Histone contributions to the structure of DNA in the nucleosome.

Abstract

We describe the application of the hydroxyl radical footprinting technique to examine the contribution of the core histone tails and of histones H3 and H4 to the structure of DNA in the nucleosome. We first establish that, as was previously determined for a nucleosome containing a unique sequence of DNA, mixed-sequence nucleosomes contain two distinct regions of DNA structure. The central three turns of DNA in the nucleosome have a helical periodicity of -10.7 base pairs per turn, while flanking regions have a periodicity of -10.0 base pairs per turn. Removal ofthe histone tails does not change the hydroxyl radical cleavage pattern in either mixedor unique-sequence nucleosome samples. A tetramer of histones H3 and H4, (H3/H4)2, organizes the central 120 base pairs of DNA identically to that found in the nucleosome. Moreover, "tailless" octamers and the (H3/H4)2 tetramer recognize the same nucleosome positioning signals as the intact octamer.Many studies have investigated the role of both DNA and the histone proteins in the architecture of the nucleosome (1-4). The structure of DNA in the nucleosome is altered in two ways from that found when the DNA is free in solution. First, DNA in the nucleosome is highly bent (1). This leads to the preferential association of the histone core with inherently curved DNA molecules, contributing to nucleosome positioning with respect to DNA sequence (5-9). Second, the average helical periodicity ofDNA is altered from 410.5 base pairs (bp) per turn when free in solution to an average of -10.2 bp per turn when in the nucleosome (5, 10-12). In addition, high-resolution analysis using the hydroxyl radical DNA cleavage reagent has revealed that two distinct regions of DNA with different helical periodicities exist in a nucleosome containing part of the 5S ribosomal RNA gene of Xenopus borealis (12). These changes in DNA structure upon incorporation into a nucleosome are expected to influence both the translational positioning of DNA with respect to the histone core and the way nucleosomal DNA interacts with other DNA-binding proteins.The central role of histones H3 and H4 in nucleosome structure and assembly, in vivo and in vitro, is supported by both physical (for a review, see ref. 13) and biochemical (14) evidence. Histones H3 and H4 form a tetramer, (H3/H4)2, which binds to DNA and directs the subsequent association of histones H2A and H2B (15-17). Alterations in the histone components within the nucleosome are associated with many biological processes. Deficiency of histones H2A and H2B within chromatin leads to an increase in accessibility to RNA polymerases (18)(19)(20). Modification of the highly conserved N-terminal basic domains of the histone proteins (tails) is also correlated with increased transcription (21,22). Acetylation ofthe histone tails influences the organization of nucleosomal DNA in some unknown way (23,24). In spite of these observations, the influence of the core histone tails on the actual structure of DNA within a nucleosome and the extent to whic...

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