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 plasm ids by single-strand-specific nucleases was investigated. Mapping of P 1 cleavage sites in pBR322, achieved by EcoRI digestion after the orig inal P1 attack, showed an intriguing phenomenon: preexisting susceptib le sites became ''protected,'' whereas some new sites appeared at high levels of H1. Similar results were obtained with another single-stran d-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 k nown to adopt an altered P1-sensitive conformation. On the other hand, H1 titration of a dimerized version of the d(AT)(18)-containing plasm id led to protection of all preexisting sites except the d(AT)(18) ins erts, which were still cut even at high H1 levels; in this plasmid no new sites appeared. The protection of preexisting sites is best explai ned by long-range effects of histone H1 binding on the superhelical to rsion of the plasmid. The appearance of new sites, on the other hand, probably also involves a local effect of stabilization of specific seq uences in P1-sensitive conformation, due to direct H1 binding to such sequences. That such binding involves linker histone N- and/or C-termi nal tails is indicated bq the fact that titration with the globular do main of H5, while causing disappearance of preexisting sites, does not lead to the appearance of any new sites.