Structural features of a phased nucleosome core particle.

Chicken erythrocyte inner histones associate with a cloned 260-base-pair (bp) segment of Lytechinus variegatus DNA in a unique location. The fragment contains a 120-bp segment encoding 5S rRNA, a 90-bp flanking sequence to the 5' side of the transcribed segment, and a 50-bp downstream flanking sequence. Association of DNA, uniquely labeled at one end or the other and at either the 3' or the 5' terminus of a given strand, with histones at 0.1 M ionic strength leads to formation of a compact complex which sediments at about 13 S. Analysis of cutting of the complex by DNase I shows that protection from the nuclease is confined to a region beginning 20 bp from the left end of the segment and extending to about 165 bp from the left end. Within the protected region, the two DNA strands differ in their susceptibilities to the nuclease, the precise location of nuclease cutting sites and the spacing between these sites, and the relative susceptibilities of specific cutting locations. It seems that information present in DNA and the histone octomer is sufficient to create a precisely phased nucleosome in which interactions of the two DNA strands with histones are not the same. The structure of this unique nucleosome is not predicted by the intellectual model based on studies of mixed populations of nucleosome core particles.     

Histone packing in the nucleosome core particle of chromatin

The chromatin core particle DNA conformation deduced in broad outline by Finch et al. [Finch, J. T., Lutter, L. C., Rhodes, D., Brown, R. S., Rushton, B., Levitt, M. & Klug, A. (1977) Nature 269, 29-36] can be described in detail using other available experimental results. Histone binding sites compatible with the pattern of pancreatic DNase I digestion (Simpson, R. T. & Whitlock, J. P., Jr. (1976) Cell 9, 347-353; Noll, M. (1977) J. Mol. Biol. 116, 49-71; Lutter, L. C. (1977) J. Mol. Biol. 117, 53-69] lend to core particle DNA pseudosymmetry characteristic of molecular point group D(3). DNA symmetry and pseudosymmetry, in turn, imply equivalence and quasi-equivalence properties of the histone packing arrangement that support the following deductions: (i) One and only one alpha(2)beta(2) histone tetramer, presumably (H3)(2)(H4)(2), can serve as a stable subassembly within the histone octamer. (ii) There is a unique, strand-specific way to assign DNA binding domains to the arginine-rich histones (H3 and H4). (iii) Histones H3 and H4 alone should suffice to impose a supercoiled structure on DNA, as is observed experimentally, because only the tetramer can mimic a screw dislocation and thereby complement the screw symmetry of the DNA supercoil. (iv) The two slightly lysine-rich histones H2A and H2B are probably responsible, each in a different way, for dividing the eukaryotic chromatin fiber into discrete subunits. (v) The proposed arrangement of four distinct proteins appears to be a minimum formal requirement for making nucleosomes; that is, for introducing regularly spaced supercoiled DNA folds without also allowing formation of an indefinitely long (and genetically inert) DNA superhelix.     

DNA-dependent divalent cation binding in the nucleosome core particle

Sequence-specific binding of divalent cations to nucleosomal DNA can potentially influence nucleosome position and mobility, as well as modulate interactions with nuclear factors. We define the bonding and specificity of divalent cation interaction with nucleosomal DNA by characterizing Mn2+ binding in the x-ray structure of the nucleosome core particle at 1.9-A resolution. Manganese ions are found ligated with high occupancy in the major groove to 12 of 22 GG and GC base pair steps. The specific location and mode of metal binding is the consequence of unambiguous conformational differences between dinucleotide sites, owing to their sequence context and orientation in the nucleosome core.     

Primary organization of nucleosome core particle of chromatin: sequence of histone arrangement along DNA.

A high-resolution map for the arrangement of histones along DNA in the nucleosome core particle has been determined by a sequencing procedure based on crosslinking histones to the 5'-terminal DNA fragments produced by scission of one DNA strand at the point of crosslinking. The position of histones on DNA has been identified by measuring the length of crosslinked DNA fragments. The results demonstrate that each of the histones is arranged within several adjacent or dispersed DNA segments of a little less than 10 nucleotides in length. Histone-free intervals are located between these segments at the regular distances of about (10)n nucleotides from the 5' end of the DNA and are likely to face one side of the DNA helix. Histones appear to be arranged in a similar manner on both DNA strands and do not form "locks" around DNA. A linearized model of the core particle is proposed.     

Binding of ethidium bromide causes dissociation of the nucleosome core particle.

The binding of ethidium bromide to chicken erythrocyte core particles results in a step-wise dissociation of the structure that involves the initial release of one copy each of histones H2A and H2B. Quantitation of the dissociated DNA reveals that a critical amount of drug is required for the dissociation. Above the critical value, the dissociation is time dependent, reversible, and independent of DNA concentration.     

In vitro core particle and nucleosome assembly at physiological ionic strength.

Nucleosome core particles have been efficiently assembled in vitro by direct interaction of histones and DNA at physiological ionic strength, as assayed by digestion with DNases, supercoiling of relaxed circular DNA, and electron microscopy. Reconstitution was achieved either by the simultaneous addition of all core histones, or by the sequential binding of H3 . H4 tetramer and H2A . H2B dimer to DNA. Micrococcal nuclease digestion and electron microscopy studies indicated that there is heterogeneity in the spacings at which core particles are assembly on the DNA. Length measurements of oligomeric DNA produced during the course of the digestion suggest that the core histone octamer can organize 167 (+/- 4) rather than 145 base pairs of DNA, the extra 20 base pairs being quickly digested. Binding of histone H1 to core particles resulted in the protection of about 165 base pairs of DNA from nuclease attack. Because the core histone octamer is fully dissociated into H3 . H4 tetramer and H2A . H2B dimer at physiological ionic strength, our results would suggest that in vivo core particle assembly may also occur by interaction of these two complexes on the nascent DNA.     

DNA and DNA polymerase in the core of the Dane particle of hepatitis B.

The core of the Dane particle was shown to contain a DNA polymerase and a circular double stranded DNA with a molecular weight of 1.6 X 10(6) daltons which served as the primer-template for the enzyme. The product of the DNA polymerase reaction was in a base paired form and was covalently attached to the circular DNA. Neither the circular DNA nor the attached DNA product of the enzyme reaction was attacked by the DNase or released from intact cores until the cores were disrupted with sodium dodecyl sulfate, suggesting that they are internal components of the core. The DNA polymerase is a specific marker for Dane particles and can be used to distinguish sera with high and low concentrations of Dane particles. The DNA polymerase reaction can also be used to radiolabel Dane particle cores for a specific and sensitive radioimmunoprecipitation assay for antibody against the hepatitis B core antigen (anti-HBc).     

Internal motions in DNA.

We have measured the 31P and 1H NMR parameters of fractionated double-stranded DNA fragments 600, 300, and 150 base pairs long. The results indicate that, inside an intact double helix, both the deoxyribose and the sugar-phosphate backbone of the helix fluctuate substantially from their equilibrium geometry. The time constant for those coupled motions is of the order of 1 nsec.     

Rapid DNA-protein cross-linking and strand scission by an abasic site in a nucleosome core particle

Apurinic/apyrimidinic (AP) sites are ubiquitous DNA lesions that are highly mutagenic and cytotoxic if not repaired. In addition, clusters of two or more abasic lesions within one to two turns of DNA, a hallmark of ionizing radiation, are repaired much less efficiently and thus present greater mutagenic potential. Abasic sites are chemically labile, but naked DNA containing them undergoes strand scission slowly with a half-life on the order of weeks. We find that independently generated AP sites within nucleosome core particles are highly destabilized, with strand scission occurring ～60-fold more rapidly than in naked DNA. The majority of core particles containing single AP lesions accumulate DNA-protein cross-links, which persist following strand scission. The N-terminal region of histone protein H4 contributes significantly to DNA-protein cross-links and strand scission when AP sites are produced approximately 1.5 helical turns from the nucleosome dyad, which is a known hot spot for nucleosomal DNA damage. Reaction rates for AP sites at two positions within this region differ by ～4-fold. However, the strand scission of the slowest reacting AP site is accelerated when it is part of a repair resistant bistranded lesion composed of two AP sites, resulting in rapid formation of double strand breaks in high yields. Multiple lysine residues within a single H4 protein catalyze double strand cleavage through a mechanism believed to involve a templating effect. These results show that AP sites within the nucleosome produce significant amounts of DNA-protein cross-links and generate double strand breaks, the most deleterious form of DNA damage.     

Human immunodeficiency virus integrase directs integration to sites of severe DNA distortion within the nucleosome core.

We have examined the consequences of DNA distortion and specific histone-DNA contacts within the nucleosome for integration mediated by the human immunodeficiency virus (HIV)-encoded integrase enzyme. We find that sites of high-frequency integration cluster in the most severely deformed, kinked DNA regions within the nucleosome core. This may reflect either a preference for a wide major groove for association of the integrase or a requirement for target DNA distortion in the DNA strand transfer mechanism. Both the distortion and folding of the target DNA through packaging into nucleosomes may influence the selection of HIV integration sites within the chromosome.     

Crosslinked histone octamer as a model of the nucleosome core.

When histones in chromatin core particles were crosslinked with dimethylsuberimidate, the resulting particles had properties closely similar to those of native core particles. A crosslinked octameric histone complex was isolated from these particles under nondenaturing conditions. Upon reaction with DNA, this octameric protein folded the DNA into a structure closely resembling that of native core particles as verified by various techniques; protein denaturants were necessary for reassociation. The histone octamer is useful as a model of the nucleosome protein core and for studying histone-DNA interactions that occur in nucleosomes.     

The structure of DNA in a nucleosome.

We describe the application of the hydroxyl radical footprinting technique to examine the histone-DNA interactions of a nucleosome that includes part of the 5S ribosomal RNA gene of Xenopus borealis. We establish that two distinct regions of DNA with different helical periodicities exist within the nucleosome and demonstrate a change in the helical periodicity of this DNA upon nucleosome formation. In particular, we find that on average the helical periodicity of DNA in this nucleosome is 10.18 +/- 0.05 base pairs per turn. The same DNA, when bound to a calcium phosphate surface, has a periodicity of 10.49 +/- 0.05 base pairs per turn, similar to that of random sequence DNA. Modulations in minor groove width within the naked DNA detected by the hydroxyl radical are maintained and exaggerated in nucleosomal DNA. These features correlate with regions in the DNA previously suggested to be important for nucleosome positioning.     

Histone contributions to the structure of DNA in the nucleosome.

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 approximately 10.7 base pairs per turn, while flanking regions have a periodicity of approximately 10.0 base pairs per turn. Removal of the histone tails does not change the hydroxyl radical cleavage pattern in either mixed- or 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.     

Ribonucleotide-induced helical alteration in DNA prevents nucleosome formation.

Several polynucleotides that assume an A-form helical structure in solution are unable to form nucleosomes. We attempted to establish a relationship between the ease of the A-form----B-form helix transition and ease of nucleosome formation by reconstituting nucleosomes using ribosubstituted DNA containing various levels of ribonucleotides. Instead we discovered that, when riboadenosine is substituted for deoxyriboadenosine, even one ribonucleotide per 125 base pairs of DNA reduces nucleosome formation and that DNA containing greater than 5% ribonucleotide is completely unable to form nucleosomes. Ribosubstituted DNA restriction fragments exhibited altered mobility on native 6% polyacrylamide gels, indicating an altered helical structure (probably bending). The effects on both nucleosome formation and gel mobility are nucleotide specific and are correlated, being greatest for riboadenosine and decreasing in the order riboadenosine greater than riboguanosine greater than ribocytosine. The results are consistent with the hypothesis that the rate of nucleosome formation can be drastically reduced by isolated local perturbations, such as kinking or bending, in the helical structure of DNA.     

UV-induced formation of pyrimidine dimers in nucleosome core DNA is strongly modulated with a period of 10.3 bases.

We have determined the distribution of the major UV-induced photoproducts in nucleosome core DNA using the 3'----5' exonuclease activity of T4 DNA polymerase, which has been shown to stop digestion immediately 3' to UV-induced pyrimidine dimers. This assay is extremely sensitive since all DNA fragments without photoproducts (background) are reduced to small oligonucleotides, which can be separated from those fragments containing photoproducts. The results show that the distribution of UV-induced photoproducts (primarily cyclobutane dipyrimidines) is not uniform throughout core DNA but displays a striking 10.3 (+/- 0.1) base periodicity. Furthermore, this characteristic distribution of photoproducts was obtained regardless of whether nucleosome core DNA was isolated from UV-irradiated intact chromatin fibers, histone H1-depleted chromatin fibers, isolated mononucleosomes, or cells in culture. The yield of pyrimidine dimers along the DNA seems to be modulated in a manner that reflects structural features of the nucleosome unit, possibly core histone-DNA interactions, since this pattern was not obtained for UV-irradiated core DNA either free in solution or bound tightly to calcium phosphate crystals. Based on their location relative to DNase I cutting sites, the sites of maximum pyrimidine dimer formation in core DNA mapped to positions where the phosphate backbone is farthest from the core histone surface. These results indicate that within the core region of nucleosomes, histone-DNA interactions significantly alter the quantum yield of cyclobutane dipyrimidines, possibly by restraining conformational changes in the DNA helix required for formation of these photoproducts.     

Reaction of nucleosome DNA with dimethyl sulfate.

We have measured the effect of the histones in the nucleosome core particle on methylation of purines in nucleosome DNA by dimethyl sulfate. By using 32P terminally labeled nucleosome cores, we have examined the pattern of strand cleavage at methylated sites in the nucleosome DNA and compared it to the pattern observed in histone-free DNA. We are unable to detect any significant difference between the reactivity of N7 of guanines in nucleosome DNA and of that in naked DNA, with the exception of a single site of enhanced reactivity at approximately nucleotide 62 from the 5' end of the nucleosome. Contrary to our expectation, there is no detectable periodic modulation of reactivity corresponding to the twist of the DNA on the nucleosome surface. We are able to place a low upper limit on the extent to which the histones of the nucleosome can protect N7 of guanine in the large groove. With somewhat less precision, we also conclude that the N3 of adenine in the small groove is largely unprotected. These results indicate that in nucleosome DNA the bases are nearly as accessible to solvent as they are in DNA free of protein.     

Changing nucleosome positions through modification of the DNA rotational information.

The effects of the rotational information of DNA in determining the in vitro localization of nucleosomal core particles (ncps) have been studied in the Saccharomyces cerevisiae 5S rRNA repeat gene. We have altered the distribution of the phased series of flexibility signals present on this DNA by inserting a 25-bp tract, and we have analyzed the effects of this mutation on the distribution and on the frequencies of ncps, as compared with the wild type and a reference 21-bp insertion mutant. The variation of the standard free energy of nucleosome reconstitution was determined. The results show that the DNA rotational information is a major determinant of ncps positioning, define how many rotationally phased signals are required for the formation of a stable particle, and teach how to modify their distribution through the alteration of the rotational signals.     

Nucleosome core particles suppress the thermal untwisting of core DNA and adjacent linker DNA.

Covalently closed circular DNA is known to undergo a temperature-dependent change in helical twist. We have reconstituted nucleosome core particles onto closed circular DNA and measured the thermal untwisting of the DNA as a function of nucleosome density. The results demonstrate that the DNA associated with the nucleosome core particle does not alter its twist when the temperature is varied between 4 degrees C and 37 degrees C, and that the length of DNA prevented from thermal untwisting includes the linker as well as the core DNA.     

Observations on the core particle of hepatitis B virus and the DNA polymerase associated with hepatitis B antigen.

Several methods are presented for the purification of core particles of hepatitis B virus (HBV) from nuclei of infected human hepatocytes. No endogenous DNA polymerase activity was found in any of the preparations of core particles even when circular double and single stranded DNAs were used as exogenous templates. The DNA polymerase activity associated with serum HB Ag was not stimulated by circular DNAs. Sodium dodecyl sulfate (SDS) at concentrations of greater than or equal to 0.1% inhibited the DNA polymerase activity of serum HB Ag. Exogenous templates such as native and activated calf thymus and Micrococcus lysodeikticus DNAs did not stimulate the DNA polymerase of serum HB Ag even in the presence of low concentrations of SDS. It is suggested that the DNA polymerase associated the HB Ag is specific for its own DNA as template.