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J Langowski

Publications and source records attributed to J Langowski.

At least 37 records · Page 2Linked to original sources
PubMed1998

Chromatin structure and chromosome aberrations: modeling of damage induced by isotropic and localized irradiation.

Various models for the nuclear architecture in interphase cell nuclei have been presented, proposing a territorial or a non-territorial organization of chromosomes. To better understand the correlation between nuclear architecture and the formation of chromosomal aberrations, we applied computer simulations to model the extent of radiation induced chromosome damage under certain geometrical constraints. For this purpose, chromosomes were described by different models, which approximate the chromatin fiber by a polymer chain, folded in different ways. Corresponding to the different condensation levels, a territorial or a non-territorial organization of chromosomes was obtained. To determine the relative frequencies of radiation induced damage, the effects of isotropic ionizing radiation and of a focused laser UV-beam were studied. For isotropic ionizing radiation, the calculated translocation frequencies showed no differences between territorial and non-territorial models except for one special case. For localized irradiation, the results of both organizations were clearly different, with respect to the total number of damaged chromosomes per cell. The predictions agreed well with the experimental data available.

Animals↗
PubMed1998

DNA curvature in solution measured by fluorescence resonance energy transfer.

The sequence-induced curvature of DNA fragments free in solution was characterized by measurements of the end-to-end distance using fluorescence resonance energy transfer (FRET). The 31 bp oligonucleotides were labeled at their 5' ends with fluorescein as the donor and rhodamine X as the acceptor. We compared a curved oligonucleotide with three phased A6 blocks and a control containing (AT)3 instead of the A6 blocks. The increased efficiency of energy transfer of the A6-containing DNA indicates the existence of a permanent sequence-induced curvature, the magnitude of which is in good agreement with estimates from theoretical curvature predictions. Energy transfer efficiency and correspondingly curvature increases with NaCl concentration.

Circular Dichroism↗
PubMed1998

DNA binding and oligomerization of NtrC studied by fluorescence anisotropy and fluorescence correlation spectroscopy.

Fluorescence anisotropy and fluorescence correlation spectroscopy measurements of rhodamine-labeled DNA oligonucleotide duplexes have been used to determine equilibrium binding constants for DNA binding of the prokaryotic transcription activator protein NtrC. Measurements were made with wild-type NtrC from Escherichia coli and the constitutively active mutant NtrCS160Ffrom Salmonella using DNA duplexes with one or two binding sites. The following results were obtained: (i) the dissociation constant K d for binding of one NtrC dimer to a single binding site was the same for the wild-type and mutant proteins within the error of measurement. (ii) The value of K d decreased from 1.4 +/- 0.7 x 10(-11) M at 15 mM K acetate to 5.8 +/- 2.6 x 10(-9) M at 600 mM K acetate. From the salt dependence of the dissociation constant we calculated that two ion pairs form upon binding of one dimeric protein to the DNA. (iii) Binding of two NtrC dimers to the DNA duplex with two binding sites occured with essentially no cooperativity. Titration curves of NtrCS160Fbinding to the same duplex demonstrated that more than two protein dimers of the mutant protein could bind to the DNA.

Amino Acid Sequence↗
PubMed1998

Superhelix organization by DNA curvature as measured through site-specific labeling.

For determining the position of a defined site in a superhelical DNA we have developed a method for introducing a covalent biotin label at a specific sequence while preserving the superhelicity. This is done by first introducing a specific nick, labeling the DNA by limited nick translation and sealing the nick with ligase. The superhelicity is controlled by including ethidium in the ligation reaction. Using scanning force of microscopy on DNAs labeled by this method, we have then compared the position of streptavidin markers at a specific site relative to the end loop of the superhelix. We found that in DNAs with permanently curved inserts the label is located preferentially at a defined distance from the end loop, while in controls without curved inserts the label position was random. This indicates that curves are located in or near the end loops in a superhelix.

Amino Acid Substitution↗
PubMed1998

Looping dynamics of linear DNA molecules and the effect of DNA curvature: a study by Brownian dynamics simulation.

A Brownian dynamics (BD) model described in the accompanying paper (Klenin, K., H. Merlitz, and J. Langowski. 1998. A Brownian dynamics program for the simulation of linear and circular DNA, and other wormlike chain polyelectrolytes. Biophys. J. 74:000-000) has been used for computing the end-to-end distance distribution function, the cyclization probability, and the cyclization kinetics of linear DNA fragments between 120 and 470 basepairs with optional insertion of DNA bends. Protein-mediated DNA loop formation was modeled by varying the reaction distance for cyclization between 0 and 10 nm. The low cyclization probability of DNA fragments shorter than the Kuhn length (300 bp) is enhanced by several orders of magnitude when the cyclization is mediated by a protein bridge of 10 nm diameter, and/or when the DNA is bent. From the BD trajectories, end-to-end collision frequencies were computed. Typical rates for loop formation of linear DNAs are 1.3 x 10(3) s(-1) (235 bp) and 4.8 x 10(2) s(-1) (470 bp), while the insertion of a 120 degree bend in the center increases this rate to 3.0 x 10(4) s(-1) (235 bp) and 5.5 x 10(3) s(-1) (470 bp), respectively. The duration of each encounter is between 0.05 and 0.5 micros for these DNAs. The results are discussed in the context of the interaction of transcription activator proteins.

Biophysics↗
PubMed1998

A Brownian dynamics program for the simulation of linear and circular DNA and other wormlike chain polyelectrolytes.

For the interpretation of solution structural and dynamic data of linear and circular DNA molecules in the kb range, and for the prediction of the effect of local structural changes on the global conformation of such DNAs, we have developed an efficient and easy way to set up a program based on a second-order explicit Brownian dynamics algorithm. The DNA is modeled by a chain of rigid segments interacting through harmonic spring potentials for bending, torsion, and stretching. The electrostatics are handled using precalculated energy tables for the interactions between DNA segments as a function of relative orientation and distance. Hydrodynamic interactions are treated using the Rotne-Prager tensor. While maintaining acceptable precision, the simulation can be accelerated by recalculating this tensor only once in a certain number of steps.

Biophysics↗
PubMed1998

Salt-dependent DNA superhelix diameter studied by small angle neutron scattering measurements and Monte Carlo simulations.

Using small angle neutron scattering we have measured the static form factor of two different superhelical DNAs, p1868 (1868 bp) and pUC18 (2686 bp), in dilute aqueous solution at salt concentrations between 0 and 1.5 M Na+ in 10 mM Tris at 0% and 100% D2O. For both DNA molecules, the theoretical static form factor was also calculated from an ensemble of Monte Carlo configurations generated by a previously described model. Simulated and measured form factors of both DNAs showed the same behavior between 10 and 100 mM salt concentration: An undulation in the scattering curve at a momentum transfer q = 0.5 nm-1 present at lower concentration disappears above 100 mM. The position of the undulation corresponds to a distance of approximately 10-20 nm. This indicated a change in the DNA superhelix diameter, as the undulation is not present in the scattering curve of the relaxed DNA. From the measured scattering curves of superhelical DNA we estimated the superhelix diameter as a function of Na+ concentration by a quantitative comparison with the scattering curve of relaxed DNA. The ratio of the scattering curves of superhelical and relaxed DNA is very similar to the form factor of a pair of point scatterers. We concluded that the distance of this pair corresponds to the interstrand separation in the superhelix. The computed superhelix diameter of 16.0 +/- 0.9 nm at 10 mM decreased to 9.0 +/- 0.7 nm at 100 mM salt concentration. Measured and simulated scattering curves agreed almost quantitatively, therefore we also calculated the superhelix diameter from the simulated conformations. It decreased from 18.0 +/- 1.5 nm at 10 mM to 9.4 +/- 1.5 nm at 100 mM salt concentration. This value did not significantly change to lower values at higher Na+ concentration, in agreement with results obtained by electron microscopy, scanning force microscopy imaging in aqueous solution, and recent MC simulations, but in contrast to the observation of a lateral collapse of the DNA superhelix as indicated by cryo-electron microscopy studies.

Biophysical Phenomena↗
PubMed1997

Superhelix dimensions of a 1868 base pair plasmid determined by scanning force microscopy in air and in aqueous solution.

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.

Air↗
PubMed1997

Structural analysis of mouse rDNA: coincidence between nuclease hypersensitive sites, DNA curvature and regulatory elements in the intergenic spacer.

We have analyzed the chromatin structure of mouse ribosomal RNA genes (rDNA) by partial digestion of genomic DNA with micrococcal nuclease (MNase), DNase I and identified hypersensitive sites by indirect end-labeling. This analysis has revealed defined regions of nuclease hypersensitivity in the intergenic spacer which in turn coincide with regulatory elements. Hypersensitive sites map to the transcription initiation site, the enhancer repeats, the spacer promoter and two sequence elements which coincide with amplification-promoting sequences. Analysis of the DNA curvature by computer modeling uncovered a striking correlation between sequence-directed structural features of regulatory regions and the position of nuclease hypersensitive sites. Moreover, we demonstrate that nucleosomes are specifically positioned upstream and downstream of the transcription start site. In vitro studies using chromatin assembled in the presence of Drosophila embryo extracts show that binding of the transcription termination factor TTF-I to the upstream terminator mediates this specific nucleosome positioning at the rDNA promoter in an ATP- dependent fashion.

3T3 Cells↗
PubMed1997

Salt effects on the structure and internal dynamics of superhelical DNAs studied by light scattering and Brownian dynamics.

Using laser light scattering, we have measured the static and dynamic structure factor of two different superhelical DNAs, p1868 (1868 bp) and simian virus 40 (SV40) (5243 bp), in dilute aqueous solution at salt concentrations between 1 mM and 3 M NaCl. For both DNA molecules, Brownian dynamics (BD) simulations were also performed, using a previously described model. A Fourier mode decomposition procedure was used to compute theoretical light scattering autocorrelation functions (ACFs) from the BD trajectories. Both measured and computed autocorrelation functions were then subjected to the same multiexponential decomposition procedure. Simulated and measured relaxation times as a function of scattering angle were in very good agreement. Similarly, computed and measured static structure factors and radii of gyration agreed within experimental error. One main result of this study is that the amplitudes of the fast-relaxing component in the ACF show a peak at 1 M salt concentration. This nonmonotonic behavior might be caused by an initial increase in the amplitudes of internal motions due to diminishing long-range electrostatic repulsions, followed by a decrease at higher salt concentration due to a compaction of the structure.

Biophysical Phenomena↗
PubMed1997

Curvature and sequence analysis of eukaryotic promoters.

We have calculated the curvature of 504 eukaryotic promoters predicted by the bent A-tract model of Bolshoy et al. (Proc. Natl. Acad. Sci. USA, 88(6), pp. 2312-16) and the bent non-A-tract models of Calladine et al. (J. Mol. Biol., 201, pp. 127-37) and Satchwell et al. (J. Mol. Biol., 191, pp. 659-75) and found in each case a correlation between TBP binding sites and DNA curvature. Characterizing the TBP binding sites revealed that in addition to the classical TATA box (TATAAA) five more elements occur significantly often in the promoters, nearly all of them being one point mutations of the classical TATA box element. Separate curvature calculations for promoters with canonical and non-canonical TATA boxes have shown that in both cases the strong curvature of the helix axes in the domain of the binding sites is maintained (classical TBP binding sites: + 64-135%, non-classical TBP binding sites: + 27-49%). These results support the proposition that beside DNA flexibility and DNA-protein interactions intrinsic curvature of DNA is one further important criterion for the recognition of different DNA elements by TBP.

Animals↗
PubMed1997

A Brownian dynamics model for the chromatin fiber.

MOTIVATION: We describe a Brownian dynamics model for the folding of the chromatin fiber based on the model of Woodcock et al. (Proc Natl Acad Sci USA, 90, 9021-9025, 1993). The model takes into account the elastic properties of the DNA as well as the electrostatic interaction and nucleosomal excluded-volume interaction. The solvent is described as a viscous medium, the electrostatic interactions by a screened Coulomb potential. RESULTS: The hydrodynamic properties and their dependence on the solvent's ionic strength are accurately reproduced by the model for nucleosome di- and tetramers. Ionic strength-dependent changes in mobility can be attributed to partial screening of the electrostatic repulsion between different segments of linker DNA. Formation of fiber-like structures occurs on time scales of several hundred microseconds for a linear configuration of 25 nucleosomes. The model was implemented by creating user-defined data types. Use of this so-called object-oriented paradigm allowed for a high degree of component reuse in simulation, analysis and visualization contexts. AVAILABILITY: The described software is available on request from the authors. Additional information can be found on the WWW at http:/(/)www.dkfz-heidelberg.de/Macromol/ehrlich /chromatin.htm/.

Chromatin↗
PubMed1996

Rod-like shape of vesicular stomatitis virus matrix protein.

The shape of purified matrix protein (M) of vesicular stomatitis virus was determined using biophysical techniques like analytical centrifugation, dynamic light scattering, and small-angle neutron scattering. The data obtained are consistent with a rod-like model for M protein with a length of about 100 +/- 10 A and a radius of 9 +/- 1 A. These dimensions are in agreement with the substructure of M protein aggregates and with the fine morphology of the axial channel material found inside the viral nucleocapsid coil. This morphological information was combined with CD measurements and secondary structure predictions on four vesiculovirus M proteins leading to a proposal for the structure of M protein.

Amino Acid Sequence↗
PubMed1996

Sequence-specific labeling of superhelical DNA by triple helix formation and psoralen crosslinking.

Site-specific labeling of covalently closed circular DNA was achieved by using triple helix-forming oligonucleotides 10, 11 and 27 nt in length. The sequences consisted exclusively of pyrimidines (C and T) with a reactive psoralen at the 5'-end and a biotin at the 3'-end. The probes were directed to different target sites on the plasmids pUC18 (2686 bp), pUC18/4A (2799 bp) and pUC1 8/4A-H 1 (2530 bp). After triple helix formation at acid pH the oligonucleotides were photocrosslinked to the target DNAs via the psoralen moiety, endowing the covalent adduct with unconditional stability, e.g. under conditions unfavorable for preservation of the triplex, such as neutral pH. Complex formation was monitored after polyacrylamide gel electrophoresis by streptavidin-alkaline phosphatase (SAP)-induced chemiluminescence. The yield of triple helix increased with the molar ratio of oligonucleotide to target and the length of the probe sequence (27mer > 11mer). The covalent adduct DNA were visualized by scanning force microscopy (SFM) using avidin or streptavidin as protein tags for the biotin group on the oligonucleotide probes. We discuss the versatility of triple helix DNA complexes for studying the conformation of superhelical DNA.

Base Sequence↗
PubMed1996

Brownian dynamics simulations of supercoiled DNA with bent sequences.

The recently presented Brownian dynamics model for superhelical DNA is extended to include local curvature of the DNA helix axis. Here we analyze the effect of a permanent bend on the structure and dynamics of an 1870-bp superhelix with delta Lk = -10. Furthermore, we define quantitative expressions for computing structural parameters such as loop positions, superhelix diameter, and plectonemic content for trajectories of superhelical DNA, and assess the convergence toward global equilibrium. The structural fluctuations in an interwound superhelix, as reflected in the change in end loop positions, seem to occur by destruction/creation of loops rather than by a sliding motion of the DNA around its contour. Their time scale is on the order of 30-100 microseconds. A permanent bend changes the structure and the internal motions of the DNA drastically. The position of the end loop is fixed at the permanent bend, and the local motions of the chain are enhanced near the loops. A displacement of the bend from the end loop to a position inside the plectonemic part of the superhelix results in the formation of a new loop and the disappearance of the old one; we estimate the time involved in this process to be about 0.5 ms.

Biophysical Phenomena↗
PubMed1996

Nuclear architecture and the induction of chromosomal aberrations.

Progress in fluorescence in situ hybridization, three dimensional microscopy and image analysis has provided the means to study the three-dimensional structure and distribution of chromosome territories within the cell nucleus. In this contribution, we summarize the present state of knowledge of the territorial organization of interphase chromosomes and their topological relationships with other macromolecular domains in the human cell nucleus, and present data from computer simulations of chromosome territory distributions. On this basis, we discuss models of chromosome territory and nuclear architecture and topological consequences for the formation of chromosome exchanges.

Animals↗