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The role of DNA sequence in telomeric chromatin features
No full textThe structure of human telomeres is not yet completely characterized. Several proteins involved in the formation of the human telomeric complex have been identified, such as hTRF1 and hTRF2 which bind to GGGTAA duplex telomeric repeats. Notwithstanding most human telomeric DNA is organized into tightly spaced nucleosomes, the role of nucleosomes in telomeric chromatin is largely unknown. We previously found that telomeric nucleosomes are less stable than bulk nucleosomes and occupy multiple isoenergetic positions spaced every telomere repeat, due to the peculiar features of telomeric repeated sequences. We will present data on some important sequence-dependent features of telomeric nucleosomes, such as intrinsic nucleosome mobility and the assembly in the 30 nm fiber, by means of an in vitro model system, AFM visualization and theoretical calculations. Data on the interaction of telomeric nucleosomes with the proteins hTRF1 and hTRF2 will be also presented
Studio di una struttura dell’Appenninico inserita nelle fortificazioni del sito di Coppa Nevigata (Manfredonia-FG).
No full textCircular dichroism of complexes between acetylated poly-L-lysine and deoxyribonucleic acid.
No full textPoly-l-lysine (poly(Lys)) was acetylated to various extents and the conformation of the polypeptide with different degrees of acetylation was studied by using the circular dichroism (CD) technique. It was found that by increasing the percent of acetyl groups in the side chain, the polymer assumes an increasing percent of α-helix conformation. The CD of complexes between acetylated poly(Lys) and deoxyribonucleic acid (DNA) was also studied. The effect due to the polypeptide on the optical properties of nucleic acid in the region 245-300 nm, at a constant ratio of neutralization, decreases by increasing the percent of acetylation. Comparison with the CD spectra of DNA-poly(Lys) complexes at different ratios of neutralization suggests that the same type of perturbation on the electronic transitions of DNA is involved. The optical properties of DNA are not influenced by the polypeptide α-helical regions, whereas these regions are involved in the complex formation according to the results of the thermal stability of the complex
Conformational studies on deoxyribonucleic acid-polylysine complexes.
No full textThe structure of the complex between DNA and polylysine is analyzed on the basis of conformational calculations and experimental findings and possible connections with the local structure of nucleohistones are discussed. A molecular model characterized by alternant peptide sequences of right-handed and left-handed α-helix-type conformations, winding along the narrow groove of DNA in the B-type form, is proposed. This model is stabilized by hydrophobic, electrostatic, and hydrogen-bond interactions between the ζ-amino groups and the phosphate residues of both the strands of DNA and by hydrogen bonds between the NH peptide groups and the bases of one strand of the double helix
Structural and dynamic features of telomeric chromatin
No full textTelomeres are the specialized nucleoprotein structures that protect the ends of eukaryotic chromosomes. Vertebrate telomeres consist of several kb of double-stranded TTAGGG repeats, ending in 100-200 bases of single-stranded TTAGGG, named 3’-overhang. Protection of chromosome termini is achieved through the establishment of specialized structures, whose precise organization is not known yet. Telomeres are organized in nucleosomal arrays, with an unusually shorter spacing than bulk chromatin. Little is known about their role in the higher order telomeric chromatin structure.
In order to investigate structural features of telomeric chromatin, in this thesis a model system has been developed and studied at single molecule level by Atomic Force Microscopy (AFM) imaging. Human telomeric nucleosomal arrays, reconstituted on a 1500 bp long telomeric DNA have been analyzed. The same nucleosomal arrays have also been studied by a theoretical method, which derives nucleosomal positioning from the sequence-dependent DNA mechanical properties (curvature and flexibility). A satisfactory correlation between the theoretical and experimental results has been obtained, suggesting a relevant role of DNA sequence in telomeric chromatin basic organization. Both the experimental and theoretical telomeric nucleosome positioning appear characterized by nucleosome dyad axis multiple positioning with the six bp periodicity of human telomeric DNA. Moreover, the barriers between adjacent free energy minima of nucleosome formation are so low that it is possible to consider the nucleosomal organization on human telomeric DNA as almost continuous and the nucleosomes as free moving along DNA. These features suggest a higher mobility of telomeric nucleosomes with respect to that of bulk nucleosomes.
Since the intrinsic mobility of telomeric nucleosomes could be a relevant feature in telomeres dynamics, a model system has been set up to study nucleosome mobility. A construct has been realized, in which nucleosomes, initially located at the end of an 800 bp long DNA fragment, are adjacent to a strong nucleosome positioning sequence that should act as a nucleosome trap. By a restriction enzyme assay and AFM imaging it has been found a significant higher mobility of telomeric nucleosomes than average sequence nucleosomes. In fact, the mobility of telomeric nucleosome increases rising the temperature and the ionic strength. Moreover, the telomeric nucleosome mobility is influenced by specific telomeric protein, hTRF1, since hTRF1 binding to telomeric nucleosome gives rise to significant nucleosome repositioning
SELECTIVE BINDING OF ACTINOMYCIN-D INDUCES A REVERSIBLE CONFORMATIONAL TRANSITION OF NUCLEOSOMES
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