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1.
Thermodynamics of Long Supercoiled Molecules: Insights from Highly Efficient Monte Carlo Simulations
Supercoiled DNA polymer models for which the torsional energy depends on the total twist of molecules (Tw) are a priori well suited for thermodynamic analysis of long molecules. So far, nevertheless, the exact determination of Tw in these models has been based on a computation of the writhe of the molecules (Wr) by exploiting the conservation of the linking number, Lk = Tw + Wr, which reflects topological constraints coming from the helical nature of DNA. Because Wr is equal to the number of times the main axis of a DNA molecule winds around itself, current Monte Carlo algorithms have a quadratic time complexity, O(L2), with respect to the contour length (L) of the molecules. Here, we present an efficient method to compute Tw exactly, leading in principle to algorithms with a linear complexity, which in practice is O(L1.2). Specifically, we use a discrete wormlike chain that includes the explicit double-helix structure of DNA and where the linking number is conserved by continuously preventing the generation of twist between any two consecutive cylinders of the discretized chain. As an application, we show that long (up to 21 kbp) linear molecules stretched by mechanical forces akin to magnetic tweezers contain, in the buckling regime, multiple and branched plectonemes that often coexist with curls and helices, and whose length and number are in good agreement with experiments. By attaching the ends of the molecules to a reservoir of twists with which these can exchange helix turns, we also show how to compute the torques in these models. As an example, we report values that are in good agreement with experiments and that concern the longest molecules that have been studied so far (16 kbp). 相似文献
2.
《Seminars in cell biology》1995,6(2):65-72
A DNA structure checkpoint can be defined as any checkpoint which responds to changes in the structure of the DNA either through the cell cycle, or in response to outside events such as DNA damage. Genetic analysis of DNA structure checkpoints in fission yeast has identified several distinct pathways responding to different circumstances. Three checkpoints have been identified which inhibit the onset of mitosis. (1) A radiation checkpoint which prevents mitosis after DNA damage. (2) A checkpoint linking S phase and mitosis (the S-M checkpoint) that prevents mitosis when DNA synthesis is incomplete. (3) A checkpoint linking G1 to mitosis (the G1-M checkpoint) that prevents the onset of mitosis in cells which are arrested in the G1 period of the cycle. A large number of genetic loci that are required for these checkpoints have been identified through mutant analysis, and the involvement of the relevant genes with the individual checkpoint pathways has been investigated. The largest class of checkpoint genes, known as the ‘checkpoint rad’ genes, are required for all the DNA structure checkpoints and the evidence suggests that they may also be involved in regulating DNA synthesis following precursor deprivation (hydroxyurea treatment) or when the replication fork encounters DNA damage. In this review, the available genetic and physiological evidence has been interpreted to suggest a close association between the ‘checkpoint rad’ class of gene products and the DNA-protein complexes that regulate and perform DNA synthesis. Biochemical evidence will be required in order to prove or disprove this hypothesis. 相似文献
3.
Do the two chains of the DNA molecule coil round one another plectonemically ? If so, what is the approximate value of Lk (the linking number) for any closed, circular DNA molecule? Experiments using gel electrophoresis have shown that supercoiled DNA molecules usually migrate in a series of discrete bands. The only tenable explanation for this quantized behavior is that the molecules in one band all have the same value of Lk and that this value differs by unity from that of the adjacent bands. Various experiments in which circular DNA is unwound by known amounts show that (given this assumption) Lk for relaxed DNA is very roughly equal to (where N is the number of base-pairs), as expected from the classical double helix.The original model for the double helix was right-handed. The experimental evidence for this feature is suggestive but not yet completely compelling. 相似文献
4.
A Stein 《Nucleic acids research》1980,8(20):4803-4820
Chromatin was assembled in vitro from relaxed closed circular DNA (SV40) and core histones at histone to DNA ratios of 0.2 to 0.3 (g/g) and incubated with topoisomerase I to relax supercoils in DNA regions not constrained by protein. Addition of histones H1 + H5 to the chromatin at an ionic strength of 0.1 M, in the presence of the solubilizing agent, polyglutamic acid, and topoisomerase I, increased the magnitude of the DNA linking number change, relative to protein-free DNA. No change in the linking number distribution occurred for relaxed protein-free DNA under these conditions. Control experiments indicated that the increase in the absolute value of the DNA linking number change in the chromatin could not be attributed to an increase in the number of nucleosomes per DNA molecule. These data suggest a solution to the linking number problem associated with models of chromatin structure. 相似文献
5.
Treatment of mammalian cells with DNA intercalating agents produces protein-associated DNA strand breaks. These breaks have been proposed to represent the action of a topoisomerase, which would alter the DNA linking number. Changes in DNA linking number in cells treated with the intercalating agent 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) were studied by ethidium titration of nucleoid sedimentation. m-AMSA treatment was found to produce an increase in DNA linking number. Previously, we had proposed that intercalator-induced protein-associated DNA breaks act to reduce DNA torsional strain that results from the intercalator-induced decrease in DNA twist. In such a model, linking number would be expected to decrease. The finding that the DNA linking number increased following m-AMSA treatment suggests that intercalators may block enzymes that normally decrease linking number. Such enzymes would have DNA gyrase like properties. Consistent with this possibility, a DNA gyrase inhibitor, novobiocin, inhibited the restoration of normal linking number and, to a lesser degree, the reversal of protein-associated strand breaks after removal of intercalator. 相似文献
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7.
Transformation of the Lyme Disease Spirochete Borrelia burgdorferi with Heterologous DNA 总被引:1,自引:0,他引:1
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Brian Stevenson James L. Bono Abdallah Elias Kit Tilly Patricia Rosa 《Journal of bacteriology》1998,180(18):4850-4855
Studies of the spirochete Borrelia burgdorferi have been hindered by the scarcity of genetic tools that can be used in these bacteria. For the first time, a method has been developed by which heterologous DNA (DNA without a naturally occurring B. burgdorferi homolog) can be introduced into and persistently maintained by B. burgdorferi. This technique uses integration of circular DNA into the bacterial genome via a single-crossover event. The ability to transform B. burgdorferi with heterologous DNA will now permit a wide range of experiments on the biology of these bacteria and their involvement in the many facets of Lyme disease. 相似文献
8.
Supercoiling in prokaryotic and eukaryotic DNA: changes in response to topological perturbation of plasmids in E. coli and SV40 in vitro, in nuclei and in CV-1 cells. 总被引:8,自引:2,他引:8
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Changes in DNA linking number have been observed in plasmid DNA purified from E. coli cells after the cells were treated with chloroquine. Chloroquine, a DNA intercalating drug, unwinds the DNA, decreasing the levels of negative supercoiling. Following this in vivo topological perturbation, within minutes DNA gyrase decreases DNA linking number producing more negatively supercoiled DNA topoisomers. Following the removal of the drug from cells, within minutes topoisomerase 1 or DNA gyrase increases the linking number restoring the original level of supercoiling. Analogous changes in DNA linking number after addition of chloroquine are observed in purified plasmid DNA, and in purified SV40 minichromosomes in the presence of exogenous topoisomerase. Changes in linking number are also observed in SV40 chromosomes in isolated nuclei and in SV40 DNA purified from CV-1 cells following topological perturbation with chloroquine. These results suggest that eukaryotic cells may have mechanisms to maintain a defined level of DNA supercoiling. 相似文献
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11.
In vitro display methods are superior tools for obtaining monoclonal antibodies. Although totally in vitro display methods, such as ribosome display and mRNA display, have the advantages of larger library sizes and quicker selection procedures compared with phage display, their applications have been limited to single-chain Fvs due to the requirement for linking of the mRNA and the nascent protein on the ribosome. Here we describe a different type of totally in vitro method, DNA display, that is applicable to heterodimeric Fab fragments: in vitro compartmentalization in water-in-oil emulsions allows the linking of an oligomeric protein and its encoding DNA with multiple ORFs. Since previously used emulsions impaired the synthesis of functional Fab fragments, we modified conditions for preparing emulsions, and identified conditions under which it was possible to enrich Fab fragments 106-fold per three rounds of affinity selection. Furthermore, we confirmed that genes encoding stable Fab fragments could be selected from a Fab fragment library with a randomized hydrophobic core in the constant region by applying heat treatment as a selection pressure. Since this method has all advantages of both phage display and totally in vitro display, it represents a new option for many applications using display methods. 相似文献
12.
Linking-number changes in the DNA substrate during Cre-mediated loxP site-specific recombination 总被引:4,自引:0,他引:4
We have examined the linking-number changes that occur during phage P1 Cre-mediated recombination in vitro between two loxP sites. Such recombination reactions can be divided into three types: intramolecular inversion, in which recombination occurs between two loxP sites in opposite orientations on the same DNA substrate; intramolecular excision, where recombination occurs between two loxP sites that are in the same orientation on the DNA substrate; and intermolecular recombination, which occurs between two loxP sites on separate DNA molecules. Our results indicate that inversion changes the linking number of the substrate DNA by two topological turns. With a negatively supercoiled substrate, the product is changed by +2 turns. A relaxed substrate yields products that have been changed by either +2 or -2 turns. For intermolecular reactions, the sum of the linking numbers of each of the two starting circles is equal to the linking number of the dimer circle generated by recombination, and no change occurs in linking number. For intramolecular excision reactions, the data are most consistent, with no change in linking number during recombination. These results are discussed in terms of models for alignment and synapsis of the recombining sites and the mechanism of strand exchange. 相似文献
13.
Robert K. Moyzis Jacques Bonnet David W. Li Paul O.P. Tso 《Journal of molecular biology》1981,153(4):841-864
The biochemical and biophysical techniques originally introduced by Davidson et al. (1973) and Graham et al. (1974) for the determination of the general organization and length of repetitive and non-repetitive sequences in eukaryotic DNA have been extended and modified. Improvements in the experimental methods employed in these pioneering works have led to novel interpretations and conclusions about mammalian DNA sequence organization. In what is commonly referred to as an interspersion experiment, the average spacing of repetitive DNA regions is inferred from the length dependence of hydroxyapatite binding of radio-labeled tracer DNAs reassociated with an excess of short 200 nucleotide repetitive sequence driver DNA. Studies on Syrian hamster DNA, using an improved procedure for conducting interspersion experiments, suggest that either a frequent cluster in the distribution of non-repetitive DNA sequence lengths occurs at 7200 (±2000) nucleotides or that repetitive sequences are randomly spaced on a number average basis. In contrast, measurements obtained using the traditional methods suggest that a frequent cluster in the distribution of non-repetitive DNA sequence lengths occurs at approximately 1000 nucleotides. When reassociations were conducted at elevated temperatures, to allow only well-matched repetitive sequences to hybridize, the amount of DNA operationally observed as “repetitive” was reduced. Interspersion experiments conducted with Syrian hamster DNA at a reassociation temperature of 75 °C yielded data similar to those obtained by Manning et al. (1975) for Drosophila melanogaster DNA reassociated at 60 °C. 相似文献
14.
Practical problems of handling large numbers of samples limit the application of molecular genetic procedures in clinical settings and in research. In the present review we describe a multipronged manifold support, coated with streptavidin, that offers distinct advantages in preparative and diagnostic applications. In order to increase the surface available on the manifold, porous Sepharose particles conjugated with streptavidin were attached to the plastic support. This procedure increased the surface by almost three orders of magnitude, permitting sufficient streptavidin to be coupled to the support for most routine applications. The manifold supports have been used for sample preparation and in a number of genetic assays, including allele discrimination assays and DNA sequencing, In all these assay formats the manifold supports allow large numbers of samples to be processed in parallel. 相似文献
15.
V. V. Anshelevich A. V. Vologodskii M. D. Frank-Kamenetskii 《Journal of biomolecular structure & dynamics》2013,31(2):247-259
Abstract The development of statistical mechanical models of the formation of noncanonical structures in circular DNA and the finding of the energy parameters for these models made it possible to predict the appearance of such structures in a DNA with any given sequence. It does not seem feasible, however, to perform such calculations for DNA sequences of considerable length by allowing for all the possible states. We propose a special algorithm for calculating the thermodynamic characteristics of various conformational rearrangements in DNA that occur under negative supercoilings, allowing for several possible states of each base pair in the chain. Calculations have been performed for a number of natural DNAs. According to these calculations, the most likely noncanonical structures in DNA under normal conditions are cruciform structures and the Z form. The results of the calculations are compared with the experimental data reported in the literature. State diagrams have been computed for a number of inserts in circular DNA that can adopt both the cruciform conformation and the left-handed helical Z form. 相似文献
16.
Paolo U. Giacomoni 《Biopolymers》1982,21(1):117-129
A melting experiment was performed on the whole set of populations of the replicative form of ?X174 DNA, which can be obtained treating this DNA with rat liver nicking-closing enzyme in the presence of ethidium bromide. Gel electrophoresis performed by loading the DNA samples at neutral and alkaline pH allows separation of these populations in discrete sets of bands, which can then be compared. The outcome of the experiments indicates that in the range of electrophoretic mobilities which can be explored, no band is formed exclusively by circular complementary strands which can be separated by alkaline denaturation. These results are compared with what would be expected if double-stranded closed circular DNA had structures other than the canonical double helix. Under nonrestrictive hypotheses, the experiments reported allow one to obtain a minimum estimate of the absolute value of the linking number of a closed circular double-stranded DNA: for native ?X174 RF DNA, the linking number appears to be greater than 12 (in absolute value). Some data on the electrophoretic mobility of denatured closed circular duplexes are reported, which still wait for a physicochemical interpretation. 相似文献
17.
The mathematical basis of the hypothesis that type-2 topoisomerases recognize and act at specific DNA juxtapositions has been investigated by coarse-grained lattice polymer models, showing that selective segment passages at hooked juxtapositions can result in dramatic reductions in catenane and knot populations. The lattice modeling approach is here extended to account for the narrowing of variance of linking number (Lk) of DNA circles by type-2 topoisomerases. In general, the steady-state variance of Lk resulting from selective segment passages at a specific juxtaposition geometry j is inversely proportional to the average linking number, 〈Lk〉j, of circles with the given juxtaposition. Based on this formulation, we demonstrate that selective segment passages at hooked juxtapositions reduce the variance of Lk. The dependence of this effect on model DNA circle size is remarkably similar to that observed experimentally for type-2 topoisomerases, which appear to be less capable in narrowing Lk variance for small DNA circles than for larger DNA circles. This behavior is rationalized by a substantial cancellation of writhe in small circles with hook-like juxtapositions. During our simulations, we uncovered a twisted variation of the hooked juxtaposition that has an even more dramatic effect on Lk variance narrowing than the hooked juxtaposition. For an extended set of juxtapositions, we detected a significant correlation between the Lk narrowing potential and the logarithmic decatenating and unknotting potentials for a given juxtaposition, a trend reminiscent of scaling relations observed with experimental measurements on type-2 topoisomerases from a variety of organisms. The consistent agreement between theory and experiment argues for type-2 topoisomerase action at hooked or twisted-hooked DNA juxtapositions. 相似文献
18.
A rapid filter method for the microfluorometric analysis of DNA is reported in this communication. Cells collected on cellulose filters are subject to an assay sequence developed from a fluorometric method initially described by J. M. Kissane and E. Robins ((1958) J. Biol. Chem.233, 184–188) for DNA quantitation. The assay is one of high specificity requiring no separation of DNA from other major cellular components. Samples containing as little as 0.2–0.3 μg of DNA have been analyzed by this filter method with a coefficient of variation for replicate standard samples of 3.3%. The DNA content of a number of cell types having different physiological characteristics has been determined by the use of this filter technique. The data obtained for Chlorella, Chlamydomonas, Ochromonas, and Stronglyocentrotus eggs were well within the values reported for these cells by other investigators using classical macromethods. We have taken advantage of the high specificity, sensitivity, and accuracy of this filter assay to determine DNA content during the synchronous growth cycle of the wall-less alga Olisthodiscus luteus using a single small volume culture as a sample source. 相似文献
19.
Xu Y Gao S Bruno JF Luft BJ Dunn JJ 《Biochemical and biophysical research communications》2008,375(4):522-525
Zoonotic pathogens including those transmitted by insect vectors are some of the most deadly of all infectious diseases known to mankind. A number of these agents have been further weaponized and are widely recognized as being potentially significant biothreat agents. We describe a novel method based on multiply-primed rolling circle in vitro amplification for profiling genomic DNAs to permit rapid, cultivation-free differential detection and identification of circular plasmids in infectious agents. Using Phi29 DNA polymerase and a two-step priming reaction we could reproducibly detect and characterize by DNA sequencing circular DNA from Borrelia burgdorferi B31 in DNA samples containing as little as 25 pg of Borrelia DNA amongst a vast excess of human DNA. This simple technology can ultimately be adapted as a sensitive method to detect specific DNA from both known and unknown pathogens in a wide variety of complex environments. 相似文献
20.
The state of cellular chromatin in response to DNA damage has been examined by monitoring the change in the linking number of circular episomes. COS cells transfected with an SV40-based vector were treated with camptothecin (CPT), a eukaryotic DNA topoisomerase I (TOP1) poison which induces TOP1-mediated DNA damage. Within minutes, a large increase in the linking number (over 10 linking number) of a small fraction (5-15%) of the episomal DNA was observed. A similar CPT-induced increase in plasmid DNA linking number was observed in Saccharomyces cerevisae expressing human DNA TOP1. In this case, the majority of the plasmid DNA can undergo rapid relaxation. The large increase in the plasmid linking number suggests major chromatin structural reorganization in response to TOP I-mediated DNA damage. 相似文献