New insights into unwrapping DNA from the nucleosome from a single-molecule optical tweezers method.

T hierarchical, multidimensional assemblies of protein–DNA structures known as the eukaryotic chromosome present both challenges and opportunities for biophysicists interested in chromatin structure (1, 2). One of the most interesting aspects of chromatin structure to come to light in recent years is the discovery that the core histone proteins alone can effectively direct the formation of multiple initial levels of this hierarchy (3). The core histones are involved in the assembly of individual nucleosomes, mediate folding of nucleosome arrays, and direct interfiber interactions important for assembly of arrays into higher-order structures (3). Secondly, the nucleosome itself has come to be viewed as a dynamic entity, undergoing transitions, which result in exposure of DNA sites contained within (2). However, the details of these dynamic transitions and the mechanism by which DNA spontaneously unravels or is forcibly unraveled from the nucleosome has not been adequately defined. An elegant application of a single-molecule optical trapping technique to the study of nucleosome structure and dynamics in a model system has provided exciting new insights into the unwinding of DNA from the nucleosomes within a nucleosomal array (4). Because of the diversity and heterogeneity of chromatin structures, the focus of most biophysical characterization has been the primary subunit of chromatin, the nucleosome. Nucleosomes are assembled by the wrapping of two 80-bp superhelical turns of DNA around a central spool of proteins consisting of an octameric assembly of the four core histone proteins, H2A, H2B, H3, and H4 (Fig. 1). Apart from DNA, these proteins heterodimerize and, in the case of H3, undergo homotypic interactions to form stable H2A H2B dimers and (H3, H4)2 tetramers in solutions containing physiological ionic strengths (5, 6). Nucleosome assembly is initiated by the wrapping of 100–120 bp of DNA around the (H3 H4)2 tetramer (7). Subsequently, H2A H2B dimers bind to either side of the tetramer– DNA complex and extend the wrapping of DNA within the nucleosome up to 160 bp (5, 7). This creates a left-handed superhelical ramp of protein onto which the DNA is wrapped, essentially consisting of four histone dimers linked end-to-end: (H2A H2B)–(H4 H3)–(H3 H4)–(H2B H2A) (8, 9). The H3:H3 and H2B:H4 dimer–dimer interfaces are comprised of structurally similar four-helix bundles; however the latter does not remain stably associated in the absence of DNA in aqueous solutions containing physiological ionic strengths (5). Thus the H2B:H4 interface is a likely site for initial disruption of histone–histone interactions on unfolding of the nucleosome core in vivo (5, 10). Wrapping the DNA onto the lefthanded spiral formed by the histone fold domains requires the helix to be severely distorted into approximately two 80-bp superhelical loops (9). Given that the persistence length of DNA is about 150 bp (11), one would expect a tremendous energetic cost to bend DNA into the conformation found in the nucleosome (see ref. 12). Of course, the cost of bending nucleosomal DNA is more than offset by favorable electrostatic interactions between basic side chains on the histone proteins and the polyanionic backbone of the DNA. Histone contacts to DNA occur every 10 bp on each strand (see Fig. 1) and involve an arginine residue penetrating the minor groove; several main polypeptide chain amide interactions with two consecutive phosphates on each DNA strand and, surprisingly, substantial hydrophobic interactions with the faces of the deoxyribose sugars in the DNA (9). As mentioned above, nucleosomes are not structurally inert entities, but rather undergo several conformational transitions that are likely to be important in facilitating interactions between transacting factors and DNA in chromatin in vivo (2, 13). Widom and colleagues have demonstrated that DNA binding sites in nucleosomal DNA are exposed with low probability but at sufficiently rapid rates to allow physiologically significant site accessibility (13, 14). DNA probably unwraps from the edge of the nucleosome because sites within nucleosomal DNA are transiently exposed apart from histones with a probability of about 1 in 103-105 as one moves from the periphery of the nucleosome toward the center (13, 14). Thus given the dynamic nature of this system, factors present at sufficient concentrations and having high enough affinities for naked DNA will be able to efficiently compete with histone proteins and effect significant loading of their cognate DNA elements in chromatin (13). However, many issues regarding the dynamics of the association of DNA with core histone remain unresolved. Moreover, the response of nucleosomal DNA to external stresses has not been adequately studied. This latter issue is of critical importance because nucleosomes are disassembled and invaded by many different DNA-dependent processive enzymes (2, 15–17). The new work by Brower-Toland et al. (4) addresses this issue directly in vitro. A key to their success is the use of a well defined nucleosomal array model system reconstituted from purified core histones and a DNA template consisting of tandemly repeated nucleosome positioning sequences (3, 18). By carefully applying tension to the ends of the oligonucleosomal structure using an