Two DNA-binding sites on the globular domain of histone H5 are required for binding to both bulk and 5 S reconstituted nucleosomes

We have previously shown the existence of two DNA-binding sites on the glob ular domain of H5 (termed GH5), both of which are required for nucleosome o rganisation, as judged by the protection of a 166 bp chromatosome intermedi ate during micrococcal nuclease digestion of chromatin. This supports a mod el in which GH5 contacts two duplexes on the nucleosome. However, studies o f a nucleosome assembled on the 5 S rRNA gene have argued against the requi rement for two DNA-binding sites for chromatosome protection, which has imp lications for the role of linker histones. We have used this proposed diffe rence in the requirement for a second site on the globular domain in the tw o models as a means of investigating whether bulk and reconstituted 5 S nuc leosomes are indeed fundamentally different. GH5 protects a 166 bp chromato some in both "bulk" and 5 S systems, and in both cases protection is abolis hed when all four basic residues in site II are replaced by alanine. Bindin g to four-way DNA junctions, which present a pair of juxtaposed duplexes, i s also abolished. Single mutations of the basic residues did not abolish ch romatosome protection in either system, or binding to four-way junctions, s uggesting that the residues function as a cluster. Both bulk and 5 S nucleo somes thus require a functional second DNA-binding site on GH5 in order to bind properly to the nucleosome. This is likely to reflect a similar mode o f binding in each case, in which two DNA duplexes are contacted in the nucl eosome. There is no indication from these experiments that linker histones bind fundamentally differently to 5 S and bulk nucleosomes. (C) 2000 Academ ic Press.