Folding of the DNA double helix in chromatin-like structures from simian virus 40.

Abstract

Relaxed circular, covalently closed simian virus 40 DNA molecules were associated with the four histones that are present in virions. In electron micrographs the resulting complexes appear twisted, with globular structures (nucleosomes) along the DNA. Incubation with an untwisting extract converts the twisted complexes to relaxed structures. Extraction of the DNA from the relaxed complexes yields supercoiled molecules. The number of superhelical turns in these molecules corresponds to the number of nucleosomes per DNA molecule in the complexes.In eukaryotic nuclei, the fundamental structure of chromatin fibers appears to be a flexible chain composed of globular particles connected by DNA filaments (1, 2). In these particles, termed nucleosomes (2), about 200 base pairs of DNA are associated with the four histonies F2a,, F2a2, F2b, and F3 (2-7). Such a repeating unit structure can be formed in vitro by association of the four histonies and linear l)NAs (2).In the nucleosomes the DNA is under constraint, since it is compacted about 5-fold compared to its length in the extended double helical form (2). The nature of this constraint can be studied by the association of histones to covalently closed circular DNA molecules, since supercoiling or unwinding of the DNA within the nucleosome (luring its formation would alter the supercoiling of the rest of the molecule (8). In addition, from the known thermodynamic prolerties of superhelical DNAs (8-10), the influence of the degree of superhelicity on the formation of nucleosome structures can provide information on whether the formation of a nucleosome is equivalent to an unwinding or winding of the double helix. Simian virus 40 (SV40) D)NA is particularly attractive for such a study for two reasons. First, two circular covalently closed allomorphic forms of this DNA are available, the superhelical DNA I and the relaxed circular DNA Ir which results from the incubation of DNA I with an untwisting extract (FE) (l1). This extract is thought to introduce a single-strand nick into superhelical DNA and to reseal the nick after the torsional tension in the double helix has been relieved. Second, SV40 DNA and the four histones are associated in vivo anld the complexes can be isolated from virions (12, 13) and from infected cells. In the latter case, the complex appears as a compactecl structure with about 20 nueleosomes (14).We

Accumulation of the FACT complex, as well as histone H3.3, serves as a target marker for somatic hypermutation

“…Indeed, Kodgire et al reported that insertion of the nucleosome positioning sequence, which is tightly folded by the nucleosome, into the variable region of the Ig light chain gene reduces SHM in and around the inserted region (30). Another explanation involves the fact that unwinding DNA from nucleosome produces one negative supercoil (31). It is known that excessive negative supercoil behind the transcription elongation complex tends to form irregular DNA structures called non-B DNA (3,32), which is proposed to be the substrate for DNA-cleaving enzymes such as DNA topoisomerase 1 (6).…”

The helical periodicity of DNA on the nucleosome

“…The X-ray data showed that the double helix of DNA in a nucleosome is wound (about a histone core) into about two turns of a (shallow) superhelix, whereas measurements on closed, circular DNA extracted from SV40 chromatin gave a degree of supercoiling nearer one superhelical turn per nucleosome (2). However, as first realised by Crick (3), what was being measured in the physico-chemical experiments was not the "number of supercoils", but the change in the linkage number of DNA (i.e.…”

The SV40 transcription complex. II. Non-dissociation of protein from SV40 chromatin during transcription

“…4) The present study demonstrates that the SV40 viral transcription complex (VTC) can synthesize long RNA transcripts under conditions in which protein does not dissociate from the transcriptionally active chromatin. Electron microscopic and biochemical studies have revealed a close similarity between the structure of SV40 and cellular chromatin (4)(5)(6)(7)(8)(9)(10). On the basis of sedimentation rate (23) and density in CsCl (Fig.…”

“…The viral complex has been shown by electron microscopic (4)(5)(6) and biochemical (5,(7)(8)(9)(10) analyses to possess a structure which closely resembles that of cellular chromatin. There is an average of 21 beads (nucleosomes) per SV40 genome, each having a diameter of ca.…”

“…There is an average of 21 beads (nucleosomes) per SV40 genome, each having a diameter of ca. 110A (4)(5)(6). Nucleosomes are comprised of about 200 base pairs of DNA wound around a complex containing two each of the four major histones (11)(12)(13)(14)(15)(16)(17).…”