Biomedical subjects

Jan Bednar

Publications and source records attributed to Jan Bednar.

6 recordsLinked to original sources

PubMed2006

3D reconstruction and comparison of shapes of DNA minicircles observed by cryo-electron microscopy.

We use cryo-electron microscopy to compare 3D shapes of 158 bp long DNA minicircles that differ only in the sequence within an 18 bp block containing either a TATA box or a catabolite activator protein binding site. We present a sorting algorithm that correlates the reconstructed shapes and groups them into distinct categories. We conclude that the presence of the TATA box sequence, which is believed to be easily bent, does not significantly affect the observed shapes.

Algorithms↗

PubMed2006

Dissection of the unusual structural and functional properties of the variant H2A.Bbd nucleosome.

The histone variant H2A.Bbd appeared to be associated with active chromatin, but how it functions is unknown. We have dissected the properties of nucleosome containing H2A.Bbd. Atomic force microscopy (AFM) and electron cryo-microscopy (cryo-EM) showed that the H2A.Bbd histone octamer organizes only approximately 130 bp of DNA, suggesting that 10 bp of each end of nucleosomal DNA are released from the octamer. In agreement with this, the entry/exit angle of the nucleosomal DNA ends formed an angle close to 180 degrees and the physico-chemical analysis pointed to a lower stability of the variant particle. Reconstitution of nucleosomes with swapped-tail mutants demonstrated that the N-terminus of H2A.Bbd has no impact on the nucleosome properties. AFM, cryo-EM and chromatin remodeling experiments showed that the overall structure and stability of the particle, but not its property to interfere with the SWI/SNF induced remodeling, were determined to a considerable extent by the H2A.Bbd docking domain. These data show that the whole H2A.Bbd histone fold domain is responsible for the unusual properties of the H2A.Bbd nucleosome.

Amino Acid Sequence↗

PubMed2005

Assembly of the full-length recombinant mouse prion protein I. Formation of soluble oligomers.

The conversion of a monomeric alpha-helix-rich isoform to multimeric beta-sheet-rich isoforms is a prominent feature of the conversion between PrP(C) and PrP(SC). We mimicked this process in vitro by exposing an unglycosylated recombinant form of the full-length mouse prion protein ((Mo)PrP(23-231)) to an acidic pH, at 37 degrees C, and we monitored the kinetics of conformational change and assembly. In these conditions, monomeric (Mo)PrP(23-231) converts slowly to two ensembles of soluble oligomers that are separated by size exclusion chromatography. The larger oligomers (I) are unstable, and their formation involves almost no change in secondary structure content. The smaller oligomers (II) form stable spherical or annular particles containing between 8 and 15 monomers as determined by multi-angle laser light scattering (MALLS). Their formation is concomitant with the main, thought limited, change in the secondary structure content (10%) seen by Fourier Transform Infrared (FTIR) spectroscopy. Even if these oligomers conserve a large part of the secondary structure of monomeric PrP, they exhibit amyloid features with the appearance of intermolecular beta-structure as revealed by the appearance of an IR band below 1620 cm(-1).

Animals↗

PubMed2005

Magneto-optical tweezers built around an inverted microscope.

We present a simple experimental setup of magneto-optical tweezers built around an inverted microscope. Two pairs of coils placed around the focal point of the objective generate a planar-rotating magnetic field that is perpendicular to the stretching direction. This configuration allows us to control the rotary movement of a paramagnetic bead trapped in the optical tweezers. The mechanical design is universal and can be simply adapted to any inverted microscope and objective. The mechanical configuration permits the use of a rather large experimental cell and the simple assembly and disassembly of the magnetic attachment.

DNA↗

PubMed2005

Histone octamer instability under single molecule experiment conditions.

We have studied the sample concentration-dependent and external stress-dependent stability of native and reconstituted nucleosomal arrays. Whereas upon stretching a single chromatin fiber in a solution of very low chromatin concentration the statistical distribution of DNA length released upon nucleosome unfolding shows only one population centered around approximately 25 nm, in nucleosome stabilizing conditions a second population with average length of approximately 50 nm was observed. Using radioactively labeled histone H3 and H2B, we demonstrate that upon lowering the chromatin concentration to very low values, first the linker histones are released, followed by the H2A-H2B dimer, whereas the H3-H4 tetramer remains stably attached to DNA even at the lowest concentration studied. The nucleosomal arrays reconstituted on a 5 S rDNA tandem repeat exhibited similar behavior. This suggests that the 25-nm disruption length is a consequence of the histone H2A-H2B dimer dissociation from the histone octamer. In nucleosome stabilizing conditions, a full approximately 145 bp is constrained in the nucleosome. Our data demonstrate that the nucleosome stability and histone octamer integrity can be severely degraded in experiments where the sample concentration is low.

Animals↗

PubMed2004

The histone octamer is invisible when NF-kappaB binds to the nucleosome.

The transcription factor NF-kappaB is involved in the transcriptional control of more than 150 genes, but the way it acts at the level of nucleosomal templates is not known. Here we report on a study examining the interaction of NF-kappaB p50 with its DNA recognition sequence in a positioned nucleosome. We demonstrate that NF-kappaB p50 was able to bind to the nucleosome with an apparent association constant close to that for free DNA. In agreement with this, the affinity of NF-kappaB p50 binding does not depend on the localization of its recognition sequence relative to the nucleosome dyad axis. In addition, the binding of NF-kappaB p50 does not induce eviction of histones and does not perturb the overall structure of the nucleosome. The NF-kappaB p50-nucleosome complex exhibits, however, local structural alterations within the NF-kappaB p50 recognition site. Importantly, these alterations were very similar to those found in the NF-kappaB p50-DNA complex. Our data suggest that NF-kappaB p50 can accommodate the distorted, bent DNA within the nucleosome. This peculiar property of NF-kappaB p50 might have evolved to meet the requirements for its function as a central switch for stress responses.

Animals↗