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  • Hazel Teague posted an update 2 years, 1 month ago

    It is characterized by DNA end-to-end distance of the closed (d1) and open (d2) conformations of the nucleosome and by the relative population of the closed-state nucleosome. The fraction of the nucleosomes in the closed state (f1) reflects the equilibrium GSK2656157 of the DNA end regions. An apparent equilibrium constant, accounting for the DNA breathing motion, can be calculated as in Eq. 3 equation(3) Kapparent=1?f1f1.WT and ��H3 mononucleosomes undergo a similar compaction process at low monovalent cationic strengths (<200?mM) as illustrated in Fig.?2. This compaction is reflected by an increase in the fraction of nucleosomes in the closed state and a slight reduction in the DNA end-to-end distance of the closed state. At concentrations between 100 and 200?mM K+ both the WT and ��H3 nucleosomes reach maximum compaction. Under the range of monovalent cationic concentrations studied here, the two conformations observed GS-1101 chemical structure for ��H3 nucleosomes differ by 2.3�C3.9?nm in the DNA end-to-end distance, as depicted in Fig.?2, a and b ( Fig.?S12, a and b). Based on this distance, an estimated 4�C7 basepairs are involved in the DNA breathing motion for each side of WT nucleosomes and 5�C8 basepairs are involved for ��H3 nucleosomes. This estimation is based on a model in which DNA unwrapping occurs symmetrically, with the same number of basepairs being involved on both sides of the nucleosome. There is some evidence, however, that suggests DNA unwraps asymmetrically ( 27?and?28). If this is the case, then the number of basepairs involved would be different than what is reported here. The number of basepairs reported here is smaller than what has been previously reported in protein-binding and single-molecule experiments, but agrees with recent findings using small-angle x-ray scattering ( 29). Comparing the conformations of WT and ��H3 mononucleosomes, both the closed state and the open state of ��H3 mononucleosomes show a larger DNA end-to-end distance. As KCl concentration increases, the population of open-state nucleosomes increases further. Multiple conformational states can contribute to the open state conformation including DNA breathing motion, dimer destabilization, and dimer dissociation ( 30). Although our labeling strategy cannot discriminate between these mechanisms, they all play a similar role in enhancing DNA accessibility. We further evaluated the effects of H3 tails with the presence of a divalent cation, i.e., Mg2+. Mg2+ is found in cell nucleus and is known to facilitate the compaction of a nucleosome array or a chromatin fiber. Understanding how Mg2+ mediates the nucleosomal interaction can help to elucidate the role of histone tails during the folding process. The in?vivo Mg2+ concentration varies from tissue to tissue and the reported values range from 0.2�C1.5?mM (31).