SEARCH · Search PubMed
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
An assay for the cytosine methylation level in the eukaryotic DNA CCWGG sequence is proposed. The method is based on the ability of DNA methylase BstNI to methylate DNA containing in a CCWGG site a nonmodified or 5-methylated cytosine to yield N4-methyl- or N4,5'-dimethylcytosine, respectively.
DNA containing 5-azacytosine is an irreversible inhibitor of DNA(cytosine-5)methyltransferase. This paper describes the binding of DNA methyltransferase to 32P-labeled fragments of DNA containing 5-azacytosine. The complexes were identified by gel electrophoresis. The EcoRII methyltransferase specified by the R15 plasmid was purified from Escherichia coli B(R15). This enzyme methylates the second C in the sequence CCAGG and has a molecular mass of 60,000 Da. Specific binding of enzyme to DNA fragments could be detected if either excess unlabeled DNA or 0.8% sodium dodecyl sulfate was added to the reaction mixture prior to electrophoresis. Binding was dependent upon the presence of both the CCAGG sequence and azacytosine in the DNA fragment. S-Adenosylmethionine stimulated the formation of the complex. The complex was stable to 6 M urea but could be digested with pronase. These DNA fragments could be used to detect the presence of several different methyltransferases in crude extracts of E. coli. No DNA protein complexes could be detected in E. coli B extracts, a strain that contains no DNA(cytosine-5)methyltransferases. The chromosomally determined methylase with the same specificity as the purified EcoRII methylase could be detected in crude extracts of E. coli K12 strains. The MspI methylase cloned in E. coli HB101 could also be detected in crude extracts. These enzymes are the only proteins that bind azacytosine-containing DNA in crude extracts of E. coli.
Condensation of single molecules from solution into crystals represents a transition between distinct energetic states. In solution, the atomic interactions within the molecule dominate. In the crystalline state, however, a set of additional interactions are formed between molecules in close contact in the lattice--these are the packing interactions. The crystal structures of d(CCCT), d(TAACCC), d(CCCAAT), and d(AACCCC) have in common a four-stranded intercalated cytosine segment, built by stacked layers of cytosine.cytosine+ (C.C+) base pairs coming from two parallel duplexes that intercalate into each other with opposite polarity. The intercalated cytosine segments in these structures are similar in their geometry, even though the sequences crystallized in different space groups. In the crystals, adenine and thymine residues of the sequences are used to build the three-dimensional crystal lattice by elaborately interacting with symmetry-related molecules. The packing elements observed provide novel insight about the copious ways in which nucleic acid molecules can interact with each other--for example, when folded in more complicated higher order structures, such as mRNA and chromatin.
The changes in the ion signals in the isotope cluster, mass resolution, signal-to-noise ratio and mass accuracy for matrix-assisted laser desorption/ionization (MALDI) of DNA oligonucleotides (dGGATC, dCAGCt, and dAACCGTT) and their fragment ions were evaluated, and these data were compared with those obtained using 3-hydroxypicolinic acid. Mass spectra obtained by using 2,5-dihydroxybenzoic acid (2,5-DHB) appear to have differences from the theoretical isotopic clusters, which arise by reductive hydrogenation producing a second peak at the M + 2 isotope of the native oligonucleotide. Based on the patterns of the isotopic envelope observed in the in-source decay fragments, we propose that cytosine is the site of reduction. We do not find evidence of reduction of oligonucleotides, viz. dTGGGGTT, that do not contain cytosine; however, 2'-deoxycytidine and 2'-deoxycytidine-5'-monophosphate undergo reductive hydrogenation. Several experiments were carried out in an effort to determine whether the reductive hydrogenation occurs during sample preparation or as a result of laser irradiation. The results of these experiments suggest that it occurs during sample preparation. The relative intensities of ion signals corresponding to the reduced base can be altered by using different matrix additives (aminonaphthalenes) or a different substrate (copper). Also, the oxidized form of 2,5-DHB is trapped by reaction with the side chain of cysteine in glutathione, providing evidence that the reaction occurs in solution as the matrix crystallizes.
A preorganized cationic receptor 2 for cytosine (C) is described which is composed of trans-a2PtII (a= NH3 or CH3NH2) cross-linked modules with adenine (A), guanine (G), and uracil (U) or thymine (T) model nucleobases. The functions of these three modules are as follows: i) Adenine orientates the two other bases at right angles, thus producing the L-shape of the receptor. ii) Guanine is the primary receptor. iii) Uracil or thymine act as coreceptors. Compared with the normal Watson-Crick pair between G and C, the association constant between 2 and C increases by a factor of 3 (in DMSO). As deduced from 1H NMR spectroscopy and confirmed by the X-ray crystal structure of the C adduct 4b, cytosine is fixed through five hydrogen bonds to the receptor, one of which involves the aromatic H(5) of C. A comparison of C binding is made with a structurally related linkage isomer receptor as well as the precursor molecule trans[alpha2PtAG]2+. The potential of modular, cationic receptors is illustrated.
Large changes in the Raman spectra of calf thymus DNA are observed upon lowering the pH. In order to gain a better insight into these effects, several simulations of the Raman spectra of the guanine-cytosine (GC) Watson-Crick and Hoogsteen base pairs are performed. By comparing the Raman bands of GC base pairs in calf thymus DNA at high and low pH with the theoretical simulations of GC base pairs, it is found that the intensity changes in the theoretical bands located between 400 and 1000 cm(-1) are small compared to the experimental ones. The behavior of the cytosine band at 1257 cm(-1) upon lowering the pH is not reproduced in the GC theoretical spectra. The bands located above 1300 cm(-1) in the theoretical spectra display intensity changes that are similar to those found for GC base pairs in calf thymus DNA spectra.
A complete scan of the potential and free-energy surfaces of monohydrated and dihydrated guanine...cytosine and 9-methylguanine...1-methylcytosine base pairs was realized by the molecular dynamics/quenching technique using the force field of Cornell et al. implemented in the AMBER7 program. The most stable and populated structures localized were further fully reoptimized at the correlated ab initio level employing the resolution of identity Møller-Plesset method with a large basis set. A systematic study of microhydration of these systems using a high-level correlated ab initio approach is presented for the first time. The different behavior of guanine...cytosine and adenine...thymine complexes is also discussed. These studies of nucleic acid base pairs are important for finding binding sites of water molecules around bases and for better understanding of the influence of the solvent on the stability of the structure of DNA.
Monoclonal antibodies prepared against DNA methyltransferase from human placenta undergo immune complex formation also with DNA methyltransferase from P815 mouse mastocytoma cells. One of these monoclonal antibodies, M2B10, was used for the immunoaffinity purification of this enzyme. Complexes of the immunoaffinity-purified mouse DNA methyltransferase with DNA were visualized by electron microscopy. DNA methyltransferase was found to be distributed along linearized plasmid DNA with a higher incidence of enzyme molecules at the terminal segments. This binding to strand ends was significantly increased after dG- or dGdC-tailing of the DNA, which is compatible with a preferred binding of the enzyme to single-stranded DNA. Sequence specificity analysis using methyl-sensitive restriction enzymes showed that the mouse DNA methyltransferase transferred methyl groups to the internal cytosines in 5'CCGG and 5'GCGC sequences, however, the external cytosine in 5'CCGG sequences was also methylated.
Yeast cytosine deaminase (yCD) was cloned, expressed, and purified by affinity chromatography. We have characterized the products resulting from covalent attachment of 2-4 PEG chains on yCD and determined the major and minor isomers for each respective conjugate. The results show that for non-covalently associated homodimers, it is possible to characterize and deduce PEGylation levels on individual subunits through the concurrent use of size exclusion chromatography (SEC), MALDI-TOF MS, and SDS-PAGE gels. The results also show that contrary to what we expected, attaching more than two PEG chains to yCD decreased its stability. Enzymatic activity studies revealed that the fusion of an N-terminus purification tag on yCD has no significant effect on 5-fluorocytosine or cytosine affinity, with apparent turnover rates remaining within 10(5) M(-1) . s(-1). Stability studies at 37 degrees C revealed that t1/2 = 8-9 h for yCD and 2mPEG(5K)-yCD, whereas for 3-, 4mPEG(5K)-yCD and yCD/BSA, t(1/2) < 2 h. Incubation of BSA with yCD also decreased enzyme stability over prolonged incubation at 37 degrees C. This finding is important if yCD is to be used in a pretargeting strategy.
Both ultraviolet irradiation and oxidation of DNA produce a variety of pyrimidine base damages. A human endonuclease recognizes such altered bases on these DNA substrates. This human endonuclease incises ultraviolet-irradiated DNA exclusively at sites of photochemically modified cytosines. The precise sites of incision by the human enzyme were determined by DNA sequencing. Chemically oxidized DNA was incised exclusively at thymine loci. The degree of enzymic cleavage at cytosine photoproducts was identical at each site. However, the extent of incision at selected oxidized thymine residues varied within the DNA sequence. These results indicate that the distribution of thymine oxidative modifications is influenced by the neighboring DNA bases.
The major ion formed in Negative Ion Chemical Ionization Mass Spectrometry of Adenine, Guanine, Cytosine, Uracil and Thymine is the dehydrogenated anion. The CURES EC procedure for optimizing Austin Model-1 Multiconfigurational Configuration Interaction semi-empirical calculations is applied to the electron affinities of the corresponding dehydrogenated bases and N-H bond dissociation energies. These calculated values will be compared with literature values of the gas phase acidities of the purines and pyrimidines. The N-H bond dissociation energies are about 3.95 eV for Guanine, Adenine, and Thymine and 4.08 eV for Cytosine and Uracil. The electron affinities of the radicals are AMinH = 3.50 eV, GMinH = 3.46 eV, CMinH = 3.38 eV, UMinH = 3.48 eV, TMinH = 3.46 eV.
The pyrimidine nucleobase analogue 6H,8H-3,4-dihydropyrimido[4,5-c]- [1,2]oxazin-7-one (P) is a mimic both of cytosine and thymine, since it can form stable hydrogen-bonded base-pairs with either guanine or adenine. To investigate the geometric properties of pairing with guanine in a DNA double helix, the structure of d(CGCGPG)2 has been determined by single crystal X-ray analysis. The oligonucleotide crystallised as a left-handed Z-DNA duplex in the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 18.23 A, b = 30.63 A, c = 43.78 A. Refinement using NUCLSQ with 51 water molecules included in the final model converged at R = 0.179 (Rw = 0.159) for 2798 reflections (F > 2 sigma (F)) in the range 8 A to 1.7 A. Remarkably, the two P.G pairs in the hexamer duplex are different: Watson-Crick and wobble types separately illustrate both cytosine-like and thymine-like behaviour. The result suggests that mutagenesis experiments involving P and other analogues which display pronounced base-pairing ambivalence can be used to examine the structural basis of substrate discrimination by polymerases that is essential to accurate genetic replication.
Although DNA methylation is a fundamental mechanism for repressing genetic activity, the influence of methyl groups on DNA conformation is found to be small. In this study, the role of cytosine methylation is analysed in the context of DNA condensation by examining its influence on DNA-DNA recognition processes. Previously CpG sites were found to act as sequence determinants for the close and specific self-fit of B-DNA helices into cross-overs. In the present study, the crystal structure of the B-DNA dodecamer d(ACCGCCGGCGCC) methylated at its central CpG sequence shows that the methyl groups do not interfere with DNA self-fitting. In contrast, the two methyl groups form a clamp, which traps the incoming phosphate in the groove-backbone interaction. This geometry allows the formation of two new C-H...O hydrogen bonds between the methyl groups and the anionic oxygen atoms of the phosphate, which may further stabilize the interaction. This finding relates cytosine methylation to the formation of higher-order DNA structures and could provide new insights for understanding the mode of action of DNA methylation in genetic inactivation.
Cytosine deaminase (CD) catalyzes the deamination of cytosine, producing uracil. This enzyme is present in prokaryotes and fungi (but not multicellular eukaryotes) and is an important member of the pyrimidine salvage pathway in those organisms. The same enzyme also catalyzes the conversion of 5-fluorocytosine to 5-fluorouracil; this activity allows the formation of a cytotoxic chemotherapeutic agent from a non-cytotoxic precursor. The enzyme is of widespread interest both for antimicrobial drug design and for gene therapy applications against tumors. The structure of Escherichia coli CD has been determined in the presence and absence of a bound mechanism-based inhibitor. The enzyme forms an (alphabeta)(8) barrel structure with structural similarity to adenosine deaminase, a relationship that is undetectable at the sequence level, and no similarity to bacterial cytidine deaminase. The enzyme is packed into a hexameric assembly stabilized by a unique domain-swapping interaction between enzyme subunits. The active site is located in the mouth of the enzyme barrel and contains a bound iron ion that coordinates a hydroxyl nucleophile. Substrate binding involves a significant conformational change that sequesters the reaction complex from solvent.
DNA from six strains of Fusobacterium nucleatum was examined for possible methylation of adenine or cytosine in the restriction site GATC. F. nucleatum DNA was digested with the restriction enzymes Sau3AI, DpnI and MboI, subjected to electrophoresis in agarose gels and Southern blotting. The blots were hybridized with a polymerase chain reaction (PCR)-amplified DNA probe. Both radioactive and non-radioactive labeling methods were used. Some strains of F. nucleatum were found to be dam methylated or dcm methylated in the sequence GATC; the adenine residue is methylated in strain ATCC 10953, while the cytosine residue is methylated in the strains F1 and F3. Strains F6, ATCC 25586 and Fev1 are probably not methylated in this nucleotide sequence.
We have measured the effects of cytosine-beta-D-arabinofuranoside (AraC) on cell survival and neurite outgrowth in cultures of dissociated 4-6 day embryonic chick neural tube cells. High concentrations of AraC (greater than 100 microM) reduced neuronal cell survival and neurite outgrowth from viable cells. Concentrations normally used to inhibit mitotic cell division (1-10 microM) were toxic to the neurones cultured in serum free medium on a poly-DL-ornithine/laminin substrate. AraC does not appear to have a neurite promoting effect on dissociated neurones that are cultured in the presence of low numbers of non-neuronal cells. This suggests that the neurite promoting effects of AraC reported by others is likely to be through the non-neuronal cells that were an inherent feature of the culturing systems in these studies. AraC cytotoxicity was completely blocked by the addition of the competitive antagonist: 2'deoxycytidine (2'DC) but not by its metabolic precursor cytosine (cyt). We suggest that the acute effects of AraC on neurones which are actively growing neurites are the result of interference with lipid metabolism.
We have detected sequence-specific non-symmetrical cytosine methylation within a 140 bp region of the promoter for the tobacco auxin-binding protein gene T85 in pollen DNA. Direct sequencing of the population of bisulphite reaction products showed that, in this region. 10 out of a possible 49 cytosine residues were methylated at a high frequency in pollen whereas the corresponding region from somatic cells (leaf DNA) did not show a detectable level of methylation. The context of these sites was 1 x m5CpTpC, 1 x m5CpGpT, 1 x m5CpCpT, 2 x m5CpTpT, 2 x m5CpGpG, and 3 x m5CpApT of which only m5CpGpG and m5CpGpT fitted the consensus sequence for symmetrical methylation in plants.
Cytosine deaminase (CD), produced by prokaryotes but not by higher eukaryotes including plants, deaminates cytosine to uracil. The enzyme likewise converts 5-fluorocytosine (5FC), which by itself is not toxic, to 5-fluorouracil (5FU), which is toxic. The Escherichia coli codA-coding sequence encoding CD, together with appropriate regulatory elements, was introduced into Arabidopsis. Neither untransformed controls, nor transgenic plants expressing no CD mRNA, were sensitive to 5FC. Conversely, for most transgenic plants expressing CD mRNA, in the presence of 5FC calli and seedlings failed to proliferate, and seeds failed to germinate. A few transgenic plants with many codA copies expressed less CD mRNA and remained insensitive to 5FC, which likely reflected epigenetic repeat-induced gene silencing. Thus 5FC, presumably through conversion by the enzyme to 5FU, can be used to select against plants that express CD.