Biophysical characterization of the association of histones with single-stranded DNA.

BACKGROUND: Despite the profound current knowledge of the architecture and dynamics of nucleosomes, little is known about the structures generated by the interaction of histones with single-stranded DNA (ssDNA), which is widely present during replication and transcription. METHODS: Non-denaturing gel electrophoresis, transmission electron microscopy, atomic force microscopy, magnetic tweezers. RESULTS: Histones have a high affinity for ssDNA in 0.15M NaCl ionic strength, with an apparent binding constant similar to that calculated for their association with double-stranded DNA (dsDNA). The length of DNA (number of nucleotides in ssDNA or base pairs in dsDNA) associated with a fixed core histone mass is the same for both ssDNA and dsDNA. Although histone-ssDNA complexes show a high tendency to aggregate, nucleosome-like structures are formed at physiological salt concentrations. Core histones are able to protect ssDNA from digestion by micrococcal nuclease, and a shortening of ssDNA occurs upon its interaction with histones. The purified (+) strand of a cloned DNA fragment of nucleosomal origin has a higher affinity for histones than the purified complementary (-) strand. CONCLUSIONS: At physiological ionic strength histones have high affinity for ssDNA, possibly associating with it into nucleosome-like structures. GENERAL SIGNIFICANCE: In the cell nucleus histones may spontaneously interact with ssDNA to facilitate their participation in the replication and transcription of chromatin.

Nanomechanics of Fluorescent DNA Dyes on DNA Investigated by Magnetic Tweezers.

Fluorescent DNA dyes are broadly used in many biotechnological applications for detecting and imaging DNA in cells and gels. Their binding alters the structural and nanomechanical properties of DNA and affects the biological processes that are associated with it. Although interaction modes like intercalation and minor groove binding already have been identified, associated mechanic effects like local elongation, unwinding, and softening of the DNA often remain in question. We used magnetic tweezers to quantitatively investigate the impact of three DNA-binding dyes (YOYO-1, DAPI, and DRAQ5) in a concentration-dependent manner. By extending and overwinding individual, torsionally constrained, nick-free dsDNA molecules, we measured the contour lengths and molecular forces that allow estimation of thermodynamic and nanomechanical binding parameters. Whereas for YOYO-1 and DAPI the binding mechanisms could be assigned to bis-intercalation and minor groove binding, respectively, DRAQ5 exhibited both binding modes in a concentration-dependent manner.

Nucleocytoplasmic distribution of the Arabidopsis chromatin-associated HMGB2/3 and HMGB4 proteins.

High mobility group (HMG) proteins of the HMGB family are chromatin-associated proteins that as architectural factors are involved in the regulation of transcription and other DNA-dependent processes. HMGB proteins are generally considered nuclear proteins, although mammalian HMGB1 can also be detected in the cytoplasm and outside of cells. Plant HMGB proteins studied so far were found exclusively in the cell nucleus. Using immunofluorescence and fluorescence microscopy of HMGB proteins fused to the green fluorescent protein, we have examined the subcellular localization of the Arabidopsis (Arabidopsis thaliana) HMGB2/3 and HMGB4 proteins, revealing that, in addition to a prominent nuclear localization, they can be detected also in the cytoplasm. The nucleocytoplasmic distribution appears to depend on the cell type. By time-lapse fluorescence microscopy, it was observed that the HMGB2 and HMGB4 proteins tagged with photoactivatable green fluorescent protein can shuttle between the nucleus and the cytoplasm, while HMGB1 remains nuclear. The balance between the basic amino-terminal and the acidic carboxyl-terminal domains flanking the central HMG box DNA-binding domain critically influences the nucleocytoplasmic distribution of the HMGB proteins. Moreover, protein kinase CK2-mediated phosphorylation of the acidic tail modulates the intranuclear distribution of HMGB2. Collectively, our results show that, in contrast to other Arabidopsis HMGB proteins such as HMGB1 and HMGB5, the HMGB2/3 and HMGB4 proteins occur preferentially in the cell nucleus, but to various extents also in the cytoplasm.

Binding kinetics of bisintercalator Triostin a with optical tweezers force mechanics.

The binding kinetics of the intercalative binding of Triostin A to lambda-DNA was investigated by measuring the force extension response of the DNA-ligand complexes with an optical tweezers system. These force response curves, containing the information about different binding properties, were analyzed based on a recent method (put forth by another research group) for monointercalators that was extended to bisintercalators. Our binding analysis reveals an exponential dependence of the association constant on the applied external force as well as a decreasing binding site size. In general, our results are in agreement with those for the monointercalator ethidium. However, to explain the high-force binding site size, a new model for bisintercalation of Triostin A at high forces is proposed.

Single beam optical tweezers setup with backscattered light detection for three-dimensional measurements on DNA and nanopores.

We introduce a versatile and high precision three-dimensional optical tweezers setup with minimal optical interference to measure small forces and manipulate single molecules in the vicinity of a weak reflective surface. Our tweezers system integrates an inverted optical microscope with a single IR-laser beam that is spatially filtered in an appropriate way to allow force measurements in three dimensions with remarkably high precision when operated in backscattered light detection mode. The setup was tested by overstretching a lambda-DNA in x and z directions (perpendicular and along the optical axis), and by manipulating individual lambda-DNA molecules in the vicinity of a nanopore that allowed quantitative single molecule threading experiments with minimal optical interference.

Interfacial ternary complex DNA/Ca/lipids at anionic vesicle surfaces.

The electroporative transfer of gene DNA and other bioactive substances into tissue cells by electric pulses gains increasing importance in the new disciplines of electrochemotherapy and electrogenetherapy. The efficiency of the electrotransfer depends crucially on the adsorption of the gene DNA and oligonucleotides to the plasma cell membranes. Here it is shown that the adsorption of larger oligonucleotides such as fragments (ca. 300 bp) of sonicated calf-thymus DNA, to anionic lipids of unilamellar vesicles (diameter Phi=300+/-90 nm) is greatly enhanced by divalent cations such as Ca(2+)-ions. Applying centrifugation, bound and free DNA are monitored optically at the wavelength lambda=260 nm. Using arsenazo III as a Ca(2+)-indicator and atomic absorption spectroscopy (AAS), Ca(2+)-titrations of DNA and vesicles yield the individual equilibrium constants of Ca(2+)- and DNA-binding not only for the binary complexes: Ca/lipids, Ca/DNA and DNA/lipids, respectively, but also for the various processes to form the ternary complex DNA/Ca/lipids. The data provide the basis for goal-directed optimization protocols for the adsorption and thus efficient electrotransfer of oligonucleotides and polynucleotides into cells.

The novel desmin mutant p.A120D impairs filament formation, prevents intercalated disk localization, and causes sudden cardiac death.

BACKGROUND: The intermediate filament protein desmin is encoded by the gene DES and contributes to the mechanical stabilization of the striated muscle sarcomere and cell contacts within the cardiac intercalated disk. DES mutations cause severe skeletal and cardiac muscle diseases with heterogeneous phenotypes. Recently, DES mutations were also found in patients with arrhythmogenic right ventricular cardiomyopathy. Currently, the cellular and molecular pathomechanisms of the DES mutations leading to this disease are not exactly known. METHODS AND RESULTS: We identified the 2 novel variants DES-p.A120D (c.359C>A) and DES-p.H326R (c.977A>G), which were characterized by cell culture experiments and atomic force microscopy. Family analysis indicated a broad spectrum of cardiomyopathies with a striking frequency of arrhythmias and sudden cardiac deaths. The in vitro experiments of desmin-p.A120D reveal a severe intrinsic filament formation defect causing cytoplasmic aggregates in cell lines and of the isolated recombinant protein. Model variants of codon 120 indicated that ionic interactions contribute to this filament formation defect. Ex vivo analysis of ventricular tissue slices revealed a loss of desmin staining within the intercalated disk and severe cytoplasmic aggregate formation, whereas z-band localization was not affected. The functional experiments of desmin-p.H326R did not demonstrate any differences from wild type. CONCLUSIONS: Because of the functional in vivo and in vitro characterization, DES-p.A120D has to be regarded as a pathogenic mutation and DES-p.H326R as a rare variant with unknown significance. Presumably, the loss of the desmin-p. A120D filament localization at the intercalated disk explains its clinical arrhythmogenic potential.

Video-based and interference-free axial force detection and analysis for optical tweezers.

For measuring the minute forces exerted on single molecules during controlled translocation through nanopores with sub-piconewton precision, we have developed a video-based axial force detection and analysis system for optical tweezers. Since our detection system is equipped with a standard and versatile CCD video camera with a limited bandwidth offering operation at moderate light illumination with minimal sample heating, we integrated Allan variance analysis for trap stiffness calibration. Upon manipulating a microbead in the vicinity of a weakly reflecting surface with simultaneous axial force detection, interference effects have to be considered and minimized. We measured and analyzed the backscattering light properties of polystyrene and silica microbeads with different diameters and propose distinct and optimized experimental configurations (microbead material and diameter) for minimal light backscattering and virtually interference-free microbead position detection. As a proof of principle, we investigated the nanopore threading forces of a single dsDNA strand attached to a microbead with an overall force resolution of ±0.5 pN at a sample rate of 123 Hz.