Anna Bergia

Plzf Mediates Transcriptional Repression of HoxD Gene Expression through Chromatin Remodeling

The molecular mechanisms that regulate coordinated and colinear activation of Hox gene expression in space and time remain poorly understood. Here we demonstrate that Plzf regulates the spatial expression of the AbdB HoxD gene complex by binding to regulatory elements required for restricted Hox gene expression and can recruit histone deacetylases to these sites. We show by scanning forced microscopy that Plzf, via homodimerization, can form DNA loops and bridge distant Plzf binding sites located within HoxD gene regulatory elements. Furthermore, we demonstrate that Plzf physically interacts with Polycomb proteins on DNA. We propose a model by which the balance between activating morphogenic signals and transcriptional repressors such as Plzf establishes proper Hox gene expression boundaries in the limb bud.

A Polymeric Molecular “Handle” for Multiple AFM‐Based Single‐Molecule Force Measurements

Get a grip! The strategy of single-molecule mechanical unfolding of multimodular proteins is extended to the investigation of any chemical bond by an appropriately tailored, comblike polymer bearing multiple instances of any desired chemical interaction or bond to be studied along its linear chain. This approach is used to study the Ni-(His) n-NTA complex (see picture: red, Ni-NTA; green, His tag; His = histidine; NTA = nitrilotriacetate).

Recognition of the DNA sequence by an inorganic crystal surface

The sequence-dependent curvature is generally recognized as an important and biologically relevant property of DNA because it is involved in the formation and stability of association complexes with proteins. When a DNA tract, intrinsically curved for the periodical recurrence on the same strand of A-tracts phased with the B-DNA periodicity, is deposited on a flat surface, it exposes to that surface either a T- or an A-rich face. The surface of a freshly cleaved mica crystal recognizes those two faces and preferentially interacts with the former one. Statistical analysis of scanning force microscopy (SFM) images provides evidence of this recognition between an inorganic crystal surface and nanoscale structures of double-stranded DNA. This finding could open the way toward the use of the sequence-dependent adhesion to specific crystal faces for nanotechnological purposes.

Sequence-Dependent DNA Dynamics by Scanning Force Microscopy Time-Resolved Imaging

Scanning force microscopy was used to study in fluid the conformational fluctuations of two double-stranded DNA molecules resulting from differently cut pBR322 circular DNAs. A new approach was conceived to monitor the thermodynamic equilibrium of the chain dynamics on different scale lengths. This method made it possible to demonstrate that both the observed DNA molecules were allowed to equilibrate only on their local small-scale dynamics during the time of the experiment. This capability of monitoring the length scale and the time scale of the equilibration processes in the dynamics of a DNA chain is relevant to give an insight in the thermodynamics of the DNA binding with proteins and synthetic ligands. It was also shown that the small-scale equilibration of the DNA chain during surface-restricted dynamics is enough to allow a valid measurement of the local sequence-dependent curvature.

Scanning force microscopy study on a single-stranded DNA: the genome of parvovirus B19.

The genome of parvovirus B19 is a 5600‐base‐long single‐stranded DNA molecule with peculiar sequence symmetries. Both complementary forms of this single‐stranded DNA are contained in distinct virions and they hybridize intermolecularly to double‐stranded DNA if extracted from the capsids with traditional methods, thus losing some of their native structural features. A scanning force microscopy analysis of these double‐stranded DNA molecules after thermal denaturation and renaturation gave us the chance to study the possible states that this DNA can assume in both its single‐stranded and double‐stranded forms. A novel but still poorly reproducible in situ lysis experiment that we have conducted on single virions with the scanning force microscope made it possible to image the totally unpaired state that the single‐stranded DNA molecule most likely assumes inside the viral particle. Structural considerations on single molecules offer the opportunity for the formulation of plausible hypotheses on the interaction between the DNA and the viral structural proteins that could prove important for the DNA packaging in the capsid and, possibly, the viral infection mechanisms.

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

DNA On Surfaces: Adsorption, Equilibration And Recognition Processes From A Microscopist’s View

The availability of a number of methods to controllably adsorb DNA on solid surfaces is useful to researchers working in different fields, such as structural biology, biophysical chemistry, diagnostics, sensorics, and nanotechnology. In this paper, we review some of the methods that have been devised in the last years to solve this problem. Thanks to the Scanning Force Microscope, we have recently been able to study the dynamics of DNA molecules adsorbed on mica. From the statistical analysis of the shapes of properly designed DNA molecules, we have also discovered an unexpected base‐sequence specificity in the adsorption of intrinsically curved DNA molecules on mica.

Scanning Force Microscopy Studies of the Adsorption and Desorption of DNA at Solid‐Liquid Interfaces

Both the processes of adsorption of double‐stranded DNA and its desorption from the surface of freshly cleaved mica can be studied thanks to the Scanning Force Microscope (SFM). The adsorption can be investigated under different conditions with methods ranging from the simple count of adsorbed DNA molecules to the more elaborate study of the local curvature of the adsorbed DNA chains: this high‐resolution information is shedding light on the process of structure‐dependent DNA adsorption on crystal surfaces. SFM‐based Single‐Molecule Force Spectroscopy is very useful for studying the desorption process of DNA from the same surfaces, with the same level of environmental control.