Linker histones affect patterns of digestion of supercoiled plasmids by single-strand-specific nucleases

The effect of histone H1 binding on the cleavage of superhelical plasmids by single-strand-specific nucleases was investigated. Mapping of P1 cleavage sites in pBR322, achieved by EcoRI digestion after the original P1 attack, showed an intriguing phenomenon: preexisting susceptible sites became ''protected,'' whereas some new sites appeared at high levels of H1. Similar results were obtained with another single-strand-specific nuclease, S1. Disappearance of cutting at preexisting sites and appearance of new sites was also observed in a derivative plasmid that contains a 36-bp stretch of alternating d(AT) sequence that is known to adopt an altered P1-sensitive conformation. On the other hand, H1 titration of a dimerized version of the d(AT)(18)-containing plasmid led to protection of all preexisting sites except the d(AT)(18) inserts, which were still cut even at high H1 levels; in this plasmid no new sites appeared. The protection of preexisting sites is best explained by long-range effects of histone H1 binding on the superhelical torsion of the plasmid. The appearance of new sites, on the other hand, probably also involves a local effect of stabilization of specific sequences in P1-sensitive conformation, due to direct H1 binding to such sequences. That such binding involves linker histone N- and/or C-terminal tails is indicated bq the fact that titration with the globular domain of H5, while causing disappearance of preexisting sites, does not lead to the appearance of any new sites.