Structural and dynamic features of telomeric chromatin

Telomeres 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.