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1.
A model of possible conformational transitions of supercoiled DNA in vitro in the absence of proteins under the conditions of increasing degree of compaction was developed. A 3993-bp pGEMEX supercoiled DNA immobilized on various substrates (freshly cleaved mica, standard amino mica, and modified amino mica with a hydrophobicity higher than that of standard amino mica) was visualized by atomic force microscopy in air. On the modified amino mica, which has an increased density of surface positive charges, single molecules with an extremely high degree of compaction were visualized in addition to plectonemic DNA molecules. As the degree of DNA supercoiling increased, the length of the first-order superhelical axis of molecules decreased from 570 to 370 nm, followed by the formation of second- and third-order superhelical axes about 280 and 140 nm long, respectively. The compaction of molecules ends with the formation of minitoroids about 50 nm in diameter and molecules of spherical shape. It was shown that the compaction of single supercoiled DNA molecules immobilized on amino mica to the level of minitoroids and spheroids is due to the shielding of mutually repulsing negatively charged phosphate groups of DNA by positively charged amino groups of the amino mica, which has a high charge density of its surface. 相似文献
2.
Limanskiĭ A 《Biofizika》2007,52(2):252-260
Supercoiled DNA pGEMEX with a length of 3993 nucleotides was immobilized on various substrates (freshly cleaved mica, standard amino mica, and modified amino mica) and visualized by atomic force microscopy. Plectonemically supercoiled DNA molecules and molecules with an extremely high level of compaction were visualized on modified amino mica, which was characterized by increased surface charge density. It was found that the length of the superhelix axis decreases two and four times to form superhelix axes of the second and third orders as the DNA compaction level increases because of the twice folding of DNA molecules. In this case, the length of the superhelix axis decreases from L approximately 470 nm to L approximately 140 nm (which corresponds to 10% contour length of a relaxed molecule on assumption of B-DNA) to form minitoroids and spheroids of approximately 50 nm diameter. Note that the previously reported experimentally measured length of the superhelix axis was equal to 35% contour length of the relaxed DNA molecule at the maximal density of the superhelix. Our data show that the significant decrease in the length of superhelix axis and the compaction of single supercoiled DNA molecules to the level of spheroids and minitoroids are caused by the screening of negatively charged DNA phosphate groups by positively charged amino groups of the modified amino mica because of its high surface charge density and increased hydrophobicity compared with standard amino mica. 相似文献
3.
Supercoiled 3993-bp pGEMEX DNA immobilized on four substrates (freshly cleaved mica, standard amino mica, and modified amino mica with an increased or decreased surface charge density in comparison to standard amino mica) has been visualized by atomic force microscopy in the air. Plectonomically supercoiled DNA molecules, as well as single molecules with an extremely high compaction level (i.e., with a significantly higher superhelix density compared to those previously observed experimentally or estimated theoretically), have been visualized on modified amino mica with an increased surface charge density. The distance between nucleotide pairs along the duplex axis has been determined by measuring the contour length of individual oversupercoiled DNA molecules. The estimated rise per base pair varies from 1.94 to 2.19 Å. These supercoiled DNA molecules, which are compressed like a spring and have a decreased rise per base pair compared to previously known DNA forms are considered to be a new form of DNA, S-DNA. A model of S-DNA has been constructed. Molecules of S-DNA may be an intermediate in the course of the compaction of single supercoiled DNA molecules into spheroids and minitoroids. The DNA oversupercoiling, followed by the compression of the supercoiled molecules, has been shown to be accounted for by a high surface charge density of amino mica on which DNA molecules are immobilized. 相似文献
4.
Supercoiled pGEMEX DNA with length of 3993 nucleotides was immobilized on four substrates (freshly cleaved mica, standard amino mica, modified amino mica with increased and decreased surface charge density compared with standard amino mica) and it was visualized by atomic force microscopy (AFM) in air. Plectonomically supercoiled DNA molecules as well as single molecules with extremely high level of compaction (i.e. molecules with significantly higher superhelix density values on comparison with previously experimentally measured and theoretically investigated ones) were visualized on modified amino mica which was characterized by increased surface charge density. Distance between base pairs along duplex axis was determined by measurements of contour length of single oversupercoiled DNA molecules. Determined rise per base pair was varied from 1.94 to 2.19 A. These compressed supercoiled DNA molecules like a spring with decreased rise/base pair on comparison with well-known DNA forms were called new DNA form--S-DNA. A model of S-DNA was built. Formation of the S-DNA molecules was suggested to be an intermediate stage on the compaction of the single supercoiled DNA molecules up to the spheroids and minitoroids. Oversupercoiling and further compression of the supercoiled DNA molecules was shown to cause by high surface charge density of amino mica on which DNA molecules were immobilized. 相似文献
5.
Lymans'kyĭ O 《T?Sitologii?a i genetika》2005,39(2):64-71
Supercoiled DNA pGEMEX with length of 3993 nucleotides was immobilized on the different substrates (freshly cleaved mica, standard aminomica and modified aminomica) and visualized by atomic force microscopy. Plectonomically supercoiled DNA molecules as well as molecules with extremely high level of compactization (i.e. molecules with considerably higher supercoiled density values in comparing with experimentally measured and theoretically investigated ones) were visualized on modified aminomica. At the further increasing of the compactization level an axis length of oversupercoiled molecules was decreased from approximately 390 nm to approximately 140 nm and formation of minitoroids of approximately 50 nm diameter and molecules in sphere conformation were observed. Model of possible conformational transitions of supercoiled DNA was proposed basing on the analysis of captured AFM images at the increasing of supercoiling density. 相似文献
6.
Lymans'kyĭ O 《T?Sitologii?a i genetika》2006,40(1):70-80
Functionalized by bovine serum albumin (BSA) probes for atomic force microscopy (AFM) which can be used for molecular recognition studies has been obtained. Modification and functionalization procedure of AFM probe includes three stages. First, amino probes were obtained by modification in vapors of amino silane derivative. Then surface amino groups of the amino probe interacted with homobifunctional amino reactive crosslinker. And finally, the probe with covalently attached crosslinker was functionalized by BSA molecules. Obtained AFM probes were characterized on the different stages of the modification by force measurements and the adhesion forces were determined. Process of modification was confirmed by visualization of BSA and supercoiled pGEMEX DNA molecules immobilized on the standard amino mica and amino mica modified by crosslinker. 相似文献
7.
A. P. Limanskii 《Biophysics》2006,51(2):186-195
The probes for atomic force microscopy (AFM) functionalized with bovine serum albumin (BSA) were obtained; they can be used for molecular recognition studies. The procedure of modification and functionalization of the AFM probe included three stages. First, amino probes were obtained by modification in vapors of an amino silane derivative. Then, a covalent bond was formed between the surface amino groups of the probe and a homobifunctional aminoreactive crosslinker. Finally, the probe with a covalently attached crosslinker was functionalized with BSA molecules. The AFM probes were characterized by force measurements at different stages of the modification; the adhesion force and the work of adhesion force were determined. The modification process was confirmed by visualization of BSA and supercoiled pGEMEX DNA molecules immobilized on the standard amino mica and on amino mica modified with a crosslinker. 相似文献
8.
Limanskiĭ AP 《Biofizika》2006,51(2):225-235
Probes for atomic force microscopy functionalized by bovine serum albumin were obtained, which may be used for molecular recognition studies. The procedure of the modification and functionalization of probes includes three stages. First, amino probes are obtained by modification in vapors of amino silane derivative. Then a homobifunctional amino reactive cross-linker is covalently linked to surface amino groups of the amino probe. And finally, the probe with the covalently attached cross-linker is functionalized by bovine serum albumin molecules. The probes obtained were characterized at different stages of the modification by atomic force microscopy: the adhesion force and the work of adhesion force were determined from histograms. The modification of probe surface was confirmed by visualization of bovine serum albumin and supercoiled pGEMEX DNA molecules immobilized on the amino mica and amino mica modified by cross-linker. 相似文献
9.
Immobilization of biomolecules on surfaces while keeping the maximum conformational flexibility of the molecules is one of the most important techniques for atomic force microscopy imaging. We have developed two methods of controlling adsorption of DNA molecules on mica surfaces. The first method is the use of a mica surface modified with diluted 3-aminopropyltriethoxysilane (APS). Here we named this a "diluted APS-treated mica (AP-mica)" technique. The second method is the use of a mica surface modified with mixed self-assembled monolayers of organosilanes. In both of the techniques, the number of DNA molecules immobilized on a mica surface was controlled. Further, a conformational change of circular DNA, from a supercoiled to a relaxed form was observed for the molecules immobilized on a diluted AP-mica surface, when 254-nm UV light was irradiated. This observation demonstrated that flexibility of circular DNA molecules was kept on a diluted AP-mica surface. 相似文献
10.
Atomic force microscopy imaging of DNA covalently immobilized on a functionalized mica substrate. 总被引:7,自引:0,他引:7
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L S Shlyakhtenko A A Gall J J Weimer D D Hawn Y L Lyubchenko 《Biophysical journal》1999,77(1):568-576
A procedure for covalent binding of DNA to a functionalized mica substrate is described. The approach is based on photochemical cross-linking of DNA to immobilized psoralen derivatives. A tetrafluorphenyl (TFP) ester of trimethyl psoralen (trioxalen) was synthesized, and the procedure to immobilize it onto a functionalized aminopropyl mica surface (AP-mica) was developed. DNA molecules were cross-linked to trioxalen moieties by UV irradiation of complexes. The steps of the sample preparation procedure were analyzed with x-ray photoelectron spectroscopy (XPS). Results from XPS show that an AP-mica surface can be formed by vapor phase deposition of silane and that this surface can be derivatized with trioxalen. The derivatized surface is capable of binding of DNA molecules such that, after UV cross-linking, they withstand a thorough rinsing with SDS. Observations with atomic force microscopy showed that derivatized surfaces remain smooth, so DNA molecules are easily visualized. Linear and circular DNA molecules were photochemically immobilized on the surface. The molecules are distributed over the surface uniformly, indicating rather even modification of AP-mica with trioxalen. Generally, the shapes of supercoiled molecules electrostatically immobilized on AP-mica and those photocross-linked on trioxalen-functionalized surfaces remain quite similar. This suggests that UV cross-linking does not induce formation of a noticeable number of single-stranded breaks in DNA molecules. 相似文献
11.
The flexibility of the chromatin structure, necessary for the processing of the genomic DNA, is controlled by a number of factors where flexibility and mobility of the nucleosomes is essential. Here, the influence of DNA supercoiling on the structure of single nucleosomes is investigated. Circular supercoiled plasmid DNA sub-saturated with histones was visualized by scanning force microscopy (SFM) in aqueous solution. SFM-imaging compared with topological analysis indicates instability of nucleosomes when the salt concentration is raised from 10 mM to 100 mM NaCl. Nucleosomes were observed after the deposition to the used scanning surface, i.e. mica coated with polylysine. On the images, the nucleosomes appear with a high probability in end-loops near the apices of the superhelices. In 100 mM NaCl but not in 10 mM NaCl, a significant number of complexes present the nucleosomes on superhelical crossings mainly located adjacent to an end-loop. The morphology of these structures and statistical analysis suggest that DNA loops were formed on the histone octamers, where the loop size distribution shows a pronounced peak at 50 nm. Recently, the formation and diffusion of loops on octamers has been discussed as a mechanism of translocations of nucleosomes along DNA. The presented data likely confirm the occurrence of loops, which may be stabilized by supercoiling. Analysis of the structure of regular nucleosomes not located on crossings indicates that reducing the salt concentration leads to more conformations, where DNA is partially unwrapped from the distal ends of the octamer. 相似文献
12.
Superhelix dimensions of a 1868 base pair plasmid determined by scanning force microscopy in air and in aqueous solution. 总被引:3,自引:3,他引:0
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We have used scanning force microscopy (SFM) to study the conformation of a 1868 base pair plasmid (p1868) in its open circular form and at a superhelical density of sigma= -0.034. The samples were deposited on a mica surface in the presence of MgCl2. DNA images were obtained both in air and in aqueous solutions, and the dimensions of the DNA superhelix were analysed. Evaluation of the whole plasmid yielded average superhelix dimensions of 27 +/- 9 nm (outer superhelix diameter D), 107 +/- 51 nm (superhelix pitch P), and 54 +/-8 degrees (superhelix pitch angle alpha). We also analysed compact superhelical regions within the plasmid separately, and determined values of D = 9.2 +/- 3.3 nm, P = 42 +/- 13 nm and alpha= 63 +/- 20 degrees for samples scanned in air or rehydrated in water. These results indicate relatively large conformation changes between superhelical and more open regions of the plasmid. In addition to the analysis of the DNA superhelix dimensions, we have followed the deposition process of open circular p1868 to mica in real time. These experiments show that it is possible to image DNA samples by SFM without prior drying, and that the surface bound DNA molecules retain some ability to change their position on the surface. 相似文献
13.
The Xenopus laevis mitochondrial protein mtDBP-C cooperatively folds the DNA in vitro. 总被引:2,自引:0,他引:2
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The binding of the Xenopus laevis mitochondrial protein mtDBP-C to DNA was studied by equilibrium density banding, agarose gel electrophoresis and electron microscopy. The results obtained show that the mtDBP-C binds cooperatively to DNA irrespective of whether the DNA is supercoiled, relaxed or linear and it induces the formation of superhelical turns locally leading to the formation of a highly folded structure. It appears that this protein could be involved in the compaction of DNA in the mitochondrial nucleoid. 相似文献
14.
Computer simulation of DNA supercoiling 总被引:8,自引:0,他引:8
K V Klenin A V Vologodskii V V Anshelevich A M Dykhne M D Frank-Kamenetskii 《Journal of molecular biology》1991,217(3):413-419
We treat supercoiled DNA within a wormlike model with excluded volume. A modified Monte Carlo approach has been used, which allowed computer statistical-mechanical simulations of moderately and highly supercoiled DNA molecules. Even highly supercoiled molecules do not have a regular shape, though with an increase in writhing the chains look more and more like branched interwound helixes. The averaged writhing (Wr) approximately 0.7 delta Lk. The superhelical free energy F is calculated as a function of the linking number. Lk. The calculations have shown that the generally accepted quadratic dependence of F on Lk is valid for a variety of conditions, though it is by no means universal. Significant deviations from the quadratic dependence are expected at high superhelical density under ionic conditions where the effective diameter of DNA is small. The results are compared with the available experimental data. 相似文献
15.
Unpaired structures in SCA10 (ATTCT)n.(AGAAT)n repeats 总被引:4,自引:0,他引:4
Potaman VN Bissler JJ Hashem VI Oussatcheva EA Lu L Shlyakhtenko LS Lyubchenko YL Matsuura T Ashizawa T Leffak M Benham CJ Sinden RR 《Journal of molecular biology》2003,326(4):1095-1111
A number of human hereditary diseases have been associated with the instability of DNA repeats in the genome. Recently, spinocerebellar ataxia type 10 has been associated with expansion of the pentanucleotide repeat (ATTCT)(n).(AGAAT)(n) from a normal range of ten to 22 to as many as 4500 copies. The structural properties of this repeat cloned in circular plasmids were studied by a variety of methods. Two-dimensional gel electrophoresis and atomic force microscopy detected local DNA unpairing in supercoiled plasmids. Chemical probing analysis indicated that, at moderate superhelical densities, the (ATTCT)(n).(AGAAT)(n) repeat forms an unpaired region, which further extends into adjacent A+T-rich flanking sequences at higher superhelical densities. The superhelical energy required to initiate duplex unpairing is essentially length-independent from eight to 46 repeats. In plasmids containing five repeats, minimal unpairing of (ATTCT)(5).(AGAAT)(5) occurred while 2D gel analysis and chemical probing indicate greater unpairing in A+T-rich sequences in other regions of the plasmid. The observed experimental results are consistent with a statistical mechanical, computational analysis of these supercoiled plasmids. For plasmids containing 29 repeats, which is just above the normal human size range, flanked by an A+T-rich sequence, atomic force microscopy detected the formation of a locally condensed structure at high superhelical densities. However, even at high superhelical densities, DNA strands within the presumably compact A+T-rich region were accessible to small chemicals and oligonucleotide hybridization. Thus, DNA strands in this "collapsed structure" remain unpaired and accessible for interaction with other molecules. The unpaired DNA structure functioned as an aberrant replication origin, in that it supported complete plasmid replication in a HeLa cell extract. A model is proposed in which unscheduled or aberrant DNA replication is a critical step in the expansion mutation. 相似文献
16.
The dependence of the crusiciform structure formation on superhelical density was studied by means of high resolution gel-electrophoresis. A short pAO3 DNA plasmid (1683 b. p.) which is a quarter of the ColE1 DNA plasmid and contains the main palindrome of ColE1 DNA was used. The excellent resolution of all topoisomers of pAO3 DNA in gel-electrophoresis made it possible to observe a sharp abruption in the pattern of pAO3 DNA topoisomers separation. The two-dimensional gel-electrophoresis data showed that observed abruption is caused by a sharp decrease of writhing in the molecules with superhelical density--sigma approximately equal to 0,05. An analysis of S1-nuclease digestion products of DNA with different superhelical density was accomplished and these data showed that a sharp structural transition in supercoiled DNA pAO3 is caused by formation of a cruciform structure in the main palindrome. 相似文献
17.
The potential of atomic force microscopy (AFM) for the investigation of peculiarities of microorganisms genome structure is demonstrated. AFM images of phage lambda DNA linear molecules and supercoiled mica in buffer solution was imaged in air. New experimental method of DNA stretching based on using amino-modified mica with a decreased surface density of active amino-groups is proposed. Stretched molecules of phage lambda DNA were imaged by AFM. 相似文献
18.
Elisa Caffarelli Carlo Franzini Luisa Leoni Maria Savino 《Cell biochemistry and biophysics》1984,6(1):23-31
Cruciform structures have been detected in pBR322 supercoiled DNA, both in its naked state and when complexed with histone
octamer, using S1 endonuclease cleavage and EcoRI restriction. An inspection of the DNA sequence shows that the S1-hypersensitive
sites are very near to AT-rich regions of pBR322 genome.
A nucleosome “phasing” in these regions, as found on AT-rich regions of SV40 DNA (15), has been shown by restriction enzymes analysis.
On the basis of these results it can be proposed that cruciform structures protrude on the nucleosome surface. This model
explains the reason why these structures, which need high superhelical density, can exist in supercoiled DNA partially relaxed
by nucleosome formation. 相似文献
19.
Mechanism of mitochondrial DNA replication in mouse L-cells: introduction of superhelical turns into newly replicated molecules 总被引:1,自引:0,他引:1
The first closed circular product of mouse L-cell mitochondrial DNA synthesis is a zero superhelix density molecule. Both of the asynchronously synthesized mitochondrial DNA daughter molecules pass through the zero superhelix density state. These molecules have a mean lifetime of approximately one hour before conversion to supercoiled molecules containing approximately 100 superhelical turns. A low frequency of intermediates in the conversion of these two closed circular forms is demonstrable by agarose gel electrophoresis. The degree of sensitivity to alkali has been used to demonstrate that newly replicated mitochondrial DNA has the same content of ribonucleotides as mass-labeled mitochondrial DNA. 相似文献
20.
M Adrian B ten Heggeler-Bordier W Wahli A Z Stasiak A Stasiak J Dubochet 《The EMBO journal》1990,9(13):4551-4554
The shape of supercoiled DNA molecules in solution is directly visualized by cryo-electron microscopy of vitrified samples. We observe that: (i) supercoiled DNA molecules in solution adopt an interwound rather than a toroidal form, (ii) the diameter of the interwound superhelix changes from about 12 nm to 4 nm upon addition of magnesium salt to the solution and (iii) the partition of the linking deficit between twist and writhe can be quantitatively determined for individual molecules. 相似文献