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In a previous study, we found that metaphase chromosomes are formed by thin plates, and here we have applied atomic force microscopy (AFM) and friction force measurements at the nanoscale (nanotribology) to analyze the properties of these planar structures in aqueous media at room temperature. Our results show that high concentrations of NaCl and EDTA and extensive digestion with protease and nuclease enzymes cause plate denaturation. Nanotribology studies show that native plates under structuring conditions (5 mM Mg2+) have a relatively high friction coefficient (μ ≈ 0.3), which is markedly reduced when high concentrations of NaCl or EDTA are added (μ ≈ 0.1). This lubricant effect can be interpreted considering the electrostatic repulsion between DNA phosphate groups and the AFM tip. Protease digestion increases the friction coefficient (μ ≈ 0.5), but the highest friction is observed when DNA is cleaved by micrococcal nuclease (μ ≈ 0.9), indicating that DNA is the main structural element of plates. Whereas nuclease-digested plates are irreversibly damaged after the friction measurement, native plates can absorb kinetic energy from the AFM tip without suffering any damage. These results suggest that plates are formed by a flexible and mechanically resistant two-dimensional network which allows the safe storage of DNA during mitosis.

We show that nondenaturing agarose gels can be used for the study of the structure and dynamic properties of native (uncross-linked) chromatin. In gels containing 1.7 mM Mg2+, chicken erythrocyte chromatin fragments having from about 6 to 50 nucleosomes produce well defined bands. These bands have an electrophoretic mobility that decreases only slightly with molecular weight. This surprising behavior is not observed in low ionic strength gels. Fragments with less than 6 nucleosomes and low content of histones H1-H5 give rise to broad bands in gels with Mg2+. In contrast, fragments containing only 3-4 nucleosomes but with the normal H1-H5 content are able to form associated structures with a mobility similar to that observed for high molecular weight chromatin. Electron microscopy results indicate that the associated fragments and the fragments of higher molecular weight show similar electrophoretic properties because they become very compact in the presence of Mg2+ and form cylindrical structures with a diameter of ˜33 nm. Our results suggest that the interactions involved in the self-assembly of small fragments are the same that direct the folding of larger fragments; in both cases, the resulting compact chromatin structure is formed from a basic element containing 5-7 nucleosomes.

Our results show that the noncovalent dye 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one (Nile red) can be used as a fluorescent probe to study the hydrophobic properties of proteins associated with the anionic detergent sodium dodecyl sulfate (SDS). Nile red can interact with both SDS micelles and protein-SDS complexes. The enhancement of Nile red fluorescence observed with diverse types of proteins occurs at SDS concentrations lower than the critical micelle concentration of this detergent. This is also observed using the covalent fluorophore rhodamine B isothiocyanate. Additional results obtained in studies in solution show that the fluorescence intensity and the spectral characteristics of Nile red associated with different proteins complexed with SDS are very similar. These spectroscopic similarities are probably related to the equivalent synchrotron X-ray scattering results found for various protein-SDS complexes in solution. The scattering results suggest that SDS induces the formation of complexes in which the basic structural properties are independent of the different initial structures of native proteins. We speculate that Nile red is bound to regions with equivalent hydrophobic characteristics located in the uniform structures produced by the association of SDS with proteins.

In a previous work (J.-R. Daban, M. Samso, and S. Bartolome, Anal. Biochem. 199, 162–168, 1991) we observed that, in the presence of the detergent sodium dodecyl sulfate (SDS), diverse types of proteins produced a high increase in the fluorescence intensity of the hydrophobic probe 9-diethylamino-5H-benzo[α]phenoxazine-5-one (Nile red). This enhancement of Nile red fluorescence was observed at SDS concentrations lower than the critical micelle concentration (CMC) of this detergent in the buffer (0.025 m Tris and 0.192 m glycine, pH 8.3) currently used in SDS-polyacrylamide gel electrophoresis. This observation led us to introduce a modification in the typical (U. K. Laemmli, Nature 227, 680–685, 1970) SDS-polyacrylamide gels, in which the SDS concentration in the gel after electrophoresis is lower than the CMC of this detergent but high enough to maintain the stability of the protein-SDS complexes in the bands. The staining of these modified gels with Nile red produces very high fluorescence in the protein-SDS bands and low background fluorescence. The Nile red staining method described in this paper is very rapid (i.e., the bands can be visualized and photographed within 6 min after the electrophoretic separation) and has a high sensitivity, similar to that obtained with the covalent fluorophores rhodamine B isothiocyanate and carboxytetramethylrhodamine succinimidyl ester also investigated in this work. Furthermore, our quantitative estimates indicate that most of the protein bands stained with Nile red show similar values of the fluorescence intensity per unit mass.

Previous studies showed that during mitosis chromatin filaments are folded into multilayer plates (1). These structures can be self-assembled from chromatin fragments obtained by micrococcal nuclease digestion of metaphase chromosomes (2). Chromosomes of different animal and plant species show great differences in size (which are dependent on the amount of DNA that they contain), but in all cases chromosomes are elongated cylinders that have relatively similar shape proportions (the length to diameter ratio is approximately 13). It is possible to explain this morphology by considering that chromosomes are self-organizing supramolecular structures formed by stacked layers of planar chromatin having different nucleosome-nucleosome interaction energies in different regions (3). The nucleosomes in the periphery of the chromosome are less stabilized by the attractive interactions with other nucleosomes and this generates a surface potential that destabilizes the structure. Chromosomes are smooth cylinders because this morphology has a lower surface energy than structures having irregular surfaces. The symmetry breaking produced by the different values of the surface energies in the telomeres and in the lateral surface explains the elongated structure of the chromosomes. The results obtained by other authors in nanomechanical studies of chromatin and chromosome stretching have been used to test the proposed supramolecular structure. It is demonstrated quantitatively that internucleosome interactions between chromatin layers can justify the work required for elastic chromosome stretching. Chromosomes can be considered as hydrogels with a lamellar liquid crystal organization. The good mechanical properties of this structure may be useful for the maintenance of chromosome integrity during mitosis. Furthermore this chromatin organization avoids random entanglement of the extremely long genomic DNA molecules in chromosomes.(1) Daban (2011) Micron 42:733-750.(2) Milla and Daban (2012) Biophys J 103:567-575.(3) Daban (2014) J. R. Soc. Interface 11:20131043.

The increase in the fluorescence intensity of 1-anilinonaphthalene-8-sulfonate (ANS) produced by core histones is higher than that produced by very lysine-rich histones (H1 and H5). In the presence of the anionic detergent sodium dodecyl sulfate (SDS) the enhancement of ANS fluorescence caused by these two groups of histones is roughly the same, but much lower than that observed for core histones in the absence of this detergent. However, the increase of ANS fluorescence produced by histone-SDS complexes is high enough to use it for the staining of these proteins separated in SDS-polyacrylamide gels. Histone bands are stained with ANS after electrophoresis and visualized by transillumination of the gel with a uv light source. The method described in this work allows the rapid detection of less than 0.5 μg of histone per band.

In non-denaturing low ionic strength gels, the titration of core DNA with H2A,H2B produces five well-defined bands. Quantitative densitometry and cross-linking experiments indicate that these bands are due to the successive binding of H2A,H2B dimers to core DNA. Only two bands are obtained with DNA-(H3,H4) samples. The slower of these bands is broad and presumably corresponds to two complexes containing one and two H3,H4 tetramers, respectively. In gels of higher ionic strength, DNA-(H2A,H2B) samples produce an ill-defined band, suggesting that the lifetime of the complexes containing H2A,H2B is relatively short. However, the low intensity of the free DNA band observed in these gels indicates that most of the DNA is associated with H2A,H2B. In agreement with this, our results obtained using different techniques (sedimentation, cross-linking, trypsin and nuclease digestions, and thermal denaturation) demonstrate that the association of H2A,H2B with core DNA occurs in free solution in both the absence and presence of NaCl (0.1 to 0.2 m ). The low mobilities of DNA-(H2A,H2B) complexes, together with sedimentation and DNase I digestion results, indicate that the DNA in these complexes is not folded into the compact structure found in the core particle. Furthermore, non-denaturing gels have been used to study the dynamic properties of DNA-(H2A,H2B) and DNA-(H3,H4) complexes in 0.2 m -NaCl. Our results show that: (1) H2A,H2B and H3,H4 can associate, respectively, with DNA-(H3,H4) and DNA-(H2A,H2B) to produce complexes containing the four core histones; (2) DNA-(H2A,H2B) and DNA-(H3,H4) are able to transfer histones to free core DNA; (3) an exchange of histone pairs takes place between DNA-(H2A,H2B) and DNA-(H3,H4) and produces complexes with the same histone composition as that of the normal nucleosome core particle; and (4) although both histone pairs can exchange, histones H2A,H2B show a higher tendency than H3,H4 to migrate from one incomplete core particle to another. The complexes produced in these reactions have the same compact structure as reconstituted core particles containing the four core histones. Our kinetic results are consistent with a reaction mechanism in which the transfer of histones involves direct contacts between the reacting complexes. The possible participation of these spontaneous reactions on the mechanism of nucleosome assembly is discussed.

The behaviour, upon variations in ionic strength, pH and temperature of 19F nuclear magnetic resonance signals of the trifluoroacetonylated derivative of histone H3 is compared with those of the H3·H4 complex and of the Hv fraction (an equimolar mixture of H2A, H2B, H3 and H4). The line width of the 19F-labelled histone H3 signals increases with ionic strength or pH, an effect consistent with aggregation of the protein. In the case of H3·H4 complex or Hv the line width decreases at intermediate ionic strengths (0.1–0.25 M NaCl). This effect is interpreted as the consequence of the formation of a well defined structure with ionic strength. At high salt concentrations the line width increases as a consequence of the final rigid quaternary structure or of the formation of higher aggregates.

The role of the histone pairs H2A,H2B and H3,H4 in the kinetics of core particle formation was investigated by using N -(1-pyrene)maleimide-labeled histone H3. The excimer emission intensity of a DNA-core histone complex prepared by direct mixing of DNA and histones in 0.2 m -NaCl is reduced by half when H2A,H2B is omitted. Fluorescence quenching studies and lifetime measurements indicate that the emission differences are probably due to static quenching. In a correctly folded nucleosome or a DNA-(H3,H4) complex, the two pyrene rings are buried and are held very close. DNA-(H3,H4) can interact with additional copies of H3,H4, but only when two dimers of H2A,H2B are correctly bound is there a specific twofold increase in excimer emission. The kinetics of the reaction of H3,H4 with DNA in 0.2 m -NaCl were followed by measuring the increase in 460 nm fluorescence. The apparent rate constant of the dominant kinetic component is ~ 2 × 10 −1 s −1 . If histones H2A,H2B are added immediately after the preparation of the DNA-(H3,H4) complex, an increase in excimer fluorescence is observed, with an apparent rate constant of ~ 6 × 10 −3 s −1 . However, if histones H2A,H2B are added one hour after DNA-(H3,H4) complex formation, there is no increase in excimer fluorescence. These results suggest that an intermediate involving the H3,H4 tetramer is formed first in nucleosome assembly. In the presence of H2A,H2B, this intermediate evolves to the final folded nucleosome, but in the absence of H2A,H2B it rearranges to an unmaturable dead-end complex. Additional experiments show that a very fast transfer of histone pairs (probably H2A,H2B) can take place between partially reconstituted nucleosomes.

In the present report it is shown that histone bands in urea-acetic acid or Triton-urea-acetic acid-polyacrylamide gels can be stained with the fluorescent dye 1-anilinonaphthalene 8-sulfonate and visualized by transillumination of the gel with an uv-light source. The 1-anilinonaphthalene 8-sulfonate staining method described here for urea and Triton-urea gels is rapid (it can be completed in 90 min) and allows the detection of less than 1 μg of histone per band.