Abstract
A fundamental step in the assembly of native chromatin is the specific recognition and binding of linker histones to the nucleoprotein subunit known as the nucleosome. A first step in defining this important interaction is the determination of residues within linker histones that are important for the structure-specific recognition of the nucleosome core. By combining in vitro assays for the native binding activity of linker histones and site-directed mutagenesis, we have examined a cluster of basic residues within the globular domain of H1(0), a somatic linker histone variant from Xenopus laevis. We show that these residues, which comprise a putative DNA binding surface within the globular domain, do not play an essential role in the structure-specific binding of a linker histone to the nucleosome.
Highlights
The primary unit of structure in chromatin, the nucleosome core, is comprised of two turns of DNA wound onto a spool of core histone proteins
We find that recombinant Xenopus H10 yields chromatosomes upon micrococcal nuclease digestion of reconstituted nucleosomes as efficiently as linker histones isolated from native chromatin (Fig. 2B)
Studies of linker histones have shown that these proteins bind to the exterior surface of the nucleosome, and the globular domain of H1 alone is responsible for this structure-specific recognition [6, 10, 14]
Summary
Vol 271, No 42, Issue of October 18, pp. 25817–25822, 1996 Printed in U.S.A. A Putative DNA Binding Surface in the Globular Domain of a Linker Histone Is Not Essential for Specific Binding to the Nucleosome*. By combining in vitro assays for the native binding activity of linker histones and sitedirected mutagenesis, we have examined a cluster of basic residues within the globular domain of H10, a somatic linker histone variant from Xenopus laevis We show that these residues, which comprise a putative DNA binding surface within the globular domain, do not play an essential role in the structure-specific binding of a linker histone to the nucleosome. We have developed a simple in vitro assay for detecting binding of linker histones to a model chromatin complex This complex is based on a DNA fragment that contains a Xenopus borealis 5 S rRNA gene. Based on structural homology to several sequence-specific DNA-binding proteins, a region encompassing helix III in the globular domain has been proposed to bind in the major groove of DNA when linker histones are bound to nucleosomes. We have tested the relevance of these basic residues for the specific binding of a linker histone to a nucleosome
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