Sabrina Pisano

The N-terminal domains of TRF1 and TRF2 regulate their ability to condense telomeric DNA

TRF1 and TRF2 are key proteins in human telomeres, which, despite their similarities, have different behaviors upon DNA binding. Previous work has shown that unlike TRF1, TRF2 condenses telomeric, thus creating consequential negative torsion on the adjacent DNA, a property that is thought to lead to the stimulation of single-strand invasion and was proposed to favor telomeric DNA looping. In this report, we show that these activities, originating from the central TRFH domain of TRF2, are also displayed by the TRFH domain of TRF1 but are repressed in the full-length protein by the presence of an acidic domain at the N-terminus. Strikingly, a similar repression is observed on TRF2 through the binding of a TERRA-like RNA molecule to the N-terminus of TRF2. Phylogenetic and biochemical studies suggest that the N-terminal domains of TRF proteins originate from a gradual extension of the coding sequences of a duplicated ancestral gene with a consequential progressive alteration of the biochemical properties of these proteins. Overall, these data suggest that the N-termini of TRF1 and TRF2 have evolved to finely regulate their ability to condense DNA.

One Identity or More for Telomeres

A major issue in telomere research is to understand how the integrity of chromosome ends is controlled. The fact that different types of nucleoprotein complexes have been described at the telomeres of different organisms raises the question of whether they have in common a structural identity that explains their role in chromosome protection. We will review here how telomeric nucleoprotein complexes are structured, comparing different organisms and trying to link these structures to telomere biology. It emerges that telomeres are formed by a complex and specific network of interactions between DNA, RNA, and proteins. The fact that these interactions and associated activities are reinforcing each other might help to guarantee the robustness of telomeric functions across the cell cycle and in the event of cellular perturbations. We will also discuss the recent notion that telomeres have evolved specific systems to overcome the DNA topological stress generated during their replication and transcription. This will lead to revisit the way we envisage the functioning of telomeric complexes since the regulation of topology is central to DNA stability, replication, recombination, and transcription as well as to chromosome higher-order organization.

The human telomeric protein hTRF1 induces telomere-specific nucleosome mobility

Human telomeres consist of thousands of base pairs of double-stranded TTAGGG repeats, organized by histone proteins into tightly spaced nucleosomes. The double-stranded telomeric repeats are also specifically bound by the telomeric proteins hTRF1 and hTRF2, which are essential for telomere length maintenance and for chromosome protection. An unresolved question is what role nucleosomes play in telomere structure and dynamics and how they interact and/or compete with hTRF proteins. Here we show that hTRF1 specifically induces mobility of telomeric nucleosomes. Moreover, Atomic Force Microscopy (AFM) imaging shows that hTRF1 induces compaction of telomeric DNA only in the presence of a nucleosome, suggesting that this compaction occurs through hTRF1–nucleosome interactions. Our findings reveal an unknown property of hTRF1 that has implications for understanding telomere structure and dynamics.

Dual role of sequence-dependent DNA curvature in nucleosome stability: the critical test of highly bent Crithidia fasciculata DNA tract

In spite of the knowledge of the nucleosome molecular structure, the role of DNA intrinsic curvature in determining nucleosome stabilization is still an open question. In this paper, we describe a general model that allows the prediction of the nucleosome stability, tested on 83 different DNA sequences, in surprising good agreement with the experimental data, carried out in ours as well as in many other laboratories. The model is based on the dual role of DNA curvature in nucleosome thermodynamic stabilization. A critical test is the evaluation of the nucleosome free energy relative to a Crithidia fasciculata kinetoplast DNA fragment, which represents the most curved DNA found so far in biological systems and, therefore, is generally believed to form a highly stable nucleosome.

Superstructural self-assembly of the G-quadruplex structure formed by the homopurine strand in a DNA tract of human telomerase gene promoter

Abstract AFM imaging and physico-chemical studies provide evidence of the ability of the G-quadruplex structure, formed by a homopurine DNA sequence present in the human telomerase gene promoter, to self-assemble, giving rise to periodical linear superstructures. The reported study suggests the possibility of G-rich DNA sequences to form a new type of G-wire stabilized by end-to-end stacking between terminal G-quartets and characterized by loops of adenine extruded by parallel G-quadruplex repetitive elements.

Recognition on the Nanoscale of a DNA Sequence by an Inorganic Crystal Surface

On studying by atomic force microscopy (AFM) the determination by the DNA sequence of superstructural properties, such as chain curvature and flexibility, we unexpectedly found that the surface of mica can recognize the DNA sequence. This recognition was found to be linked to a partial segregation of the A and T bases that occurs on whichever face that a highly curved DNA tract exposes to a mica surface upon its ACHTUNGTRENNUNGadsorption. In this work, we analyze the physical basis of this recognition and its dependence on the concentration of the magnesium ions that are commonly added for DNA imaging on mica by AFM. On the basis of this analysis, we propose a tentative electrostatic model that ascribes this recognition process of the DNA sequence to the capability of the bases of modulating the electrostatic field of the phosphates. The same model predicts that such a recognition effect is possibly present also in linear DNA. In this study we adopted the same strategy we had previously proposed of using palindromic dimers in order to get rid of the uncertainty regarding the orientation of the sequence in the profiles of the DNA molecules imaged by AFM. The curved tract under investigation is the Crithidia fasciculata fragment. Its tail-to-tail and head-to-head palindromic dimers were deposited on the surface of a mica crystal from solutions containing 2, 5, 6, or 10 mm Mg , under conditions as close as possible to equilibration, and then imaged in-air by AFM. The Mg ions are required to mediate the binding between the DNA and mica surfaces, whose surfaces are both negatively charged. We could not extend the range of these concentrations because at values lower than 2 mm very few molecules are deposited, and at values higher than 10 mm quasiequilibration conditions in the deposition are prevented by the appearance of some kinetic trapping. In the previous work, the same dimers were deposited only in the presence of 2 mm Mg . In this work we show that the approach we previously adopted can provide much more information than that reported earlier. The AFM traces of the individual molecules were digitalized, and the segmental curvatures of the single molecules were evaluated as previously described. Because of the palindromic symmetry of the dimers, the profiles that they can assume on the surface can be virtually classified into four classes that correspond to the trans forms we labeled S/S* or the C/C* cis ones, as illustrated in Figure 1A. However, these