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At least 865 records · Page 48Linked to original sources
PubMed2002

Rotational drag on DNA: a single molecule experiment.

Within a single-molecule configuration, we have studied rotational drag on double stranded linear DNA by measuring the force during mechanical opening and closing of the double helix at different rates. The molecule is cranked at one end by the effect of unzipping and is free to rotate at the other end. In this configuration the rotational friction torque tau on double-stranded DNA leads to an additional contribution to the opening force. It is shown that the effect of rotational drag increases with the length of the molecule, is approximately proportional to the angular velocity of cranking, and we estimate that the torque tau is of the order of 1k(B)T for 10 000 base pairs of DNA cranked at 2000 turns per second.

Chemical Phenomena↗
PubMed1988

Palindrome-hairpin linear plasmids possessing only a part of the ORF1 gene of the yeast killer plasmid pGKL1.

The yeast Kluyveromyces lactis harboring linear DNA plasmids pGKL1 and pGKL2 exhibits killer and killer-resistant phenotypes. Two new linear plasmids pK192L and pK192S were found in the weak killer mutant KUV192 induced by UV irradiation. pK192S was always accompanied by pK192L in subclones of KUV192. Both plasmids were derived from pGKL1 by deletion of the large right part of it. pK192L was 4.9 kb in size and had a palindromic structure consisting of 2.35 kb inverted terminal repetitions and a 215 base unique sequence. Analysis of denatured and renatured DNA strands suggested that pK192S was a hairpin-like form of pK192L. The pK192 plasmids were maintained only in cells haboring either pGKL1 or pGKL1Sin addition to pGKL2 and completed with pFKL1 or pGKL1S for their maintenance. Since no complete ORF1 was conserved in pK192 plasmids, these results lead to the conclusion that the ORF1 gene is necessary for the replication and/or maintenance of pGKL1.

Amino Acid Sequence↗
PubMed1981

Restriction endonuclease mapping of linear unintegrated proviral DNA of bovine leukemia virus.

A detailed restriction map was deduced for the genome of the exogenous bovine leukemia virus. The cleavage sites for nine restriction enzymes were mapped. The unintegrated linear viral DNA intermediate that is produced by infection of permissive cells with bovine leukemia virus was isolated. The linear viral DNA had a unique restriction map, indicating that it is not a set of random circular permutations of the RNA genome. From hybridization with a 3'-enriched probe, the DNA restriction map was aligned relative to the 5'-to-3' orientation of the viral RNA. Restriction enzyme analysis of integrated bovine leukemia virus information present in animals with enzootic bovine leukosis provided evidence for the existence of genetic variants of the virus.

Chromosome Mapping↗
PubMed1995

Efficient gene targeting in the filamentous fungus Alternaria alternata.

To characterize homologous recombination of transforming DNA in the filamentous fungus Alternaria alternata, we have compared the frequencies of gene targeting by circular and linear DNA fragments in the fungus. The A. alternata BRM1 gene, which is an essential gene for melanin biosynthesis, was selected as a target locus. BRM1 targeting events are easily identified because loss of function leads to a change in mycelial color from black to light brown. We constructed targeting vectors by inserting 0.6 to 3.1 kb internal BRM1 segments into a plasmid containing the hygromycin B phosphotransferase gene. When circular plasmids were used, melanin-deficient (Mel-) transformants accounted for 30 to 80% of hygromycin B-resistant (HyR) transformants, correlating closely with the size of the BRM1 segment in the transforming DNA. Restriction enzyme digestion within the BRM1 region greatly enhanced the frequency of gene targeting: integration of the linear plasmids was almost completely attributable to homologous recombination, regardless of the size of the BRM1 segments. Plasmids carrying both BRM1 segments and rDNA segments were transformed into the fungus to examine the effect of the number of target copies on homologous recombination. Using the circular plasmids, Mel- transformants accounted for only 5% of HyR transformants. In contrast, when the linear plasmid produced by restriction enzyme digestion within the BRM1 segment was used, almost all transformants were Mel-. These results indicate that homologous integration of circular molecules in A. alternata is sensitive to the length of homology and the number of targets, and that double-strand breaks in transforming DNA greatly enhance homologous recombination.

Alternaria↗
PubMed1999

Dynamics of long DNA confined by linear polymers.

We studied the electrophoretic behavior of long DNA molecules in a linear polymer [polyacrylamide (PA)] solution through direct observation by means of fluorescence microscopy. DNA migrates in an I-shaped conformation in concentrated polymer solutions under steady electric fields, but it is not stretched up to its natural contour length in this I-shaped conformation under such fields. The stretching of DNA is induced under alternating current fields through the entanglement effect between DNA and host polymers. We experimentally investigated the conditions required for this stretching phenomenon and found that DNA can be stretched at a concentration of around 7% PA, under a field of around 10 Hz. These conditions do not depend on the length of the DNA chains. It is expected that DNA stretching will be useful in the optical mapping of specific sites along an individual DNA chain.

Bacteriophage T4↗
PubMed1995

Visual mapping by fiber-FISH.

FISH techniques have opened new possibilities for high-resolution genome mapping. Effective utilization of these techniques for the rapid orientation and ordering of adjacent and overlapping probes as well as for the characterization of long-range genomic contigs would facilitate physical mapping and positional cloning efforts. Here, we have evaluated our recently developed improved fiber-FISH technique for the physical mapping of a 500-kb region at 1p32 as well as for the detection of genomic rearrangement affecting this region. Our fiber-FISH technique is based on the hybridization of probes to unfixed linearized DNA fibers on a microscope slide. Preparation of the target DNA from cells embedded in pulsed-field gel electrophoresis (PFGE) blocks makes it possible to obtain long intact DNA fibers that give an excellent signal-to-noise ratio in FISH. The linear range of the method reached from 2 to 500 kb with a measuring accuracy approaching that of PFGE. Fiber-FISH was used to establish the order, orientation, and distances for several probes for this region, including six large insert phage, cosmid, and P1 clones and seven genomic subclones. This has significantly facilitated our efforts to develop a genomic contig for this region, recently discovered to contain the gene for inherited neuronal ceroid lipofuscinosis (INCL). Finally, we also demonstrated how rearrangements affecting the L-myc gene at this locus in small-cell lung cancer can be visualized with fiber-FISH. In conclusion, fiber-FISH is very useful for high-resolution physical mapping and contig evaluation as well as for detecting genetic rearrangements.

Carcinoma, Small Cell↗
PubMed1999

Nucleosome dynamics. Protein and DNA contributions in the chiral transition of the tetrasome, the histone (H3-H4)2 tetramer-DNA particle.

Our laboratory has previously reported the chiral transition of DNA minicircle-reconstituted tetrasomes (the particles made of DNA wrapped around the histone (H3-H4)2tetramer). This transition was induced by DNA positive torsional constraint, generated either by initial supercoiling of the loop or by its thermal fluctuations during topoisomerase relaxation. Taking into account the wrapping of the DNA around the histones into less than a turn, and its negative crossing at the entry-exit, the transition was proposed to involve a 360 degrees rotation of the loop around the particle dyad axis, and the formation of a positive crossing. The tetramer horseshoe-shaped conformation within the octamer further suggested that this process could be mediated by a reorientation of the two sector-like H3-H4 dimers about their H3/H3 interface, which would switch the overall handedness of the proteinaceous superhelix from left to right-handed. We now provide additional evidence for such a contribution of the protein by showing, through gel electrophoresis, topoisomerase relaxation and electron microscopy, that a sterical hindrance at the H3/H3 interface, introduced by covalent linking of bulky adducts through thiol oxidation of H3 cysteine 110, interferes with the transition. Such interference varies, depending on the particular SH-reagent used; but the most remarkable effect was obtained with 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB), which displaces the preferred conformation of the tetrasomes from left-handed to semi-right-handed, and at the same time preserves a significant degree of chiral flexibility. DNA contribution was evidenced by a specific fractionation of circular tetrasomes in gel electrophoresis which, together with a different positioning of control and DTNB tetrasomes on linear DNA, pointed to an interdependence between tetrasome conformation and positions. Moreover, linear tetrasomes fluctuate between crossed and uncrossed conformations in a salt-dependent equilibrium which appears to vary with their positions on the DNA. These data suggest a modulatable role of the DNA around the dyad in the transition, depending primarily on its sequence-dependent deformability. This role is played at both levels of H3-H4 dimer reorientation and lateral opening, a mechanism by which the particle may relieve the clash between its entering and exiting DNAs. These properties make the tetrasome an attractive potential intermediate in nucleosome dynamics in vivo, in particular duringX transcriptional activation and elongation.

Animals↗
PubMed2002

Nucleosome linker proteins HMGB1 and histone H1 differentially enhance DNA ligation reactions.

We previously reported that HMGB1, which originally binds to chromatin in a manner competitive with linker histone H1 to modulate chromatin structure, enhances both intra-molecular and inter-molecular ligations. In this paper, we found that histone H1 differentially enhances ligation reaction of DNA double-strand breaks (DSB). Histone H1 stimulated exclusively inter-molecular ligation reaction of DSB with DNA ligase IIIbeta and IV, whereas HMGB1 enhanced mainly intra-molecular ligation reaction. Electron microscopy of direct DNA-protein interaction without chemical cross-linking visualized that HMGB1 bends and loops linear DNA to form compact DNA structure and that histone H1 is capable of assembling DNA in tandem arrangement with occasional branches. These results suggest that differences in the enhancement of DNA ligation reaction are due to those in alteration of DNA configuration induced by these two linker proteins. HMGB1 and histone H1 may function in non-homologous end-joining of DSB repair and V(D)J recombination in different manners.

DNA↗
PubMed1999

DNA bending induced by the archaebacterial histone-like protein MC1.

The conformational changes induced by the binding of the histone-like protein MC1 to DNA duplexes have been analyzed by dark-field electron microscopy and polyacrylamide gel electrophoresis. Visualisation of the DNA molecules by electron microscopy reveals that the binding of MC1 induces sharp kinks. Linear DNA duplexes (176 bp) which contained a preferential site located at the center were used for quantitative analysis. Measurements of the angle at the center of all duplexes, at a fixed DNA concentration, as a function of the MC1 concentration, were very well fitted by a simple model of an isotropic flexible junction and an equilibrium between the two conformations of DNA with bound or unbound MC1. This model amounts to double-folded Gaussian distributions and yields an equilibrium deflection angle of theta0=116 degrees for the DNA with bound MC1. It allowed measurements of the fraction of DNA with bound MC1 to be taken as a function of MC1 concentrations and yields an equilibrium dissociation constant of Kd=100 nM. It shows that the flexibility of DNA is reduced by the binding of MC1 and the formation of a kink. The equilibrium dissociation constant value was corroborated by gel electrophoresis. Control of the model by the computation of the reduced chi2 shows that the measurements are consistent and that electron microscopy can be used to quantify precisely the DNA deformations induced by the binding of a protein to a preferential site.

Archaeal Proteins↗
PubMed1988

Molecular basis for sequence-specific DNA alkylation by CC-1065.

CC-1065 is a potent antitumor antibiotic that binds covalently to N3 of adenine in the minor groove of DNA. The CC-1065 molecule is made up of three repeating pyrroloindole subunits, one of which (the left-hand one or A subunit) contains a reactive cyclopropyl function. The drug reacts with adenines in DNA in a highly sequence-specific manner, overlapping four base pairs to the 5'-side of the covalently modified base. Concomitant with CC-1065 covalent binding to DNA is an asymmetric effect on local DNA structure which extends more than one helix turn to the 5'-side of the covalent binding site. The DNA alkylation, sequence specificity, and biological potency of CC-1065 and a select group of trimeric synthetic analogues were evaluated. The results suggest that (a) noncovalent interactions between this series of compounds and DNA do not lead to the formation of complexes stable enough to be detected by footprinting methods, (b) sequence specificity and alkylation intensity can be modulated by the substituents on the nonreactive middle and right-hand segments, and (c) biological potency correlates well with ability to alkylate DNA. In addition, the extent and the sequence specificity of covalent adduct formation between linear DNA fragments and three analogues comprised of the CC-1065 alkylating subunit linked to zero (analogue A), one (analogue AB), or two (analogue ABC) nonreactive indole subunits were compared.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylation↗
PubMed1989

On the mechanism of topoisomerase I inhibition by camptothecin: evidence for binding to an enzyme-DNA complex.

Camptothecin, a cytotoxic antitumor compound, has been shown to produce protein-linked DNA breaks mediated by mammalian topoisomerase I. We have investigated the mechanism by which camptothecin disrupts DNA processing by topoisomerase I and have examined the effect of certain structurally related compounds on the formation of a DNA-topoisomerase I covalent complex. Enzyme-mediated cleavage of supercoiled plasmid DNA in the presence of camptothecin was completely reversed upon the addition of exogenous linear DNA or upon dilution of the reaction mixture. Camptothecin and topoisomerase I produced the same amount of cleavage from supercoiled DNA or relaxed DNA. In addition, the alkaloid decreased the initial velocity of supercoiled DNA relaxation mediated by catalytic quantities of topoisomerase I. Inhibition occurred under conditions favoring processive catalysis as well as under conditions favoring distributive catalysis. By use of [3H]camptothecin and an equilibrium dialysis assay, the alkaloid was shown to bind reversibly to a DNA-topoisomerase I complex, but not to isolated enzyme or isolated DNA. These results are consistent with a model in which camptothecin reversibly traps an intermediate involved in DNA unwinding by topoisomerase I and thereby perturbs a set of equilibria, resulting in increased DNA cleavage. By examining certain compounds that are structurally related to camptothecin, it was found that the 20-hydroxy group, which has been shown to be essential for antitumor activity, was also necessary for stabilization of the covalent complex between DNA and topoisomerase I. In contrast, no such correlation existed for UV-light-induced cleavage of DNA by Cu(II)-camptothecin derivatives.

Camptothecin↗
PubMed2006

The Deinococcus radiodurans-encoded HU protein has two DNA-binding domains.

Deinococcus radiodurans can reconstitute its genome from double-strand breaks, most likely due to unusually efficient DNA repair and recombination. Factors that may contribute to such processes include the histone-like protein HU. The D. radiodurans-encoded HU (DrHU), which binds preferentially to DNA recombination intermediates, contains a 47-amino acid extension preceding the fold characteristic of HU proteins. Here we use electrophoretic mobility shift assays and DNA footprinting to show that the DrHU N-terminal domain significantly modulates DNA binding. The truncated DrHU (deltaDrHU), comprising only the conserved DNA-binding fold, has a site size of approximately 11 bp in contrast to full-length DrHU which does not stably engage DNA shorter than approximately 50 bp. Unlike wild-type DrHU, deltaDrHU distinguishes between linear DNA and DNA with nicks or gaps. DeltaDrHU also binds preferentially to four-way junction (4WJ) DNA, with half-maximal saturation of 1.4 +/- 0.4 nM compared to 20 +/- 2 nM for 37 bp duplex DNA. However, in contrast to full-length protein which binds the junction arms, deltaDrHU primarily protects the junction crossover. Evidently, the DrHU N-terminus changes the mode of binding to both 4WJ DNA, duplex DNA, and DNA with nicks or gaps, thereby resulting in DrHU binding preferentially only to 4WJ structures. Combined with Western blots that confirm the presence of the extended form of DrHU in vivo, our data provide mechanistic insight into discrimination between 4WJ DNA and other distorted DNA constructs and suggest that an in vivo role of DrHU may be to stabilize DNA junctions.

Bacterial Proteins↗
PubMed1996

Energy coupling in Escherichia coli DNA gyrase: the relationship between nucleotide binding, strand passage, and DNA supercoiling.

Binding of the nonhydrolyzable ATP analogue 5'-adenylyl-beta, gamma-imidodiphosphate (ADPNP) to Escherichia coli DNA gyrase can lead to a limited noncatalytic supercoiling of DNA. Here we examine the efficiency of coupling between ADPNP binding and the change in linking number either of positively or negatively supercoiled plasmid DNA or of small DNA circles. The coupling efficiency varies from 100% (delta Lk = -2 per gyrase tetramer, a stoichiometry of 1) with positively supercoiled substrates under certain reaction conditions to an undetectably low value with moderately negatively supercoiled substrates (sigma = -0.046) or small circular substrates. Furthermore, the rate of ADPNP binding to the gyrase-DNA complex is also dependent on the topological state of the DNA; the previously observed slow binding of ADPNP to the complex of gyrase with linear DNA is accelerated 16-fold when the substrate DNA is negatively supercoiled, suggesting a functional interaction between the nucleotide-binding and DNA-binding domains which is independent of the strand-passage process. The implications for the normal ATP-dependent supercoiling reaction of the enzyme are considered and the results discussed in terms of current mechanistic models for DNA gyrase action and the possible in vivo roles of the enzyme.

Adenylyl Imidodiphosphate↗
PubMed2004

Binding of the DNA-dependent protein kinase catalytic subunit to Holliday junctions.

DNA-PK (DNA-dependent protein kinase) is a double-strand break sensor involved in DNA repair and signal transduction. In the present study, we constructed site-directed cross-linking probes to explore the range of DNA discontinuities that are recognized by DNA-PK(CS) (DNA-PK catalytic subunit). A comparison between different substrate architectures showed that DNA-PK(CS) associates preferentially with the crossover region of synthetic Holliday junctions. This interaction with four-way junctions was preserved when biotin-streptavidin complexes were assembled at the termini to exclude the binding of Ku proteins. The association of DNA-PK(CS) with Holliday junctions was salt-labile even in the presence of Ku proteins, but this interaction could be stabilized when the DNA probes were incubated with the endogenous enzyme in nuclear extracts of human cells. Cross-linking of the endogenous enzyme in cellular extracts also demonstrated that DNA-PK(CS) binds to DNA ends and four-way junctions with similar affinities in the context of a nuclear protein environment. Kinase assays using p53 proteins as a substrate showed that, in association with four-way structures, DNA-PK(CS) adopts an active conformation different from that in the complex with linear DNA. Our results are consistent with a structure-specific, but Ku- and DNA end-independent, recruitment of DNA-PK(CS) to Holliday junction intermediates. This observation suggests an unexpected functional link between the two main pathways that are responsible for the repair of DNA double-strand breaks in mammalian cells.

Azides↗
PubMed1994

Large-scale opening of A + T rich regions within supercoiled DNA molecules is suppressed by salt.

Large-scale cooperative helix opening has been previously observed in A + T rich sequences contained in supercoiled DNA molecules at elevated temperatures. Since it is well known that helix melting of linear DNA is suppressed by addition of salt, we have investigated the effects of added salts on opening transitions in negatively supercoiled DNA circles. We have found that localised large-scale stable melting in supercoiled DNA is strongly suppressed by modest elevation of salt concentration, in the range 10 to 30 mM sodium. This has been shown in a number of independent ways: 1. The temperature required to promote cruciform extrusion by the pathway that proceeds via the coordinate large-scale opening of an A + T rich region surrounding the inverted repeat (the C-type pathway, first observed in the extrusion of the ColE1 inverted repeat) is elevated by addition of salt. The temperature required for extrusion was increased by about 4 deg for an addition of 10 mM NaCl. 2. A + T rich regions in supercoiled DNA exhibit hyperreactivity towards osmium tetroxide as the temperature is raised; this reactivity is strongly suppressed by the addition of salt. At low salt concentrations of added NaCl (10 mM) we observe that there is an approximate equivalence between reducing the salt concentration, and the elevation of temperature. Above 30 mM NaCl the reactivity of the ColE1 sequences is completely supressed at normal temperatures. 3. Stable helix opening transitions in A + T rich sequences may be observed with elevated temperature, using two-dimensional gel electrophoresis; these transitions become progressively harder to demonstrate with the addition of salt. With the addition of low concentrations of salt, the onset of opening transitions shifts to higher superhelix density, and by 30 mM NaCl or more, no transitions are visible up to a temperature of 50 degrees C. Statistical mechanical simulation of the data indicate that the cooperativity free energy for the transition is unaltered by addition of salt, but that the free energy cost for opening each basepair is increased. These results demonstrate that addition of even relatively low concentrations of salt strongly suppress the large-scale helix opening of A + T rich regions, even at high levels of negative supercoiling. While the opening at low salt concentrations may reveal a propensity for such transitions, spontaneous opening is very unlikely under physiological conditions of salt, temperature and superhelicity, and we conclude that proteins will therefore be required to facilitate opening transitions in cellular DNA.

Base Composition↗
PubMed2006

Solubility and charge inversion of complexes of DNA and basic proteins.

The basic proteins, protamines and histones H1, are known to condense DNA in vivo. We examine here their ability to condense and solubilize in vitro linear DNA [and a synthetic polyanion, Poly(Styrene-Sulfonate) or PSS] at low ionic concentrations by varying the charge concentration ratio. Phase separation is observed in a very narrow range of ratios for short DNA and PSS; on both sides of this range, polydisperse and charged complexes are formed. A charge inversion is detected. For long DNA chains however, a different behavior is observed: the complexes are not soluble in excess of proteins.

Binding Sites↗
PubMed1999

Genome-scale cloning and expression of individual open reading frames using topoisomerase I-mediated ligation.

The in vitro cloning of DNA molecules traditionally uses PCR amplification or site-specific restriction endonucleases to generate linear DNA inserts with defined termini and requires DNA ligase to covalently join those inserts to vectors with the corresponding ends. We have used the properties of Vaccinia DNA topoisomerase I to develop a ligase-free technology for the covalent joining of DNA fragments to suitable plasmid vectors. This system is much more efficient than cloning methods that require ligase because the rapid DNA rejoining activity of Vaccinia topoisomerase I allows ligation in only 5 min at room temperature, whereas the enzyme's high substrate specificity ensures a low rate of vector-alone transformants. We have used this topoisomerase I-mediated cloning technology to develop a process for accelerated cloning and expression of individual ORFs. Its suitability for genome-scale molecular cloning and expression is demonstrated in this report.

Animals↗