Javier Arsuaga

Genomic transcriptional response to loss of chromosomal supercoiling in Escherichia coli.

BackgroundThe chromosome of Escherichia coli is maintained in a negatively supercoiled state, and supercoiling levels are affected by growth phase and a variety of environmental stimuli. In turn, supercoiling influences local DNA structure and can affect gene expression. We used microarrays representing nearly the entire genome of Escherichia coli MG1655 to examine the dynamics of chromosome structure.ResultsWe measured the transcriptional response to a loss of supercoiling caused either by genetic impairment of a topoisomerase or addition of specific topoisomerase inhibitors during log-phase growth and identified genes whose changes are statistically significant. Transcription of 7% of the genome (306 genes) was rapidly and reproducibly affected by changes in the level of supercoiling; the expression of 106 genes increased upon chromosome relaxation and the expression of 200 decreased. These changes are most likely to be direct effects, as the kinetics of their induction or repression closely follow the kinetics of DNA relaxation in the cells. Unexpectedly, the genes induced by relaxation have a significantly enriched AT content in both upstream and coding regions.ConclusionsThe 306 supercoiling-sensitive genes are functionally diverse and widely dispersed throughout the chromosome. We propose that supercoiling acts as a second messenger that transmits information about the environment to many regulatory networks in the cell.

Knotting probability of DNA molecules confined in restricted volumes: DNA knotting in phage capsids

When linear double-stranded DNA is packed inside bacteriophage capsids, it becomes highly compacted. However, the phage is believed to be fully effective only if the DNA is not entangled. Nevertheless, when DNA is extracted from a tailless mutant of the P4 phage, DNA is found to be cyclic and knotted (probability of 0.95). The knot spectrum is very complex, and most of the knots have a large number of crossings. We quantified the frequency and crossing numbers of these knots and concluded that, for the P4 tailless mutant, at least half the knotted molecules are formed while the DNA is still inside the viral capsid rather than during extraction. To analyze the origin of the knots formed inside the capsid, we compared our experimental results to Monte Carlo simulations of random knotting of equilateral polygons in confined volumes. These simulations showed that confinement of closed chains to tightly restricted volumes results in high knotting probabilities and the formation of knots with large crossing numbers. We conclude that the formation of the knots inside the viral capsid is driven mainly by the effects of confinement.

Investigation of viral DNA packaging using molecular mechanics models

A simple molecular mechanics model has been used to investigate optimal spool-like packing conformations of double-stranded DNA molecules in viral capsids with icosahedral symmetry. The model represents an elastic segmented chain by using one pseudoatom for each ten basepairs (roughly one turn of the DNA double helix). Force constants for the various terms in the energy function were chosen to approximate known physical properties, and a radial restraint was used to confine the DNA into a sphere with a volume corresponding to that of a typical bacteriophage capsid. When the DNA fills 90% of the spherical volume, optimal packaging is obtained for coaxially spooled models, but this result does not hold when the void volume is larger. When only 60% of the spherical volume is filled with DNA, the lowest energy structure has two layers, with a coiled core packed at an angle to an outer coaxially spooled shell. This relieves bending strain associated with tight curvature near the poles in a model with 100% coaxial spooling. Interestingly, the supercoiling density of these models is very similar to typical values observed in plasmids in bacterial cells. Potential applications of the methodology are also discussed.

Chromosomes are predominantly located randomly with respect to each other in interphase human cells

To test quantitatively whether there are systematic chromosome–chromosome associations within human interphase nuclei, interchanges between all possible heterologous pairs of chromosomes were measured with 24-color whole-chromosome painting (multiplex FISH), after damage to interphase lymphocytes by sparsely ionizing radiation in vitro. An excess of interchanges for a specific chromosome pair would indicate spatial proximity between the chromosomes comprising that pair. The experimental design was such that quite small deviations from randomness (extra pairwise interchanges within a group of chromosomes) would be detectable. The only statistically significant chromosome cluster was a group of five chromosomes previously observed to be preferentially located near the center of the nucleus. However, quantitatively, the overall deviation from randomness within the whole genome was small. Thus, whereas some chromosome–chromosome associations are clearly present, at the whole-chromosomal level, the predominant overall pattern appears to be spatially random.

Novel display of knotted DNA molecules by two-dimensional gel electrophoresis.

We describe a two-dimensional agarose gel electrophoresis procedure that improves the resolution of knotted DNA molecules. The first gel dimension is run at low voltage, and DNA knots migrate according to their compactness. The second gel dimension is run at high voltage, and DNA knots migrate according to other physical parameters such as shape and flexibility. In comparison with one-dimensional gel electrophoresis, this procedure segregates the knotted DNA molecules from other unknotted forms of DNA, and partially resolves populations of knots that have the same number of crossings. The two-dimensional display may allow quantitative and qualitative characterization of different types of DNA knots simply by gel velocity.

Chromosome spatial clustering inferred from radiogenic aberrations

Purpose: Analysing chromosome aberrations induced by low linear energy transfer (LET) radiation in order to characterize systematic spatial clustering among the 22 human autosomes in human lymphocytes and to compare their relative participation in interchanges. Materials and methods: A multicolour fluorescence in situ hybridization (mFISH) data set, specifying colour junctions in metaphases of human peripheral blood lymphocytes 72 h after in vitro exposure to low LET radiation, was analysed separately and in combination with previously published results. Monte Carlo computer simulations and mathematical modelling guided data analysis. Results and conclusions: Statistical tests on aberration data confirmed two clusters of chromosomes, {1, 16, 17, 19, 22} and {13, 14, 15, 21, 22}, as having their members being on average closer to each other than randomness would predict. The first set has been reported previously to be near the centre of the interphase nucleus and to be formed mainly by gene‐rich chromosomes, while the second set comprises the nucleolus chromosomes. The results suggest a possible interplay between chromosome positioning and transcription. A number of other clusters suggested in the literature were not confirmed and considerable randomness of chromosome–chromosome juxtapositions was present. In addition, and consistent with previous results, it was found that chromosome participation in interchanges is approximately proportional to the two‐thirds power of the DNA content.

Bounds for the minimum step number of knots in the simple cubic lattice

Knots are found in DNA as well as in proteins, and they have been shown to be good tools for structural analysis of these molecules. An important parameter to consider in the artificial construction of these molecules is the minimum number of monomers needed to make a knot. Here we address this problem by characterizing, both analytically and numerically, the minimum length (also called minimum step number) needed to form a particular knot in the simple cubic lattice. Our analytical work is based on improvement of a method introduced by Diao to enumerate conformations of a given knot type for a fixed length. This method allows us to extend the previously known result on the minimum step number of the trefoil knot 31 (which is 24) to the knots 41 and 51 and show that the minimum step numbers for the 41 and 51 knots are 30 and 34, respectively. Using an independent method based on the BFACF algorithm, we provide a complete list of numerical estimates (upper bounds) of the minimum step numbers for prime knots up to ten crossings, which are improvements over current published numerical results. We enumerate all minimum lattice knots of a given type and partition them into classes defined by BFACF type 0 moves.

Linking of uniform random polygons in confined spaces

In this paper, we study the topological entanglement of uniform random polygons in a confined space. We derive the formula for the mean squared linking number of such polygons. For a fixed simple closed curve in the confined space, we rigorously show that the linking probability between this curve and a uniform random polygon of n vertices is at least . Our numerical study also indicates that the linking probability between two uniform random polygons (in a confined space), of m and n vertices respectively, is bounded below by . In particular, the linking probability between two uniform random polygons, both of n vertices, is bounded below by .

Reproducibility of 3D chromatin configuration reconstructions

It is widely recognized that the three-dimensional (3D) architecture of eukaryotic chromatin plays an important role in processes such as gene regulation and cancer-driving gene fusions. Observing or inferring this 3D structure at even modest resolutions had been problematic, since genomes are highly condensed and traditional assays are coarse. However, recently devised high-throughput molecular techniques have changed this situation. Notably, the development of a suite of chromatin conformation capture (CCC) assays has enabled elicitation of contacts-spatially close chromosomal loci-which have provided insights into chromatin architecture. Most analysis of CCC data has focused on the contact level, with less effort directed toward obtaining 3D reconstructions and evaluating the accuracy and reproducibility thereof. While questions of accuracy must be addressed experimentally, questions of reproducibility can be addressed statistically-the purpose of this paper. We use a constrained optimization technique to reconstruct chromatin configurations for a number of closely related yeast datasets and assess reproducibility using four metrics that measure the distance between 3D configurations. The first of these, Procrustes fitting, measures configuration closeness after applying reflection, rotation, translation, and scaling-based alignment of the structures. The others base comparisons on the within-configuration inter-point distance matrix. Inferential results for these metrics rely on suitable permutation approaches. Results indicate that distance matrix-based approaches are preferable to Procrustes analysis, not because of the metrics per se but rather on account of the ability to customize permutation schemes to handle within-chromosome contiguity. It has recently been emphasized that the use of constrained optimization approaches to 3D architecture reconstruction are prone to being trapped in local minima. Our methods of reproducibility assessment provide a means for comparing 3D reconstruction solutions so that we can discern between local and global optima by contrasting solutions under perturbed inputs.

Computer analysis of mFISH chromosome aberration data uncovers an excess of very complicated metaphases.

Purpose : To analyse spectra of chromosome aberrations induced in vitro by low LET radiation, in order to characterize radiation damage mechanisms quantitatively. Methods : Multiplex fluorescence in situ hybridization (mFISH) allows the simultaneous identification of each homologous chromosome pair by its own colour. mFISH data, specifying number distributions for colour junctions in metaphases of human peripheral blood lymphocytes 72 hours after exposure in vitro to a 3 Gy γ-ray dose, were combined with similar, previously published results. Monte Carlo computer implementations of radiobiological models for chromosome aberration production guided quantitative analyses, which took into account distribution of cells among different metaphases and lethal effects or preferential elimination of some aberrations at cell division. Results and Conclusions : Standard models of DNA damage induction/repair/misrepair explain the main trends of the data as regards the fraction of metaphases having a particular number of colours involved in colour junctions. However, all standard models systematically under-predict the observed fraction of metaphases where a large number of different chromosomes participate in aberrations. An early appearance of chromosomal instability could explain most of the discrepancies.