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At least 595 records · Page 33Linked to original sources
PubMed1984

A new series of reductive amination derivatives of daunorubicin: syntheses, partition coefficients, and DNA binding.

A series of daunorubicin derivatives were prepared by sodium cyanoborohydride reductive amination of daunorubicin with appropriate amines. All derivatives were found to bind quite strongly to DNA and viscosity increases with linear DNA indicated that each formed an intercalation complex. A range of octanol-aqueous buffer partition coefficients was obtained, around the values of daunorubicin and dauxorubicin hydrochloride, by varying the character of the starting amine. All monoamine derivatives had activity against P388 leukemia in mice which was similar to that of daunorubicin. A diamine derivative had reduced activity against P388. Several anthracyclines administered as DNA complexes had similar activity against P388 but significantly reduced toxicity compared to the uncomplexed compounds. For anthracyclines which bind strongly to DNA, optimum activity against P388 leukemia in mice seems to be centered on compounds with octanol-buffer partition coefficients in the range of 0.5-0.8.

Animals↗
PubMed1995

DNA strand breakage, thymine glycol production, and hydroxyl radical generation induced by different samples of crystalline silica in vitro.

Five preparations of alpha-quartz [Min-U-Sil 5 (MQZ), MQZ pretreated with hydrofluoric acid (HFMQZ), Chinese standard alpha-quartz (CSQZ), and two German samples, DQ-12 and F600] and two preparations of the crystalline silica polymorphs, cristobalite and tridymite, previously characterized for surface area and surface charge, were evaluated for their relative activities in the following assays: (i) in vitro assays of short duration (< or = 15 min) for oxygen consumption and for generation of hydroxyl radicals (measured by electron spin resonance spin trapping), and (ii) in vitro assays of longer duration for DNA strand breakage (measured using linear DNA as a detector molecule) and for production of the oxidized DNA base, thymine glycol (measured by gas chromatography-mass spectrometry). Marked differences among the samples were found for their levels of oxygen consumption and of hydroxyl radicals' generation. All samples caused increased formation of thymine glycol, with wide variations in activity among samples. When normalized for equal surface area, the samples produced different levels of DNA strand breakage. Addition of hydrogen peroxide strongly accelerated DNA damage--more for cristobalite than for the alpha-quartz samples. DNA damage by quartz was enhanced by ferric chloride and inhibited by iron chelators. The order of relative activity of the samples varied with different types of in vitro assays and was not directly correlated to surface area. Electrophoretic mobility, as measured by zeta potential, was not significantly different among samples. The results suggest that the ability of different crystalline silica samples to generate a rapid burst of oxygen free radicals is distinct from their ability to induce DNA damage and DNA base oxidation over longer time periods. The relative activities of the samples in cellular assays (hemolysis of human erythrocytes; cytotoxicity and neoplastic transformation of BALB/3T3/A31-1-1 cells) were in turn markedly different from those listed above, suggesting a more critical role for surface area. The mechanisms of carcinogenesis by crystalline silica need to be further investigated in relation to the underlying physicochemical characteristics.

Animals↗
PubMed1995

Isolation of a Chlamydomonas reinhardtii telomere by functional complementation in yeast.

We attempted to determine whether Chlamydomonas reinhardtii telomeres, which do not form G-quartet structures readily in vitro, are able to nucleate telomere addition in Saccharomyces cerevisiae. Restricted C. reinhardtii genomic DNA was ligated to a linear S. cerevisiae vector lacking a telomere. A C. reinhardtii telomere ligated to this unprotected end allowed vector replication as a linear DNA molecule in S. cerevisiae. DNA sequencing revealed common [T4AG3]n and variant T6AG3 and T5AG3 C. reinhardtii telomere repeats capped by S. cerevisiae telomere repeat units. The recognition of a C. reinhardtii telomere by the telomere maintenance machinery of S. cerevisiae is consistent with a common theme for telomere structure in organisms with divergent telomere repeats.

Animals↗
PubMed2005

Endonuclease-like activity of heme proteins.

Heme proteins, metmyoglobin, methemoglobin, and metcytochrome c showed unusual affinity for double-stranded DNA. Calorimetric studies show that binding of methemoglobin to calf thymus DNA (CTDNA) is weakly endothermic, and the binding constant is 4.9+/-0.7x10(5) M(-1). The Soret absorption bands of the heme proteins remained unchanged, in the presence of excess CTDNA, but a new circular dichroic band appeared at 210 nm. Helix melting studies indicated that the protein-DNA mixture denatures at a lower temperature than the individual components. Thermograms obtained by differential scanning calorimetry of the mixture indicated two distinct transitions, which are comparable to the thermograms obtained for individual components, but there was a reduction in the excess heat capacity. Activation of heme proteins by hydrogen peroxide resulted in the formation of high valent Fe(IV) oxo intermediates, and CTDNA reacted rapidly under these conditions. The rate was first-order in DNA concentration, and this reactivity resulted in DNA strand cleavage. Upon activation with hydrogen peroxide, for example, the heme proteins converted the supercoiled pUC18 DNA into nicked circular and linear DNA. No reaction occurred in the absence of the heme protein, or hydrogen peroxide. These data clearly indicate a novel property of several heme proteins, and this is first report of the endonuclease-like activity of the heme proteins.

Animals↗
PubMed2000

Accumulation of single-strand breaks is the major cause of telomere shortening in human fibroblasts.

Telomere shortening triggers replicative senescence in human fibroblasts. The inability of DNA polymerases to replicate a linear DNA molecule completely (the end replication problem) is one cause of telomere shortening. Other possible causes are the formation of single-stranded overhangs at the end of telomeres and the preferential vulnerability of telomeres to oxidative stress. To elucidate the relative importance of these possibilities, amount and distribution of telomeric single-strand breaks, length of the G-rich overhang, and telomere shortening rate in human MRC-5 fibroblasts were measured. Treatment of nonproliferating cells with hydrogen peroxide increases the sensitivity to S1 nuclease in telomeres preferentially and accelerates their shortening by a corresponding amount as soon as the cells proliferate. A reduction of the activity of intracellular peroxides using the spin trap alpha-phenyl-t-butyl-nitrone reduces the telomere shortening rate and increases the replicative life span. The length of the telomeric single-stranded overhang is independent of DNA damaging stresses, but single-strand breaks accumulate randomly all along the telomere after alkylation. The telomere shortening rate and the rate of replicative aging can be either accelerated or decelerated by a modification of the amount of oxidative stress. Quantitatively, stress-mediated telomere damage contributes most to telomere shortening under standard conditions.

Alkylation↗
PubMed1991

Chemical cleavage of plasmid DNA by glutathione in the presence of Cu(II) ions. The Cu(II)-thiol system for DNA strand scission.

In the presence of Cu(II) ions, supercoiled DNA is cleaved in neutral solution by low concentrations of thiols. Supercoiled plasmid DNA is cleaved first to open circular DNA, which in turn produces linear DNA and eventually fragments. Cleavage is strongly temperature-dependent and is maximal at 0.10-0.25 M-NaCl concentration. In the presence of excess of either component of the Cu(II)-thiol pair, the extent of cleavage depended on the concentration of the limiting partner, and was easily detectable down to micromolar concentrations of limiting GSH. Scavengers of oxygen-derived species (such as hydrogen peroxide, superoxide radical ion and hydroxyl radical) indicated that the hydroxyl radical may be involved in the cleavage mechanism. DNA cleavage leads to some production of 2-thiobarbituric acid-reactive species and some of the cleavage sites, at least, had 5'-hydroxy and/or 3'-hydroxy groups. There was extensive base damage before cleavage. Studies with S1 nuclease indicated no gross sequence preference for Cu(II)-GSH cleavage of pSP64 plasmid DNA. The Cu(II)-thiol system did not appear to target special structural features in the DNA such as Z-DNA inserts, cruciform structures or left-handed (but non-Z) DNA. Cleavage might arise from a reagent generated either by the Cu(II)-thiol combination in free solution or by attack involving Cu(II) ions pre-bound to DNA. The attack of GSH plus Cu(II) ions on DNA may be a potential toxic lesion under physiological conditions unless special protective measures operate efficiently in the cell.

Copper↗
PubMed1977

Elimination of naturally occurring crosslinks in vaccinia virus DNA after viral penetration into cells.

Vaccinia virus DNA, extracted from purified virus or from the cytoplasmic fraction of virus-infected cells very shortly after infection, was analyzed by sedimentation in alkaline and neutral sucrose gradients. The sedimentation properties of vaccinia DNA under denaturing conditions changed, immediately after penetration into the cell, from the characteristic circular viral DNA (crosslinked double-stranded linear DNA) to nicked circular DNA or to single-stranded molecules. This transition occurred at the time of uncoating of the virus and with a slight change in the DNA size, as judged by sedimentation in neutral sucrose. These results indicate that the crosslinks, that held the complementary strands of the genome together, are removed after penetration. When vaccinia DNA was incubated with the supernatant fraction of virus-infected cells, a similar change in the sedimentation properties of the DNA under denaturing conditions was observed. It is concluded that the endonuclease present in the supernatant of infected cells eliminated the crosslinks in the DNA, and that this enzymatic hydrolysis may be the mechanism by which crosslinks are removed prior to DNA replication.

Binding Sites↗
PubMed1979

Extracts of Drosophila embryos mediate chromatin assembly in vitro.

Extracts of Drosophila embryos can mediate the assembly of a chromatinlike structure from histones and DNA under physiological conditions. The histone-DNA complex formed in vitro contains micrococcal nuclease-sensitive sites spaced at 200-base pair intervals. More extensive digestion of the complex by micrococcal nuclease generates 11S particles which cosediment with nucleosome core particles isolated from native chromatin. These particles contain 140-base pair DNA fragments which upon further cleavage with micrococcal nuclease give rise to a pattern of discretely sized DNA fragments characteristic of nucleosome core particles. We have assayed the chromatin assembly process both qualitatively by measuring the induction of supertwists into a relaxed circular DNA (a process requiring a nicking-closing enzyme) and quantitatively by measuring the formation of micrococcal nuclease-resistant DNA fragments from radioactively labeled linear DNA. The amount of chromatin formed depends primarily on the amount of histones, whereas the rate of assembly depends on the amount of extract protein added. The factors in the extract that mediate chromatin assembly appear to interact first with the DNA because preincubation of the DNA with the extract markedly increases the extent of assembly.

Animals↗
PubMed1981

Unwinding of double-stranded DNA helix by dehydration.

Conformation changes of the double-stranded DNA helix in response to dehydration were investigated by monitoring, by agarose gel electrophoresis, the linking number of covalently closed circular DNA generated by ligation of linear DNA in the presence of different organic solvents or different temperatures. It was found that: (i) The DNA helix unwinds upon addition of certain organic solvents or elevation of temperature. (ii) The conformational change observed under the experimental conditions is a continuous process in response to the organic solvent concentration. (iii) The delta H of unwinding one linking of the DNA helix is constant at approximately 12.2 +/- 0.4 kcal/mol (1 kcal = 4.184 kJ); the corresponding delta S and d(delta S)/dn are 2nkR and 2kR, in which n is the relative equivalent linking number (referred to the state of delta S = 0 for unwinding) of the DNA, R is the gas constant, and k is equal to 1117/number of base pairs. The delta H, delta S, and d(delta S)/dn for unwinding i linkings are i X 12.2 kcal/mol, 2inkR, and 2ikR, respectively. (iv) d(delta S)/dn, like k, is inversely proportional to the number of base pairs in DNA. (v) Double-stranded DNAs of different chain lengths have average delta S = 35 cal/mol.K for unwinding one linking under the experimental conditions; this corresponds to 127 +/- 14 base pairs per "relative linking."

Base Composition↗
PubMed1999

RecA polymerization on double-stranded DNA by using single-molecule manipulation: the role of ATP hydrolysis.

The polymerization of RecA on individual double-stranded DNA molecules is studied. A linear DNA (lambda DNA, 48.5 Kb), anchored at one end to a cover glass and at the other end to an optically trapped 3-micrometers diameter polystyrene bead, serves as a template. The elongation caused by RecA assembly is measured in the presence of ATP and ATP[gammaS]. By using force extension and hydrodynamic recoil, a value of the persistence length of the RecA-DNA complex is obtained. In the presence of ATP, the polymer length is unstable, first growing to saturation and then decreasing. This suggests a transient dynamics of association and dissociation for RecA on a double-stranded DNA, the process being controlled by ATP hydrolysis. Part of this dynamics is suppressed in the presence of ATP[gammaS], leading to a stabilized RecA-DNA complex. A one-dimensional nucleation and growth model is presented that may account for the protein assembly.

Adenosine Triphosphate↗
PubMed2004

Covalent DNA display as a novel tool for directed evolution of proteins in vitro.

We present a novel method for the directed evolution of polypeptides, which combines in vitro compartmentalization and covalent DNA display. A library of linear DNA fragments is co-packaged with an in vitro transcription/translation mixture in the compartments of a water-in-oil emulsion. Experimental conditions are adjusted so that, in most cases, one compartment contains one DNA molecule. The DNA fragments encode fusion proteins containing a DNA-methyltransferase (M.Hae III), which can form a covalent bond with a 5-fluorodeoxycytidine base at the extremity of the DNA fragment. The resulting library of DNA-protein fusions is extracted from the emulsion and DNA molecules displaying a protein with desired binding properties are selected from the pool of DNA-protein fusions by affinity panning on target antigens. We applied this methodology in model selection experiments, using specific ligands for the capture of peptides and globular proteins bound to DNA. We observed enrichment factors >1000-fold for selections performed in separate emulsions and up to 150-fold for selections performed using mixtures of DNA molecules. M.Hae III could be fused to small globular proteins (such as calmodulin and fibronectin domains), which are ideally suited for the generation of combinatorial libraries and for the isolation of novel binding specificities.

DNA Adducts↗
PubMed1997

The recombinant product of the Chryptomonas phi plastid gene hlpA is an architectural HU-like protein that promotes the assembly of complex nucleoprotein structures.

The HlpA protein which is encoded by the hlpA gene in the plastid genome of the cryptomonad alga Chryptomonas phi is structurally related to the non-sequence-specific DNA-binding and DNA-bending HU family of chromatin-associated proteins. The expression of the HlpA protein complements the mutant phenotype of Bacillus subtilis cells impaired in the Hbsu protein (B. subtilis HU), as measured by the resistance of the cells to methylmethane sulphonate. To analyse the interactions of HlpA with DNA, we expressed the protein in Escherichia coli and purified it to homogeneity. HlpA interacts preferentially with four-way junction DNA or DNA minicircles, when compared with linear DNA, recognising DNA structure. HlpA and E. coli HU display comparable affinities for all types of DNA tested; however, HlpA exhibits a stronger tendency to self-associate in the presence of DNA. Accordingly, HlpA oligomerises more readily than HU in protein crosslinking experiments. In the presence of topoisomerase I, HlpA constrains negative superhelical turns in closed circular plasmid DNA. The HlpA protein mediates the joining of distant recombination sites into a complex nucleoprotein structure, as judged by beta-mediated site-specific recombination. The results presented provide evidence that HlpA is a functional plastid equivalent of nuclear and mitochondrial HMG1-like proteins and bacterial HU proteins.

Bacillus subtilis↗
PubMedDate not supplied

Gene transfer and expression studies in cultured avian neural crest cells differentiating into melanocytes.

Neural crest cells obtained from explanted neural tubes take up, express, and retain exogenous DNA applied by the CaPO4 co-precipitation method during their differentiation into melanocytes. High efficiencies of gene transfer were obtained with both supercoiled DNA and intact phage particles; linear DNA or DNA from the phage yielded very low efficiencies. There is some evidence that transferred gene expression is differentiation dependent. The system should be useful for studies concerned with the analysis of cell developmental genes and their regulatory elements.

Animals↗
PubMed1998

Recognition of specific sequences in DNA by a topoisomerase I inhibitor derived from the antitumor drug rebeccamycin.

We investigated the interaction with DNA of two synthetic derivatives of the antitumor antibiotic rebeccamycin: R-3, which is a potent topoisomerase I inhibitor and contains a methoxyglucose moiety appended to the indolocarbazole chromophore, and its aglycone, R-4. Spectroscopic measurements indicate that R-3 intercalates into DNA and that its carbohydrate domain contributes significantly to reinforce the affinity for DNA. Two complementary ligation assays concur that R-3, but not its aglycone counterpart, exerts a significant effect on the curvature and/or the flexibility of DNA. The sugar moiety may be responsible for preferential binding of R-3 to circular (or bent) DNA molecules as opposed to linear DNA fragments. The sequence selectivity of binding to DNA has been studied thoroughly by footprinting with DNase I and two other nucleases. The glycosylated compound is highly selective for nucleotide sequences containing GpT (ApC) and TpG (CpA) steps. The derivative lacking the sugar moiety on the indolocarbazole chromophore binds at essentially identical sites but with considerably lower affinity, so it seems that the chromophore rather than the carbohydrate is responsible for the preferential binding to sequences surrounding GpT and TpG steps. The influence of the exocyclic substituents present on the bases at the recognition sites (i.e., the 2-amino group of guanine and the 5-methyl group of thymine) was evaluated using two series of modified DNA molecules prepared by polymerase chain reaction containing inosine and/or 2,6-diaminopurine and uridine and/or 5-methylcytosine residues. The introduction of the amino group onto purine residues or the addition of a methyl group to pyrimidine residues suffices to create new drug binding sites. Therefore, unlike most DNA-binding small molecules, the rebeccamycin analogue seems to be highly sensitive to any modification of the exocyclic substituents on the bases in both the major and minor grooves of the double helix. The footprinting profiles with the different DNA fragments bear a remarkable resemblance to those determined for nogalamycin and bisnaphthalimide compounds known to recognize their preferred GpT and TpG sites via intercalation from the major groove. The unique DNA binding characteristics of the rebeccamycin analogue correlate well with its inhibitory effects on topoisomerase I.

Aminoglycosides↗
PubMed2003

The CBP bromodomain and nucleosome targeting are required for Zta-directed nucleosome acetylation and transcription activation.

The Epstein-Barr virus (EBV)-encoded lytic activator Zta is a bZIP protein that can stimulate nucleosomal histone acetyltransferase (HAT) activity of the CREB binding protein (CBP) in vitro. We now show that deletion of the CBP bromo- and C/H3 domains eliminates stimulation of nucleosomal HAT activity in vitro and transcriptional coactivation by Zta in transfected cells. In contrast, acetylation of free histones was not affected by the addition of Zta or by deletions in the bromo or C/H3 domain of CBP. Zta stimulated acetylation of oligonucleosomes assembled on supercoiled DNA and dinucleosomes assembled on linear DNA, but Zta-stimulated acetylation was significantly reduced for mononucleosomes. Western blotting and amino-terminal protein sequencing indicated that all lysine residues in the H3 and H4 amino-terminal tails were acetylated by CBP and enhanced by the addition of Zta. Histone acetylation was also dependent upon the Zta basic DNA binding domain, which could not be substituted with the homologous basic region of c-Fos, indicating specificity in the bZIP domain nucleosome binding function. Finally, we show that Zta and CBP colocalize to viral immediate-early promoters in vivo and that overexpression of Zta leads to a robust increase in H3 and H4 acetylation at various regions of the EBV genome in vivo. Furthermore, deletion of the CBP bromodomain reduced stable CBP-Zta complex formation and histone acetylation at Zta-responsive viral promoters in vivo. These results suggest that activator- and bromodomain-dependent targeting to oligonucleosomal chromatin is required for stable promoter-bound complex formation and transcription activity.

Acetylation↗
PubMed1989

Replication of baboon endogenous virus in human cells. Kinetics of DNA synthesis and integration.

For the baboon endogenous virus to infect human cells a specific region on chromosome 6 is required for viral DNA replication and integration. In studying the kinetics of baboon endogenous virus DNA replication we show that linear DNA was synthesized as the predominant species after infection and that unintegrated DNA persisted after many cell passages. Examination of integrated DNA revealed the failure of the virus to integrate at early passages. With continuous replication, however, virus integration was observed, but at multiple sites in the host cell.

Animals↗
PubMed1996

Studies on electrotransfer of DNA into Escherichia coli: effect of molecular form of DNA.

Effects of several molecular forms of DNA were examined on voltage-pulse-mediated transfection or transformation. Among circular DNAs, the single-stranded microvirid DNA was less infective than the double-stranded replicative form, whereas transfectivity of the relaxed or nicked molecule was nearly equal to or slightly lower than that of the supercoiled DNA. The linearized double-stranded DNA derived from phage or plasmid electrotransfects Escherichia coli, albeit at a reduced efficiency. Alkaline denaturation of the linearized DNA resulted in complete loss of the infectivity. Relationship between terminal structure of the linearized DNA and efficiency of the transfection was investigated. Host recombination function did not significantly affect the infectivity of the linearized DNA.

DNA↗
PubMed1999

Mammalian mitochondrial extracts possess DNA end-binding activity.

Mammalian mitochondrial protein extracts possess DNA end-binding (DEB) activity. Protein binding to a 394 bp double-stranded DNA molecule was measured using an electrophoretic mobility shift assay. Mitochondrial DEB activity was highly specific for linear DNA. Inclusion of a vast excess of non-radioactive circular DNA did not disrupt binding to radioactive f394. In contrast, binding was abolished by the inclusion of linear competitor DNA. In mammals, nuclear DEB activity is due to Ku, a hetero-dimer composed of the Ku70 and Ku86 proteins. To determine whether mitochondrial DEB activity was also due to Ku, protein extracts were prepared from the Chinese hamster XR-V15B cell line, which lacks this protein. As anticipated, nuclear extracts prepared from these cells lacked DEB activity. In contrast, mitochondrial extracts prepared from these cells had wild-type levels of DEB activity, demonstrating that this latter activity is not a consequence of nuclear contamination. Although the nuclear and mitochondrial DEB activities are independent of each other, they are nevertheless closely related, since mitochondrial DEB activity was 'supershifted' by both anti-Ku70 and anti-Ku86 antisera. The nuclear DEB protein Ku plays an essential role in nuclear DNA double-strand break repair. The DEB activity described herein may therefore play a similar role in mitochondrial DNA repair.

Animals↗