Linker Histone-dependent Organization and Dynamics

A DNA sequence-dependent nucleosome structural and dynamic poly-morphism was recently uncovered through topoisomerase I relaxation of mononucleosomes on two homologous,350–370 bp DNA minicircle series, one originating from pBR322, the other from the 5 S nucleosome positioning sequence. Whereas both pBR and 5 S nucleosomes had access to the closed, negatively crossed conformation, only the pBR nucleosome had access to the positively crossed conformation. Simulation suggested this discrepancy was the result of a reorientation of entry/exit DNAs, itself proposed to be the consequence of specific DNA untwistings occur-ring in pBR nucleosome where H2B N-terminal tails pass between the two gyres. The present work investigates the behavior of the same two nucleosomes after binding of linker histone H5, its globular domain, GH5, and engineered H5 C-tail deletion mutants. Nucleosome access to the open uncrossed conformation was suppressed and, more surprisingly, the ability of 5 S nucleosome to positively cross was largely restored. This, together with the paradoxical observation of a less extensive crossing in the negative conformation with GH5 than without, favored an asym-metrical location of the globular domain in interaction with the central gyre and only entry (or exit) DNA, and raised the possibility of the domain physical rotation as a mechanism assisting nucleosome fluctua-tion from one conformation to the other. Moreover, both negative and positive conformations showed a high degree of loop conformational flexibility in the presence of the full-length H5 C-tail, which the simulation suggested to reflect the unique feature of the resulting stem to bring entry/exit DNAs in contact and parallel. The results point to the stem being a fundamental structural motif directing chromatin higher order folding, as well as a major player in its dynamics