Motivation: The cell nucleus is a highly organized cellular organelle that contains the genetic material. The study of nuclear architecture has become an important field of cellular biology. Extracting quantitative data from 3D fluorescence imaging helps understand the functions of different nuclear compartments. However, such approaches are limited by the requirement for processing and analyzing large sets of images.Results: Here, we describe Tools for Analysis of Nuclear Genome Organization (TANGO), an image analysis tool dedicated to the study of nuclear architecture. TANGO is a coherent framework allowing biologists to perform the complete analysis process of 3D fluorescence images by combining two environments: ImageJ (http://imagej.nih.gov/ij/) for image processing and quantitative analysis and R (http://cran.r-project.org) for statistical processing of measurement results. It includes an intuitive user interface providing the means to precisely build a segmentation procedure and set-up analyses, without possessing programming skills. TANGO is a versatile tool able to process large sets of images, allowing quantitative study of nuclear organization.Availability: TANGO is composed of two programs: (i) an ImageJ plug-in and (ii) a package (rtango) for R. They are both free and open source, available (http://biophysique.mnhn.fr/tango) for Linux, Microsoft Windows and Macintosh OSX. Distribution is under the GPL v.2 licence.Contact: thomas.boudier@snv.jussieu.frSupplementary information: Supplementary data are available at Bioinformatics online.

The restriction endonuclease EcoRV can rapidly locate a short recognition site within long non-cognate DNA using ‘facilitated diffusion’. This process has long been attributed to a sliding mechanism, in which the enzyme first binds to the DNA via nonspecific interaction and then moves along the DNA by 1D diffusion. Recent studies, however, provided evidence that 3D translocations (hopping/jumping) also help EcoRV to locate its target site. Here we report the first direct observation of sliding and jumping of individual EcoRV molecules along nonspecific DNA. Using fluorescence microscopy, we could distinguish between a slow 1D diffusion of the enzyme and a fast translocation mechanism that was demonstrated to stem from 3D jumps. Salt effects on both sliding and jumping were investigated, and we developed numerical simulations to account for both the jump frequency and the jump length distribution. We deduced from our study the 1D diffusion coefficient of EcoRV, and we estimated the number of jumps occurring during an interaction event with nonspecific DNA. Our results substantiate that sliding alternates with hopping/jumping during the facilitated diffusion of EcoRV and, furthermore, set up a framework for the investigation of target site location by other DNA-binding proteins.

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DNA triple helices offer new perspectives toward oligonucleotide-directed gene regulation. However, the poor stability of some of these structures might limit their use under physiological conditions. Specific ligands can intercalate into DNA triple helices and stabilize them.

A major challenge in the use of oligonucleotides in an anti-gene strategy is to stabilize triple helix formation under physiological conditions. A benzo[e]pyridoindole derivative was shown earlier to stabilize triple-helical better than double-helical complexes (Mergny, J. L. et al. Science 1992, 256, 1681-1684. New derivatives of the benzopyridoindole family were synthesized, and their ability to stabilize triple helices was investigated by thermal denaturation experiments using W absorption spectroscopy. The stabilizing effects of all the available derivatives were compared and allowed us to infer some general rules regarding the role of the geometry of the molecule and of its various substituents. The melting temperature (Tm) of the triplex-to-duplex transition is increased from 18 to 49 O C (ATmax = +3 1 "C) upon binding of 3-methoxy-10-methyl-7-[3-(N-methyl-N-3-aminopropyl)propyl]amino-1 lH-benzo[g]pyrido[4,3-b]indole (BgPI), in a 10 mM sodium cacodylate buffer (pH 6.2) containing 0.1 M NaC1. Sequence-specific effects were also investigated. Benzo[e]-and benzo[g]pyrido[4,3-b]indole derivatives exhibiteddifferent properties regarding the role of the alkylamine side chain attached to the pyridine ring. Effects of these compounds on the melting of duplex DNA were also sensitive to changes in the chemical nature of the alkylamine side chain. Results are discussed in terms of respective affinities for triplex and duplex structures. A model is proposed to explain the different roles played by the alkylamine side chain for both types of molecules. For the benzo[e]pyridoindole derivatives, the chain is suggested to lie in the major groove of the triple helix, whereas for the benzo[g]pyridoindole derivatives, it lies in the minor groove. These results provide an experimental and theoretical basis for understanding intercalation of dyes in triple helices and should help to conceive more specific triple helix ligands and to design oligonucleotide-intercalator conjugates for stable triple helix formation.

Fluorescence microscopy provides a powerful method to directly observe single enzymes moving along a DNA held in an extended conformation. In this work, we present results from single EcoRV enzymes labeled with quantum dots which interact with DNA manipulated by double optical tweezers. The application of quantum dots facilitated accurate enzyme tracking without photobleaching whereas the tweezers allowed us to precisely control the DNA extension. The labeling did not affect the biochemical activity of EcoRV checked by directly observing DNA digestion on the single molecule level. We used this system to demonstrate that during sliding, the enzyme stays in close contact with the DNA. Additionally, slight overstretching of the DNA resulted in a significant decrease of the 1D diffusion constant, which suggests that the deformation changes the energy landscape of the sliding interaction. Together with the simplicity of the setup, these results demonstrate that the combination of optical tweezers with fluorescence tracking is a powerful tool for the study of enzyme translocation along DNA.

BackgroundAlpha satellite is the major repeated DNA element of primate centromeres. Evolution of these tandemly repeated sequences has led to the existence of numerous families of monomers exhibiting specific organizational patterns. The limited amount of information available in non-human primates is a restriction to the understanding of the evolutionary dynamics of alpha satellite DNA.ResultsWe carried out the targeted high-throughput sequencing of alpha satellite monomers and dimers from the Cercopithecus solatus genome, an Old World monkey from the Cercopithecini tribe. Computational approaches were used to infer the existence of sequence families and to study how these families are organized with respect to each other. While previous studies had suggested that alpha satellites in Old World monkeys were poorly diversified, our analysis provides evidence for the existence of at least four distinct families of sequences within the studied species and of higher order organizational patterns. Fluorescence in situ hybridization using oligonucleotide probes that are able to target each family in a specific way showed that the different families had distinct distributions on chromosomes and were not homogeneously distributed between chromosomes.ConclusionsOur new approach provides an unprecedented and comprehensive view of the diversity and organization of alpha satellites in a species outside the hominoid group. We consider these data with respect to previously known alpha satellite families and to potential mechanisms for satellite DNA evolution. Applying this approach to other species will open new perspectives regarding the integration of satellite DNA into comparative genomic and cytogenetic studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3246-5) contains supplementary material, which is available to authorized users.

An oligonucleotide was circularized around double-stranded DNA thanks to triple helix formation. Short oligonucleotides are known to be able to form DNA triple helices by binding into the DNA major groove at an oligopurine⅐oligopyrimidine sequence. After sequence-specific recognition of a double-stranded DNA target through triple helix formation, the ends of the triplex-forming oligonucleotide were joined through the action of T4 DNA ligase, thus creating a circular DNA molecule catenated to the plasmid containing the target sequence. The labeling of the doublestranded DNA sequence has been carried out without any chemical or enzymatic modification of this sequence. These ''padlock'' oligonucleotides provide a tool to attach a noncovalent tag in an irreversible way to supercoiled plasmid or other double-stranded DNAs. Such a complex may find applications in the development of new techniques for duplex DNA detection or plasmid delivery methods for gene therapy.