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PubMed1979

Studies on 1-beta-D-arabinofuranosyl cytosine-resistant mutants of Chinese hamster fibroblasts: III. Joint resistance to arabinofuranosyl cytosine and to excess thymidine--a semidominant manifestation of deoxycytidine triphosphate pool expansion.

Variants isolated from mutagenized Chinese hamster fibroblasts by a single cycle of exposure to ara-C distributed into two classes: (1) deoxycytidine (dC) kinase deficient clones with a high level of resistance, this phenotype was recessive in hybrids; and (2) clones exhibiting joint resistance to thymidine (dT) and to "low" ara-C concentration, this phenotype was accounted for by an increased dCTP pool. The incorporation of exogenous dC into macromolecules was markedly altered in these variants. In hybrids, the phenotype of joint resistance to dT and ara-C was semidominant. Through a second selection step, variants cumulating recessive high resistance to ara-C and semidominant dT resistance were recovered. The identification of these two classes of ara-C-resistant variants suggests an interpretation of the known phenotypes of ara-C resistance as manifestations of chromosomal gene mutations. Dominant resistance mutations might contribute to the survival of cancer cells during prolonged ara-C chemotherapy.

Animals↗
PubMed1983

In vivo effects of cytosine arabinoside on deoxyribonucleic acid replication in Chinese hamster ovary cells. 2. Cytosine arabinoside affects the rate of synthesis but not the pattern of labeling of an amplified chromosomal sequence at the onset of the S period.

The effect of 1-beta-D-arabinofuranosylcytosine (ara-C) on DNA replication in methotrexate-resistant Chinese hamster ovary cells was examined under circumstances in which nuclear DNA synthesis could be distinguished from mitochondrial DNA synthesis. G1-arrested cells were induced to traverse G1 and enter the S phase in the presence of radiolabeled thymidine and various concentrations of the drug. ara-C did not affect the kinetics of G1 traverse and subsequent entry into S after release from isoleucine deprivation, as measured by autoradiography. However, the inhibitor reduced the net rate of thymidine incorporation into nuclear DNA in a dose-dependent fashion. Autoradiography of nuclear matrix-DNA halo structures suggests that the drug inhibits nuclear thymidine incorporation by slowing chain elongation and movement of newly replicated DNA through a matrix-bound replication apparatus. Southern blot analysis of restriction digests of DNA radiolabeled in early S in the presence of ara-C indicates that the synthesis of the early-replicating amplified dihydrofolate reductase domain in these cells begins at sequences identical with those observed in cells synchronized with aphidicolin or hydroxyurea. Progressively lower concentrations of ara-C permit proportionately greater extents of the amplified unit to be replicated. These results suggest that ara-C slows the rate of chain elongation without altering the site at which DNA replication is initiated within individual replicons.

Animals↗
PubMed1982

Pharmacokinetics and metabolism of cytosine arabinoside in the central nervous system.

The pharmacokinetics and metabolism of [3H] cytosine arabinoside in the central nervous system were investigated. In vitro, the ability of rabbit brain slices and the isolated choroid plexus to accumulate [3H] cytosine arabinoside from artificial cerebrospinal fluid (CSF) containing 5 nM [3H] cytosine arabinoside was studied. Although in both tissues the uptake of [3H] cytosine arabinoside was saturable, neither brain slices nor the isolated choroid plexus achieved tissue/medium ratios greater than 1.0 in 30 min. After 15-min incubations iin artificial CSF containing 5 nM [3H] cytosine arabinoside, 8 +/- 2 (S.E.M.;N = 3)% of the 3H within the choroid plexuses was [3H] cytosine arabinoside phosphates; after comparable 30-min incubations, 4 +/- 1 (N = 4)% of the 3H within brain slices was [3H] cytosine arabinoside phosphates. In vivo, 2 hr after the i.c.v. injection into rabbits of [3H] cytosine arabinoside and 0, 2 ro 20 mumol of unlabeled cytosine arabinoside, it was shown that: 1) high concentrations of cytosine arabinoside in the CSF decreased the clearance of cytosine arabinoside from the CSF; 2) cytosine arabinoside was taken up and phosphorylated by brain cells in all regions tested; and 3) although the phosphorylation of [3H] cytosine arabinoside in brain cells was saturable, a portion of the [3H] cytosine arabinoside in brain was phosphorylated to [3H] cytosine arabinoside triphosphate even when the i.c.v. injectate contained 20 mumol of unlabeled cytosine arabinoside.

Animals↗
PubMed1990

A sensitive genetic assay for the detection of cytosine deamination: determination of rate constants and the activation energy.

Previously it has not been possible to determine the rate of deamination of cytosine in DNA at 37 degrees C because this reaction occurs so slowly. We describe here a sensitive genetic assay to measure the rate of cytosine deamination in DNA at a single cytosine residue. The assay is based on reversion of a mutant in the lacZ alpha gene coding sequence of bacteriophage M13mp2 and employs ung- bacterial strains lacking the enzyme uracil glycosylase. The assay is sufficiently sensitive to allow us to detect, at a given site, a single deamination event occurring with a background frequency as low as 1 in 200,000. With this assay, we determined cytosine deamination rate constants in single-stranded DNA at temperatures ranging from 30 to 90 degrees C and then calculated that the activation energy for cytosine deamination in single-stranded DNA is 28 +/- 1 kcal/mol. At 80 degrees C, deamination rate constants at six sites varied by less than a factor of 3. At 37 degrees C, the cytosine deamination rate constants for single- and double-stranded DNA at pH 7.4 are 1 x 10(-10) and about 7 x 10(-13) per second, respectively. (In other words, the measured half-life for cytosine in single-stranded DNA at 37 degrees C is ca. 200 years, while in double-stranded DNA it is on the order of 30,000 years.) Thus, cytosine is deaminated approximately 140-fold more slowly when present in the double helix. These and other data indicate that the rate of deamination is strongly dependent upon DNA structure and the degree of protonation of the cytosine. The data suggest that agents which perturb DNA structure or facilitate direct protonation of cytosine may induce deamination at biologically significant rates. The assay provides a means to directly test the hypothesis.

Bacteriophages↗
PubMed1998

Cloning, overexpression, and purification of cytosine deaminase from Saccharomyces cerevisiae.

Cytosine deaminase is an enzyme which has been investigated for cancer chemotherapy as a result of its ability to convert the relatively nontoxic prodrug 5-fluorocytosine into the anticancer drug 5-fluorouracil. To facilitate investigations of the utility of cytosine deaminase for cancer chemotherapy, we have cloned and expressed the enzyme from Saccharomyces cerevisiae. The DNA sequence translates into a protein of 158 amino acids in length, with a predicted molecular weight of 17,563 kilodaltons. Alignment of the cytosine deaminase protein sequence from yeast with a variety of proteins defines a novel sequence motif of cytosine or cytidine binding enzymes. Recombinant expression cassettes encoding cytosine deaminase were transfected into monkey kidney COS cells, which lack endogenous cytosine deaminase, to test for production of a functional protein. Cell extracts from these transfectants contained detectable levels of enzyme activity capable of converting 5-fluorocytosine to 5-fluorouracil. Cytosine deaminase was expressed in yeast from a cDNA cassette under the control of an inducible promoter, increasing expression 250- to 300-fold relative to wild-type strains. A purification protocol has been developed which permits recovery of 60% of cytosine deaminase in active form from induced cell lysates after two purification steps. This protocol will be useful for isolating large quantities of pure enzyme which are required for the preclinical evaluation of monoclonal antibody-cytosine deaminase conjugates in combination with 5-fluorocytosine.

Amino Acid Sequence↗
PubMed1990

Hydroxyl radical induced cross-linking of cytosine and tyrosine in nucleohistone.

Hydroxyl radical induced formation of a DNA-protein cross-link involving cytosine and tyrosine in nucleohistone in buffered aqueous solution is reported. The technique of gas chromatography-mass spectrometry was used for this investigation. A gamma-irradiated aqueous mixture of cytosine and tyrosine was first investigated in order to obtain gas chromatographic-mass spectrometric properties of possible cytosine-tyrosine cross-links. One cross-link was observed, and its structure was identified as the product from the formation of a covalent bond between carbon 6 of cytosine and carbon 3 of tyrosine. With the use of gas chromatography-mass spectrometry with selected-ion monitoring, this cytosine-tyrosine cross-link was identified in acidic hydrolysates of calf thymus nucleohistone gamma-irradiated in N2O-saturated aqueous solution. The yield of this DNA-protein cross-link in nucleohistone was found to be a linear function of the radiation dose in the range of 100-500 Gy (J.kg-1). This yield amounted to 0.05 nmol.J-1. Mechanisms underlying the formation of the cytosine-tyrosine cross-link in nucleohistone were proposed to involve radical-radical and/or radical addition reactions of hydroxyl adduct radicals of cytosine and tyrosine moieties, forming a covalent bond between carbon 6 of cytosine and carbon 3 of tyrosine. When oxygen was present in irradiated solutions, no cytosine-tyrosine cross-links were observed.

Animals↗
PubMed2003

EPR and ENDOR study of crystalline cytosine x HCl doped with 5-methylcytosine. Radiation-induced radical formation and hole transfer.

Radical formation and hole transfer were investigated in crystals of cytosine.HCl (C.HCl) doped with 0-1.1 mol-% 5-methylcytosine x HCl (5MC x HCl). The doping level was determined by NMR spectroscopy. Crystals and polycrystalline samples were X-irradiated at 295 K, 77 K and 12 K and studied with EPR, ENDOR and FSE spectroscopy at these temperatures. At 295 K the dominant radicals were the so-called 3alphaH radical, formed in 5MC by a net H-abstraction from the methyl group, and the cytosine C6 H-addition (5-yl) radical. At 12 K five radicals were identified. These were the 3alphaH radical, cytosine reduction and oxidation products, and the cytosine C6 and C5 H-addition (5-yl and 6-yl, respectively) radicals. The spectroscopic parameters for the 3alphaH radical are very similar to those of a radical observed previously in the crystalline cytosine derivatives cytidine (CR), 2'deoxycytidine hydrochloride (CdR x HCl), 5'dCMP and 3'CMP as well as in the uracil derivative 2-thiouracil (2-TU). It was shown that amounts of the order of tenths of a percent 5MC x HCl doped into crystals of C.HCl give rise to a considerable yield of 3alphaH radicals after exposure to ionizing radiation both at room temperature and at lower temperatures. This supports a previous suggestion that naturally occurring 5-methylated cytosine impurities may be responsible for the formation of 3alphaH radicals in the crystalline cytosine derivatives CR, CdR.HCl, 5'dCMP and 3'CMP and suggests that the 3alphaH radical in these systems is a 5-methylated base-centered radical. The total radical yield in doped C x HCl crystals increased considerably with the doping level, both at low temperatures and at room temperature, implying that the 3alphaH radical is more stable than the primary cytosine radicals. The relative amounts of the 3alphaH radical were obtained by using simulated benchmark spectra to reconstruct experimental EPR spectra of doped polycrystalline samples. Evidence is presented suggesting that the enhanced yield of the 3alphaH radical in doped samples is due to holes originally formed at cytosine bases and transferred to 5-methylcytosine bases in addition to the 3alphaH radical being less exposed to recombination than other cytosine radicals.

5-Methylcytosine↗
PubMed1994

Proton and charge circulation through substrate symports in Saccharomyces cerevisiae: non-classical behaviour of the cytosine symport.

A yeast strain lacking cytosine deaminase activity and over-expressing the cytosine proton symport has been used to study three aspects of symport function. (1) The proton flow during cytosine uptake after depletion of cellular ATP implies that the distribution of cytosine eventually approaches equilibrium with the proton gradient, one proton being absorbed with each molecule of cytosine. After correction for the presence of a minor leak pathway for cytosine, the cytosine distribution during energy metabolism was used to assay the magnitude of delta microH. Values of about 280 mV at pH 5 were obtained in this way. (2) Certain other substrates of the cytosine symport (hypoxanthine and especially fluorocytosine) cause the uptake of more than one equivalent of protons, but nevertheless accumulate to the same extent as cytosine. This phenomenon appears to be distinct from that of proton slip and is termed pseudochannelling. (3) The recycling of symported protons through the proton pump is an ill-defined process in plants and fungi. It usually occurs only after a distinct time lag during which the change in bulk intracellular pH may be relatively small. We have found conditions where there is no apparent time lag before protons entering yeast with glycine or histidine are recycled. This behaviour is discussed in relation to the possible voltage characteristics of the proton pump, its putative regulation by delta microH and the metabolic consequences of ATP hydrolysis being accelerated.

Adenosine Triphosphate↗
PubMed2000

Reviving a dead enzyme: cytosine deaminations promoted by an inactive DNA methyltransferase and an S-adenosylmethionine analogue.

The enzymes that transfer a methyl group to C5 of cytosine within specific sequences (C5 Mtases) deaminate the target cytosine to uracil if the methyl donor S-adenosylmethionine (SAM) is omitted from the reaction. Recently, it was shown that cytosine deamination caused by C5 Mtases M.HpaII, M.SssI and M.MspI is enhanced in the presence of several analogues of SAM, and a mechanism for this analogue-promoted deamination was proposed. According to this mechanism, the analogues protonate C5 of the target cytosine, creating a dihydrocytosine intermediate that is susceptible to deamination. We show here that one of these analogues, 5'-aminoadenosine (AA), enhances cytosine deamination by the Mtase M. EcoRII, but it does so without enhancing protonation of C5. Further, we show that uracil is an intermediate in the mutational pathway and propose an alternate mechanism for the analogue-promoted deamination. The new mechanism involves a facilitated water attack at C4 but does not require attack at C6 by the enzyme. The latter feature of the mechanism was tested by using M.EcoRII mutants defective in the nucleophilic attack at C6 in the deamination assay. We find that although these proteins are defective in methyl transfer and cytosine deamination, they cause cytosine deaminations in the presence of AA in the reaction. Our results point to a possible connection between the catalytic mechanism of C5 Mtases and of enzymes that transfer methyl groups to N(4) of cytosine. Further, they provide an unusual example where a coenzyme activates an otherwise "dead" enzyme to perform catalysis by a new reaction pathway.

Adenosine↗
PubMed2004

Alanine-scanning mutagenesis reveals a cytosine deaminase mutant with altered substrate preference.

Suicide gene therapy of cancer is a method whereby cancerous tumors can be selectively eradicated while sparing damage to normal tissue. This is accomplished by delivering a gene, encoding an enzyme capable of specifically converting a nontoxic prodrug into a cytotoxin, to cancer cells followed by prodrug administration. The Escherichia coli gene, codA, encodes cytosine deaminase and is introduced into cancer cells followed by administration of the prodrug 5-fluorocytosine (5-FC). Cytosine deaminase converts 5-FC into cytotoxic 5-fluorouracil, which leads to tumor-cell eradication. One limitation of this enzyme/prodrug combination is that 5-FC is a poor substrate for bacterial cytosine deaminase. The crystal structure of bacterial cytosine deaminase (bCD) reveals that a loop structure in the active site pocket of wild-type bCD comprising residues 310-320 undergoes a conformational change upon cytosine binding, making several contacts to the pyrimidine ring. Alanine-scanning mutagenesis was used to investigate the structure-function relationship of amino acid residues within this region, especially with regard to substrate specificity. Using an E. coli genetic complementation system, seven active mutants were identified (F310A, G311A, H312A, D314A, V315A, F316A, and P318A). Further characterization of these mutants reveals that mutant F316A is 14-fold more efficient than the wild-type at deaminating cytosine to uracil. The mutant D314A enzyme demonstrates a dramatic decrease in cytosine activity (17-fold) as well as a slight increase in activity toward 5-FC (2-fold), indicating that mutant D314A prefers the prodrug over cytosine by almost 20-fold, suggesting that it may be a superior suicide gene.

Alanine↗
PubMed2001

The cytosine N4-methyltransferase M.PvuII also modifies adenine residues.

Methylation of DNA occurs at the C5 and N4 positions of cytosine and N6 of adenine. The chemistry of methylation is similar among methyltransferases specific for cytosine-N4 and adenine-N6. Moreover these enzymes have similar structures and active sites. Previously it has been demonstrated that the DNA-(adenine-N6)-methyltransferases M.EcoRV, M.EcoRI, E. coli dam and both domains of M.FokI also modify cytosine residues at the N4 position [Jeltsch et al., J. Biol. Chem. 274 (1999), 19538-19544]. Here we show that the cytosine-N4 methyltransferase M.PvuII, which modifies the second cytosine in CAGCTG sequences, also methylates adenine residues in CAGATG/CAGCTG substrates in which the target cytosine is replaced by adenine in one strand of the recognition sequence. Therefore, adenine-N6 and cytosine-N4 methyltransferases have overlapping target base specificities. These results demonstrate that the target base recognition by N-specific DNA methyltransferases is relaxed in many cases. Furthermore, it shows that the catalytic mechanisms of adenine-N6 and cytosine-N4 methyltransferases are very similar.

Adenine↗
PubMed1993

Cytosine attack by free radicals arising from bromotrichloromethane in the presence of benzoyl peroxide catalyst: a mass spectrometric study.

We and others previously reported that CCl4 reactive metabolites are able to covalently bind to liver DNA either in vivo or in vitro. However, no demonstration of the structure of resulting adducts is available in literature. That information would be of relevance, for CCl4 exhibits null or contradictory mutagenic properties and is currently considered a non-genotoxic carcinogen. In the present study we report the nature of the reaction products formed when the putative CCl4 metabolites, .CCl3 and CCl3O2. attack cytosine in a purely chemical system where they were generated from CCl3Br in a benzoyl peroxide catalyzed reaction. Reaction products formed and identified were a) under nitrogen (.CCl3 present)--5-bromo cytosine and cytosine-5-carboxylic acid; b) under air (CCl3O2. present)--5-bromo cytosine, 5-chloro cytosine, 5-hydroxy cytosine, 6-hydroxy cytosine (tentative), chloro hydroxy uracil, 5,6-dihydroxy uracil, and chloro trichloromethyl cytosine. Results from present experiments suggest that if these reaction products were also produced in vivo during either CCl4 or CCl3Br poisoning and they were not repaired in due time prior to replication, they would lead to mutagenic events. Studies directed to obtain evidence for their in vivo formation are in course in our laboratory.

Aerobiosis↗
PubMed2001

Five novel alternatively spliced transcripts of DNA (cytosine-5) methyltransferase 2 in human peripheral blood leukocytes.

Alternative splicing of RNA molecules transcribed from DNA (cytosine-5) methyltransferases has been proposed as a mechanism by which methylation is able to effect diverse biological processes in higher eukaryotes. This study has investigated transcriptional versatility of DNA (cytosine-5) methyltransferase 2, which may methylate cytosine residues within 5'-CCTGG-3' pentanucleotides in regions of the human genome devoid of 5'-CG-3' methylation. Five novel splice variants of DNA (cytosine-5) methyltransferase 2 were identified in the peripheral blood leukocytes of healthy subjects following cloning and sequencing of RT-PCR products amplified using gene specific oligodeoxyribonucleotide primers. The generation of some of these splice variants may be influenced by the formation of secondary structures within pre-mRNA due to the repetition of sequences flanking alternatively spliced exons in a reverse and complementary orientation on the same strand. These findings enable novel approaches to investigate the role of RNA secondary structures in alternative splicing. The DNA (cytosine-5) methyltransferase 2 splice variants are generated in all the major cell types of peripheral blood, as well as in neoplastic lymphoid cells indicating that they are unlikely to generate proteins involved in control of the cell cycle or cellular differentiation. Interestingly, the gene products generated by some splice variants completely or partially lack highly conserved amino acid motifs shown to be important for the catalysis of cytosine methylation. The possibility cannot be excluded, therefore, that alternative splicing of DNA (cytosine-5) methyltransferase 2 pre-mRNA may generate protein isoforms which have different methylating capabilities or which are involved in biological processes other than the catalysis of cytosine methylation.

Alternative Splicing↗