IDENTIFICATION OF 2 DNA-BINDING SITES ON THE GLOBULAR DOMAIN OF HISTONE H5

The nature of the complexes of histones H1 and H5 and their globular d omains (GH1 and GH5) with DNA suggested two DNA-binding sites which ar e likely to be the basis of the preference of H1 and H5 for the nucleo some, compared with free DNA. More recently the X-ray and NMR structur es of GH5 and GH1, respectively, have identified two basic clusters on opposite sides of the domains as candidates for these sites. Removal of the positive charge at either location by mutagenesis impairs or ab olishes the ability of GH5 to assemble cooperatively in 'tramline' com plexes containing two DNA duplexes, suggesting impairment or loss of i ts ability to bind two DNA duplexes. The mutant forms of GH5 also fail to protect the additional 20 bp of nucleosomal DNA that are character istically protected by H1, H5 and wild-type recombinant GH5. They stil l bind to H1/H5-depleted chromatin, but evidently inappropriately. The se results confirm the existence of, and identify the major components of, two DNA-binding sites on the globular domain of histone H5, and t hey strongly suggest that both binding sites are required to position the globular domain correctly on the nucleosome.