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At least 253 records · Page 14Linked to original sources
PubMed1995

Construction of trypanosome artificial mini-chromosomes.

We report the preparation of two linear constructs which, when transformed into the procyclic form of Trypanosoma brucei, become stably inherited artificial mini-chromosomes. Both of the two constructs, one of 10 kb and the other of 13 kb, contain a T.brucei PARP promoter driving a chloramphenicol acetyltransferase (CAT) gene. In the 10 kb construct the CAT gene is followed by one hygromycin phosphotransferase (Hph) gene, and in the 13 kb construct the CAT gene is followed by three tandemly linked Hph genes. At each end of these linear molecules are telomere repeats and subtelomeric sequences. Electroporation of these linear DNA constructs into the procyclic form of T.brucei generated hygromycin-B resistant cell lines. In these cell lines, the input DNA remained linear and bounded by the telomere ends, but it increased in size. In the cell lines generated by the 10 kb construct, the input DNA increased in size to 20-50 kb. In the cell lines generated by the 13 kb constructs, two sizes of linear DNAs containing the input plasmid were detected: one of 40-50 kb and the other of 150 kb. The increase in size was not the result of in vivo tandem repetitions of the input plasmid, but represented the addition of new sequences. These Hph containing linear DNA molecules were maintained stably in cell lines for at least 20 generations in the absence of drug selection and were subsequently referred to as trypanosome artificial mini-chromosomes, or TACs.

Animals↗
PubMed1995

Structure and function of transcription-repair coupling factor. II. Catalytic properties.

The transcription repair coupling factor (TRCF) of Escherichia coli has the so-called helicase motifs, is a DNA-, RNA Pol-, and UvrA-binding protein, and is required for the coupling of repair to transcription. We investigated the potential helicase, transcription termination, and transcription-repair coupling activities of TRCF on various substrates. We found that TRCF does not have a helicase activity on any of the substrates tested. However, the TRCF releases both RNA Pol and the truncated transcript from a transcriptional road block caused by a lesion, a "missing base," or a DNA-bound protein. It does not have any effect on rho-dependent or rho-independent transcriptional termination. However, some premature terminations were induced by TRCF at other sites. The coupling of transcription to repair occurs with supercoiled and relaxed circular DNA and with linear DNA. However, the coupling with linear DNA is strongly affected by the length of the DNA and does not occur with fragments in which the lesion is closer than 90 nucleotides to the 5' terminus of the template strand. Under transcription conditions the repair of lesions in the promoter region and up to the eleventh transcribed base is inhibited even in the presence of TRCF. Stimulation of repair in the transcribed strand starts at lesions at +15 nucleotides. Stimulation of repair occurs via facilitating the delivery of the A2B1 complex to the lesion site by the TCRF and can be inhibited by excess UvrA which binds to the TRCF off DNA. In vitro, strand-specific repair is not dependent on the MutL and MutS proteins which have recently been implicated in preferential repair in vivo.

Adenosine Triphosphatases↗
PubMed1984

The hepatitis B virus as a molecular model for chronic infection: synthesis of hepatitis B surface and e antigens in mouse L cells transfected with closed circular viral DNA.

Linear hepatitis B virus (HBV) DNA, excised from a recombinant plasmid with EcoR1, was purified by preparative electrophoresis on agarose gels and incubated with phage T4 ligase to form either monomeric or dimeric closed circles. Thymidine kinase deficient mouse L cells were cotransfected with thymidine kinase (tk) and circular HBV DNAs and grown in hypoxanthine medium. Colonies of tk-transformed cells, selected after 3-4 weeks of incubation and subcultured in HAT medium, synthesized either hepatitis B surface antigen (HBsAg) alone or HBsAg in combination with hepatitis B e antigen (HBeAg). The various cell colonies differed in plating efficiency, growth rates, cellular appearance, and extent of viral antigen synthesis. Southern hybridization analysis showed the presence of HBV-related sequences in high molecular weight DNA prepared from cells expressing viral antigens. Digestion of cellular DNAs with restriction endonucleases indicated integration of the entire viral genome.

Animals↗
PubMed1994

DNA binding properties of recombinant human mitochondrial transcription factor 1.

Recombinant mitochondrial transcription factor 1 (r-mtTF1) was overproduced in Escherichia coli, and purified to near homogeneity by DNA affinity chromatography. Binding affinities of r-mtTF1 to the light strand promoter (LSP) and heavy strand promoter (HSP) of human mitochondrial DNA and a non-promoter fragment derived from pUC vector were quantitatively measured by electrophoretic mobility shift assay. The order of the affinities for these DNAs estimated by competition experiments was LSP > HSP = pUC, which was essentially the same as that of transcriptional activity from each promoter in vitro. Recombinant mtTF1 bound more tightly to supercoiled or relaxed DNA than to linear DNA, moreover to single-stranded DNA with the same affinity for linear DNA. The results suggest that the mechanism of activation of HSP by mtTF1 is different from that of LSP, and that multiple binding of mtTF1 to mitochondrial DNA, in a non-specific manner, may significantly participate in activation of transcription from HSP and replication of mitochondrial DNA.

DNA, Mitochondrial↗
PubMed2001

Double-strand hydrolysis of plasmid DNA by dicerium complexes at 37 degrees C.

Significant effort has been made to develop synthetic metal complexes that hydrolyze DNA. Here we report a new dicerium complex, Ce(2)(HXTA) (HXTA = 5-methyl-2-hydroxy-1,3-xylene-alpha,alpha-diamine-N,N,N',N'-tetraacetic acid), which can hydrolyze DNA at pH 8 and 37 degrees C. This complex hydrolyzes DNA restriction fragments to give products with high regioselectivity, affording >90% 5'-OPO(3) and 3'-OH ends, like the products of DNA hydrolyzing enzymes. Ce(2)(HXTA) also hydrolyzes Litmus 29 plasmid DNA to afford both nicked and linear DNA. Analysis of the relative amounts of supercoiled, nicked, and linear DNA present show that there is one double-strand cleavage per ten single-strand cleavages, indicating that the linear DNA formed cannot be the result of two random single-strand cleavage events. The kinetics of nicked and linear DNA formation are comparable, both being associated with apparent first-order rate constants of approximately 1 x 10(-)(4) s(-)(1) for complex concentrations of 10(-)(5)-10(-)(4) M. These observations suggest that similar factors affect the hydrolysis of the first and second DNA strands and that cleaving the phosphodiester bond is likely the rate determining step in both cases. This is the first detailed study of a metal complex shown to mimic DNA hydrolases in their capability to effect double-strand DNA hydrolysis regioselectively at the 3'-O-P bond.

Cerium↗
PubMed1995

DNA cleavage at two recognition sites by the SfiI restriction endonuclease: salt dependence of cis and trans interactions between distant DNA sites.

At low ionic strength, the SfiI restriction enzyme cleaved at similar rates both supercoiled and linear DNA with two SfiI sites and linear DNA with one SfiI site. For the substrates with two sites, the majority of the DNA was converted directly to products cut at both sites; the enzyme appears to bind to two sites before catalyzing its reactions, looping out the intervening DNA. At high ionic strength, linear DNA with one SfiI site was not cut at all, linear DNA with two sites was cleaved slowly while supercoiled DNA with two sites was cleaved rapidly, though only half of the DNA with two sites was cut at both sites; the DNA that had been cut at one site was not cleaved again at the remaining site. The singly cut product must therefore have been generated by a reaction incorporating both sites. All DNA cleavage reactions by SfiI thus involve the tetrameric enzyme bound to two copies of its recognition sequence, but weakened DNA-protein interactions at high ionic strength can cause this complex to dissociate before cleaving both sites. Intramolecular interactions between distant DNA sites are generally thought to be enhanced by supercoiling and to be more stable than intermolecular interactions. The preference of SfiI at high ionic strength for substrates with two sites over substrates with one site and, in the former case, for supercoiled over linear DNA, validates this view. At low ionic strength, the similar rates with the different substrates may be due to rate-limiting product dissociation.

Base Sequence↗
PubMed1994

Mechanism of inhibition of DNA gyrase by cyclothialidine, a novel DNA gyrase inhibitor.

We investigated how cyclothialidine (Ro 09-1437), a novel DNA gyrase inhibitor belonging to a new chemical class of compounds, acts to inhibit Escherichia coli DNA gyrase. Cyclothialidine up to 100 micrograms/ml showed no effect on DNA gyrase when linear DNA was used as a substrate. Under the same conditions, quinolones, which inhibit the resealing reaction of DNA gyrase, caused a decrease in the amount of linear DNA used. No effect of cyclothialidine was observed on the accumulation of the covalent complex of DNA and the A subunit of DNA gyrase induced by ofloxacin in the absence of ATP. The effect of cyclothialidine on the DNA supercoiling reaction was antagonized by ATP, reducing the inhibitory activity 11-fold as the ATP concentration was increased from 0.5 to 5 mM. Cyclothialidine competitively inhibited the ATPase activity of DNA gyrase (Ki = 6 nM). The binding of [14C]benzoyl-cyclothialidine to E. coli gyrase was inhibited by ATP and novobiocin, but not by ofloxacin. These results suggest that cyclothialidine acts by interfering with the ATPase activity of the B subunit of DNA gyrase. Cyclothialidine was active against a DNA gyrase resistant to novobiocin, suggesting that its precise site of action might be different from that of novobiocin.

4-Quinolones↗
PubMed1999

Differential effects of cyclopolyamines on the stability and conformation of triplex DNA.

Linear polyamines are excellent promoters of triplex DNA formation. The effects of structural rigidization of polyamines on triplex DNA stability are not known at present. We wished to develop a series of polyamine analogs as secondary ligands for triplex DNA stabilization for antigene applications. To accomplish this goal, we synthesized cyclopolyamines by interconnecting the two amino or imino groups of linear polyamines with a --(CH2)n-bridge (n=3,4,5). Melting temperature (Tm) data showed that [4,3]-spermine and [4,4]-spermine stabilized poly(dA) x 2poly(dT) triplex at >25 microM concentrations (Tm = 71 degrees C at 100 microM). The dTm/dlog [polyamine] values for these compounds were 26 and 40, respectively. [4,3]-Spermine and [4,4]-spermine also stabilized triplex DNA formed by a purine-motif triplex-forming oligonucleotide, TG3TG4TG4TG3T with its target duplex, as determined by Tm, circular dichroism (CD) spectroscopy, and electrophoretic mobility shift assay (EMSA). In contrast, [4,4]-putrescine and [4,5]-putrescine as well as [4,5]-spermine had no triplex DNA stabilizing effect. CD spectra also showed triplex DNA aggregation and psi-DNA formation at >100 microM [4,3]-spermine. These data demonstrate that structural rigidization of linear polyamines has a profound effect on their ability to stabilize triplex DNA and provoke conformational transitions.

Circular Dichroism↗
PubMed1997

DNA double-strand breaks caused by replication arrest.

We report here that DNA double-strand breaks (DSBs) form in Escherichia coli upon arrest of replication forks due to a defect in, or the inhibition of, replicative DNA helicases. The formation of DSBs was assessed by the appearance of linear DNA detected by pulse-field gel electrophoresis. Processing of DSBs by recombination repair or linear DNA degradation was abolished by mutations in recBCD genes. Two E. coli replicative helicases were tested, Rep, which is essential in recBC mutants, and DnaB. The proportion of linear DNA increased up to 50% upon shift of rep recBTS recCTS cells to restrictive temperature. No increase in linear DNA was observed in the absence of replicating chromosomes, indicating that the formation of DSBs in rep strains requires replication. Inhibition of the DnaB helicase either by a strong replication terminator or by a dnaBTS mutation led to the formation of linear DNA, showing that blocked replication forks are prone to DSB formation. In wild-type E. coli, linear DNA was detected in the absence of RecBC or of both RecA and RecD. This reveals the existence of a significant amount of spontaneous DSBs. We propose that some of them may also result from the impairment of replication fork progression.

Bacterial Proteins↗
PubMed1985

Integration of linear, heterologous DNA molecules into the Bacillus subtilis chromosome: mechanism and use in induction of predictable rearrangements.

Linear DNA molecules composed of a central region nonhomologous with the Bacillus subtilis chromosome and two flanking regions homologous with the chromosome can integrate into the chromosome, provided that the homologous regions have the same relative orientation. The resulting chromosome can be maintained in a haploid or in a merodiploid cell together with a parental chromosome. This can most easily be explained by supposing that the integration occurs by crossing over at each homologous region and that a part of the chromosome between these regions is deleted and replaced by the central nonhomologous region of the integrating molecule. If no essential genes were replaced during that process a haploid cell would be obtained; if essential genes were replaced a merodiploid cell would be obtained. The use of appropriate linear molecules therefore should allow the induction of deletions, extending from a given chromosomal site in a predetermined direction, and defined duplications in the B. subtilis chromosome.

Bacillus subtilis↗
PubMed1990

The basidiomycete Lentinus edodes linear mitochondrial DNA plasmid contains a segment exhibiting a high autonomously replicating sequence activity in Saccharomyces cerevisiae.

A linear DNA plasmid, designated pLLE1, has been isolated from a mitochondrial fraction of a strain of Lentinus edodes. pLLE1(11.0 kbp) was sensitive to the 3'----5'-acting exonuclease III and resistant to the 5'----3'-acting lambda exonuclease. It showed no homology with mitochondrial and nuclear genomic DNAs of plasmidless strain as well as the pLLE1-harboring host strain of L. edodes. The 1434-bp fragment (sequences) capable of autonomous replication in the yeast Saccharomyces cerevisiae (ARSs) was cloned from pLLE1 DNA with YIp32 (pBR322 containing yeast LEU2 DNA), which displayed a high ARS activity. The cloned 1434-bp fragment was shown to lie near to the end of pLLE1 DNA (nucleotides about 800-2200) and contained three consecutive ARS consensus sequences (A/T)TTTAT(A/G)TTT(A/T) of S. cerevisiae and dispersive eight ARS consensus-like sequences. The subcloned 366-bp fragment containing the three ARSs retained original ARS activity of the 1434-bp fragment.

Base Sequence↗
PubMed1990

Analysis of the kinetic hairpin transfer model for parvoviral DNA replication.

All linear DNA molecules face special problems in replicating their 5' ends, as DNA polymerases add nucleotides only to pre-existing strands with free 3'-OH groups. Parvoviruses, a group of small animal viruses with a linear single-stranded DNA genome, cope with this problem by having palindromic terminal sequences that can fold back on themselves to form hairpin structures essential in priming DNA replication. The 3' terminal sequence that initiates replication becomes reversed in orientation during the process, and if the palindrome is imperfect, two different, reverse-complementary terminal sequences are generated. The relative abundances of the terminal sequence orientations at each end of the DNA molecules can be measured and give information about the replication process. From such clues, we developed a "kinetic hairpin transfer model" based on differential rates of hairpin formation and inversion processes depending on the conformations of the 3' termini. Numerical studies showed that this simple idea can account for the diverse pattern of DNA distributions observed in the family Parvoviridae. In this paper, we simplify the model to a set of coupled linear first-order ordinary differential equations in order to delineate its essential properties by Perron-Frobenius theory. Secondly, we examine our assumption of linear kinetics by modeling enzyme catalysis of the component steps of the hairpin transfer process. We show that the rate-determining step of the process is the binding of initiation complex to the self-priming hairpin structures. Furthermore, we find that if the replication machinery is saturated by DNA substrate late in an infection, the differential equations become non-linear but the steady-state DNA distribution is still given by the solution of our original linear equations.

Computer Simulation↗
PubMed2004

Cleavage of supercoiled circular double-stranded DNA induced by a eukaryotic cambialistic superoxide dismutase from Cinnamomum camphora.

A eukaryotic cambialistic superoxide dismutase (SOD) has been purified to homogeneity from mature seeds of the disease- and insect-resistant camphor tree (Cinnamomum camphora). Besides the known role of this SOD in protecting cells against oxidative stress, it can induce the cleavage of supercoiled double-stranded DNA into nicked and linear DNA. It can not cleave linear DNA or RNA, demonstrating there is no DNase or RNase in the purified cambialistic SOD. Furthermore, the SOD can linearize circular pGEM-4Z DNA that is relaxed by topoisomerase I. This result indicates that the DNA-cleaving activity requires substrates being topologically constrained. The supercoiled DNA-cleaving activity of the cambialistic SOD can be inhibited by either SOD inhibitor (azide) or catalase and hydroxyl radical scavengers (ethanol and mannitol). The chelator of iron, diethylenetriaminepentaacetic acid (DTPA), also inhibits the supercoiled DNA-cleaving activity. These results show that the dismutation activity is crucial for the supercoiled DNA cleavage. The modification of tryptophan residue of the cambialistic SOD with N-bromosuccinimide (NBS) shows that these two activities are structurally correlative. The reaction mechanism is proposed that the hydroxyl radical formed in a transition-metal-catalyzing Fenton-type reaction contributes to the DNA-cleaving activity. In addition, the cleavage sites in supercoiled pGEM-4Z DNA are random.

Catalase↗
PubMed1988

A nuclear gene required for the expression of the linear DNA-associated killer system in the yeast Kluyveromyces lactis.

The killer system of Kluyveromyces lactis is associated with two linear DNA plasmids, pGKL1 and pGKL2. The killer toxin and the immunity determinant are coded for by pGKL1. Mutations which block the expression of the killer character have been isolated. These mutations reside in a single chromosomal gene which we have named KEX1. The KEX1 gene of K. lactis has been cloned by complementation of kex1 mutations by using a recombinant plasmid pool containing the entire Kluyveromyces lactis genome, on a multicopy plasmid KEp6, which contains the Saccharomyces cerevisiae URA3 gene as a marker. Genetic analyses of strains carrying a disrupted kex1 allele demonstrated that the cloned DNA corresponded to the KEX1 gene. The cloned KEX1 gene of K. lactis has low but significant sequence homology with the KEX2 gene of Saccharomyces cerevisiae. In vivo complementation of the kex1 mutation of K. lactis by the KEX2 gene of S. cerevisiae, and complementation of the kex2 mutation of S. cerevisiae by the KEX1 gene of K. lactis, demonstrated that KEX1 of K. lactis is functionally related to the KEX2 gene of S. cerevisiae. K. lactis diploids homozygous for kex1 are deficient for sporulation.

Cloning, Molecular↗
PubMed1994

NMR studies and conformational analysis of a DNA four-way junction formed in a linear synthetic oligonucleotide.

A linear DNA oligomer (M(r) 14,000, 46 nucleotides) was especially designed, chemically synthesized, and studied by means of 1H NMR spectroscopy. The design of the oligomer was guided by the idea that incorporation of three short palindromic sequences, each interspersed by 5'-CTTG-3' motifs at predetermined positions in the oligomer, would give rise to the formation of three stable minihairpin loops [Ippel, J. H., et al. (1992) J. Biomol. Struct. Dyn. 9, 1-16], which in turn were expected to encourage further folding of the strand into a stable four-way junction containing three "hairpin" arms and an open-ended duplex stem as the fourth arm. Linear DNA four-way junctions constructed according to this concept can be more compact and are therefore expected to be more suitable as model compounds for conformational studies compared to junctions that are built from two or more separate strands. A stable cruciform conformation was substantiated for the 46-mer in aqueous solution in the presence of Mg2+. Complete sequential 1H NMR assignments of the nonexchangeable protons (except H4', H5', and H5") were obtained with the aid of NOESY and HOHAHA experiments. The NMR data gave evidence for the expected existence of minihairpin-loop structures at the three 5'-CTTG-3' motifs in the sequence. The complementary stem domains adopt a regular B-DNA form. Watson-Crick type base pairing is preserved for all residues in the stem domains, including the residues at the center of the junction. A systematic investigation of the interresidual NOEs observed between the protons of the eight central residues revealed the complete stacking pattern of the residues at the branch point.

Base Sequence↗
PubMed1989

Linear dichroism properties and orientations of different ultraviolet transition moments of benzo[a]pyrene derivatives bound noncovalently and covalently to DNA.

Linear dichroism and absorption methods are used to study the orientations of transition moments of absorption bands of polycyclic aromatic epoxide derivatives which overlap with those of the DNA band in the 240-300 nm region. Both the short and long axes of the pyrene residues of 1-oxiranylpyrene (1-OP) and the (+) and (-) enantiomers of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) noncovalently bound to double-stranded native DNA are oriented approximately perpendicular to the axis of the DNA helix, consistent with intercalative modes of binding. The covalent binding of these three epoxide derivatives to DNA is accompanied by reorientations of both the short and long axes of the pyrene residues. Covalent adducts derived from the highly mutagenic (+)-anti-BPDE are characterized by tilts of the short axis within 35 degrees or less, and of the long axis by more than 60-80 degrees, with respect to the planes of the DNA bases. In the adducts derived from the binding of the less mutagenic (-)-anti-BPDE and 1-OP epoxide derivatives to DNA, the long axes of the pyrenyl rings are predominantly oriented within 25 degrees of the planes of the DNA bases; however, in the case of the (-) enantiomer of BPDE, there is significant heterogeneity of conformations. In the case of the 1-OP covalent DNA adducts, the short axis of the pyrene ring system is tilted away from the planes of the DNA bases, and the pyrene ring system is not intercalated between DNA base-pairs as in the noncovalent complexes. The stereochemical properties of the saturated 7,8,9,10-ring in BPDE, or the lack of the 7 and 8 carbon atoms in 1-OP, do not seem to affect noncovalent intercalative complex formation which, most likely, is influenced mainly by the flat pyrenyl residues. These structural features, however, strongly influence the conformations of the covalent adducts, which in turn may be responsible for the differences in the mutagenic activities of these molecules.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗