the two transition pathways.

2-Aminopurine↗
PubMed2006

The mechanism of M.HhaI DNA C5 cytosine methyltransferase enzyme: a quantum mechanics/molecular mechanics approach.

The mechanism of DNA cytosine-5-methylation catalyzed by the bacterial M.HhaI enzyme has been considered as a stepwise nucleophilic addition of Cys-81-S- to cytosine C6 followed by C5 nucleophilic replacement of the methyl of S-adenosyl-L-methionine to produce 5-methyl-6-Cys-81-S-5,6-dihydrocytosine. In this study, we show that the reaction is concerted from a series of energy calculations by using the quantum mechanical/molecular mechanical hybrid method. Deprotonation of 5-methyl-6-Cys-81-S-5,6-dihydrocytosine and expulsion of Cys-81-S- provides the product DNA 5-methylcytosine. A required base catalyst for this deprotonation is not available as a member of the active site structure. A water channel between the active site and bulk water allows entrance of solvent to the active site. Hydroxide at 10(-7) mole fraction (pH = 7) is shown to be sufficient for the required catalysis. We also show that Glu-119-CO2H can divert the reaction by protonating cytosine N3 when Cys-81-S- attacks cytosine, to form the 6-Cys-81-S-3-hydrocytosine. The reactants and 6-Cys-81-S-3-hydrocytosine product are in rapid equilibrium, and this explains the observed hydrogen exchange of cytosine with solvent.

Binding Sites↗
PubMed1980

Remission maintenance for acute nonlymphocytic leukemia: cytosine arabinoside plus 6-thioguanine versus a sequence of drug regimens.

In order to determine whether the use of a sequence of chemotherapeutic regimens plus BCG could produce longer durations of remission in adult acute nonlymphocytic leukemia than maintenance therapy with cytosine arabinoside, 6-thioguanine, plus BCG, a randomized study was performed at Washington University. Upon achieving complete remissions with daunorubicin plus cytosine arabinoside, 14 patients were randomized to receive either: Regimen A--cytosine arabinoside, 6-thioguanine, plus BCG each month; or regimen B--sequential regimens consisting of: 1) azacytidine daily for five days; 2) cyclophosphamide plus cytosine arabinoside daily for four days, prednisone daily for five days, plus vincristine on the first day; 3) prednisone, 6-mercaptopurine, and methotrexate daily for five days plus vincristine on the first day; and 4) cytosine arabinoside, 6-thioguanine, plus BCG. Each of the sequential regimens was given during consecutive months, and the cycle was then repeated starting with the first regimen. Median duration of complete remission was 27 months for 8 patients randomized to receive Regimen A, compared to only seven months for 6 patients receiving Regimen B (P less than 0.05). The median survival time of patients on Regimen B was only 14 months, and has not yet been reached in Regimen A. At 40 months after diagnosis, 75% of patients on Regimen A remain alive (P less than 0.05). Toxicity was equal for the maintenance regimens. Therefore, maintenance therapy with cytosine arabinoside, 6-thioguanine, plus BCG may be superior to the sequence of chemotherapy regimens plus BCG which was employed.

Adolescent↗
PubMed1989

Cytosine photoproduct-DNA glycosylase in Escherichia coli and cultured human cells.

Ultraviolet irradiation of DNA produces a variety of pyrimidine base damages. The activities of Escherichia coli endonuclease III and a human lymphoblast endonuclease that incises ultraviolet-irradiated DNA at modified cytosine moieties were compared. Both the bacterial and human enzymes release this cytosine photoproduct as a free base. These glycosylase activities are linear with times of reaction, quantities of enzyme, and irradiation dosages of the substrates. Both enzyme activities are similarly inhibited by the addition of monovalent and divalent cations. Analysis by DNA sequencing identified loci of endonucleolytic incision as cytosines. These are neither cyclobutane pyrimidine dimers, 6-(1,2-dihydro-2-oxo-4-pyrimidinyl)-5-methyl-2,4(1H,3H)-pyrimidinediones, nor apyrimidinic sites. This cytosine photoproduct is separable from unmodified cytosine by high-performance liquid chromatography. This separation should facilitate identification of this modified cytosine and elucidation of its biological significance.

Base Sequence↗
PubMed1996

Raman signature of the four-stranded intercalated cytosine motif in crystal and solution structures of DNA deoxycytidylates d(CCCT) and d(C8).

The Raman spectral signature of the four-stranded cytosine structure formed by intercalation of two hemiprotonated and parallel-stranded oligodeoxycytidylate duplexes (so-called i motif) has been obtained from the crystal structure of d(CCCT) [Kang, C.H., Berger, I., Lockshin, C., Ratliff, R., Moyzis, R., & Rich, A. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11636-11640]. Identification of Raman markers diagnostic of the cytosine quadruplex is complemented by results obtained in a pH titration of 2'-deoxycytosine-5'-monophosphate (5'-dCMP) to show that the spectral fingerprint associated with N3 protonation of cytosine is distinct from that of quadruplex formation. The Raman spectrum thus provides a definitive basis for evaluating quantitatively both the extent of cytosine quadruplex formation and the degree of cytosine N3 protonation in DNA. Application to aqueous d(CCCT) and d(C8) demonstrates that the four-stranded intercalated structure is formed by both of these oligodeoxycytidylates in aqueous solution. Whereas both 5'-dCMP and the d(CCCT) quadruplex exhibit a midpoint of titration (apparent pKc) of 4.5 +/- 0.2 at 10 degrees C, cytosine protonation in d(C8) is shifted significantly toward the physiological range, with pKc = 5.8 +/- 0.2. The difference in pKc between the two quadruplexes is equivalent to a free energy difference of 1.7 kcal/mol at 10 degrees C. The present findings extend the library of Raman conformation markers to deoxycytidylate residues in the novel i quadruplex. The significance of these results for probing solution conformations of telomeric DNA sequences is also considered.

Base Sequence↗
PubMed1999

Combination chemotherapy for choroidal melanoma: ex vivo sensitivity to treosulfan with gemcitabine or cytosine arabinoside.

Treatment of choroidal melanoma by chemotherapy is usually unsuccessful, with response rates of less than 1% reported for dacarbazine (DTIC)-containing regimens which show 20% or more response rates in skin melanoma. Recently, we reported the activity of several cytotoxic agents against primary choroidal melanoma in an ATP-based tumour chemosensitivity assay (ATP-TCA). In this study, we have used the same method to examine the sensitivity of choroidal melanoma to combinations suggested by our earlier study. Tumour material from 36 enucleated eyes was tested against a battery of single agents and combinations which showed some activity in the previous study. The combination of treosulfan with gemcitabine or cytosine arabinoside showed consistent activity in 70% and 86% of cases, respectively. Paclitaxel was also active, particularly in combination with treosulfan (47%) or mitoxantrone (33%). Addition of paclitaxel to the combination of treosulfan + cytosine analogue added little increased sensitivity. For treosulfan + cytosine arabinoside, further sequence and timing experiments showed that simultaneous administration gave the greatest suppression, with minor loss of inhibition if the cytosine analogue was given 24 h after the treosulfan. Administration of cytosine analogue 24 h before treosulfan produced considerably less inhibition at any concentration. While we have so far been unable to study metastatic tumour from choroidal melanoma patients, the combination of treosulfan with gemcitabine or cytosine arabinoside shows activity ex vivo against primary tumour tissue. Clinical trials are in progress.

Adenosine Triphosphate↗
PubMed1975

Treatment of variola major with cytosine arabinoside.

A controlled study of the efficacy of cytosine arabinoside in the treatment of patients with variola major was performed. Cytosine aravinoside was given intravenously at a dose approximating 3 mg/kg of body weight every 24 hr for up to seven days. All nine patients receiving cytosine arabinoside and four of the 11 patients receiving placebo died. In three of the patients receiving cytosine aravinoside, death occurred late in the illness at a time when the patient's lesions began to dry up, the patient's temperature became normal, and the patient's general condition appeared to improve. The virus could be isolated from the blood at day 7 from three of four patients treated with cytosine arabinoside as compared with zero of six control patients. Hematologic data showed a depression in the number of circulating granulocytes. It is possible that the drug lowered the resistance to infection either through direct suppression of granulocytes or through interference with other immune mechanisms. Cytosine arabinside administered in the doses used in this study is not effective in the treatment of variola major.

Adult↗