LINKER HISTONE-DEPENDENT DNA-STRUCTURE IN LINEAR MONONUCLEOSOMES

We have examined the binding of the linker histone H5 (LH) to mononucl eosomes. Mononucleosomes reconstituted on short DNA fragments display a series of discrete bands on a gel corresponding to various nucleosom e positions along the DNA. When a series of engineered H5s with differ ing extents of the C-terminal tail are bound to these mononucleosomes, the electrophoretic mobilities of the resulting complexes are altered . Not only is there a general increase in mobility upon complex format ion, but there is a reduction in the differences in mobility of the mo st distal nucleosomes. The complexes were also visualized by electron microscopy. From these two complementary studies, we conclude the foll owing. (1) Entering and exiting DNAs are uncrossed in the LH-free part icles, despite a DNA wrapping of 1.65 to 1.7 turns around the histone core. This results from a bending of the entering and exiting DNA away from each other and the histone surface, presumably as a consequence of electrostatic repulsion. This confirms and extends conclusions deri ved from our recent examination of the same particles in 3D through cr yo-electron microscopy. (2) Binding of the globular domain of H5 incre ases DNA wrapping to 1.8 to 1.9 turns, but fails to induce a crossing due to an accentuation of the bends. (3) The C-terminal tail of H5 bri dges entering and exiting DNAs together into a four-stranded stem over a distance of about 30 bp. The occurrence of such a stem may introduc e constraints on models of the 30 nm chromatin fiber. (C) 1996 Academi c Press Limited