Poly[d(A.T)] and other synthetic polydeoxynucleotides containing oligoadenosine tracts form nucleosomes easily.

Synthetic double-stranded polydeoxynucleotides of the general form poly[d(AnT).d(ATn)], with n ranging from 3 to 11, have been synthesized. The conformation of the polymers was investigated by circular dichroism spectroscopy and the polymers were examined for their ability to form nucleosomes. Although spectra show that a circular dichroism band characteristic of poly[d(A.T)] appears in the polymer family for n greater than 7, we demonstrate that even polynucleotides with the longest tracts of contiguous adenosine bases (n = 11) are able to form nucleosomes when reconstituted using a histone exchange procedure. Thus resistance to nucleosome formation does not coincide with the appearance of features similar to that of poly[d(A.T)] over the bulk of the nucleosomal DNA. Furthermore, we show that an approximately 150 base-pair poly[d(A.T)] itself, long thought to be refractory to nucleosome formation, can assemble into such a protein-DNA complex when reconstituted by a low-salt exchange procedure. Competitive assays show that the homopolymer reconstitutes about as well as heterogeneous sequences DNA. Our work, therefore, suggests that highly adenosine-rich sequences in vivo apparently have a function that operates at a level other than that of nucleosome structure.     

Nucleosomes will not form on double-stranded RNa or over poly(dA).poly(dT) tracts in recombinant DNA.

We have been unable to "force" double-stranded RNA to fold into nucleosome-like structures using several different histone-RNA "reconstitution" procedures. Even if the histones are first stabilized in octameric form by dimethylsuberimidate cross-linking they are still unable to form specific complexes with the RNA. Moreover double-stranded RNA is unable to induce histones to assemble into octamers although we confirm that the non-nucleic acid homopolymer, polyglutamic acid, has this ability. We have also determined, using pyrimidine tract analysis, that nucleosomes will not form over a sufficiently long segment of poly(dA).poly(dT) in a recombinant DNA molecule. Thus nucleosomes cannot fold DNA containing an 80 base pair poly(dA).poly(dT) segment but a 20 base pair segment can be accommodated in nucleosomes fairly well. Segments of intermediate length can be accommodated but are clearly selected against. Poly(dA).poly(dT) differs only slightly from natural DNA in helix structure. Therefore either this homopolymer resists folding, or nucleosomes are very exacting in the nucleic acid steroid parameters they will tolerate. Such constraints may be relevant to nucleosome positioning in chromatin.     

Poly(dA).poly(dT) rich sequences are not sufficient to exclude nucleosome formation in a constitutive yeast promoter.

It was suggested that poly(dA).poly(dT) rich sequences in yeast Saccharomyces cerevisiae act as elements of constitutive promoters by exclusion of nucleosomes (Struhl, K. (1985). Proc. Natl. Acad. Sci. USA 82, 8419-8423). We have mapped the chromatin structure of the pet56-his3-ded1 region in minichromosomes and show that the poly(dA).poly(dT) sequences are located in nuclease sensitive regions. DNA fragments from the nuclease sensitive promoter region of DED1 were used for nucleosome reconstitution in vitro. We show that all sequences can form nucleosome cores and that the poly(dA).poly(dT) sequence can be incorporated in nucleosome cores. The results suggest that the nuclease sensitivity found in vivo is not established by poly(dA).poly(dT) mediated exclusion of nucleosomes.     

Development of a fluorescent probe for the study of nucleosome assembly and dynamics

To develop a probe for use in real-time dynamic studies of nucleosomes, core histones (from Drosophila) were conjugated to a DNA-intercalating dye, thiazole orange, by a reaction targeting Cys 110 of histone H3. In the absence of DNA, the conjugated histones are only very weakly fluorescent. However, upon reconstitution into nucleosomes by standard salt dialysis procedures, the probe fluoresces strongly, reflecting its ability to intercalate into the nucleosomal DNA. The probe is also sensitive to the nature of the DNA-histone interaction. Nucleosomes reconstituted by stepwise salt dialysis give a fluorescence signal quite different from that of the species formed when DNA and histones are simply mixed in low salt. In addition, changing either the DNA length or the type of sequence (nucleosome positioning sequences versus random DNA of the same size) used in the reconstitution alters the resulting fluorescence yield. The results are all consistent with the conclusion that a more rigid, less flexible nucleosome structure results in less fluorescence than a looser structure, presumably due to structural constraints on dye intercalation. This probe should be well suited to analyzing nucleosome dynamics and to following factor-mediated assembly and remodeling of nucleosomes in real time, particularly at the single-molecule level.     

The structure of chromatin from the pigeon brain. II. Sedimentation analysis of oligonucleosome density

Compaction of pigeon brain and rat thymus chromatin differing in the length of the linker DNA has been studied by the method of velocity sedimentation. The dependence of sedimentation coefficients of oligonucleosomes on the number of nucleosomes in the chain in solution of different ionic strength (0.005-0.085) has been analyzed. The analyses of these dependences showed that the structure of oligonucleosomes of both cell types at low ionic conditions may be described by the model of a zig-zag-shaped nucleosomal chain. The process of compaction of the oligonucleosomes at higher ionic strength (0.045-0.085) proceeds similarly for brain and thymus chromatin. The formation of a superhelical structure is determined by the interaction of no less than 6 nucleosomes; the compactness of the structure is significantly increased when the number of nucleosomes in the chain exceeds 10. The ability of the brain oligonucleosomes to form a compact structure despite the short linker allow the suggestion that in brain short chromatin the DNA chain does not form two complete turns in the nucleosome. This provides necessary flexibility of brain chromatin.     

Eukaryotic DNA does not form nucleosomes as readily as some prokaryotic DNA.

Nucleosomal-length DNA was prepared from the genomic DNA of various prokaryotic and eukaryotic organisms by limited nuclease digestion after reconstitution with core histones. The DNAs ranged in base composition from 26.5% to 72% guanosine-plus-cytosine (%GC). The nucleosomal-length DNAs were then used in a competitive reconstitution assay in order to quantitatively determine their relative abilities to form nucleosomes. The results of the assay indicate a linear dependence of the free energy of nucleosome formation on base composition and, surprisingly, show that several prokaryotic DNAs form nucleosomes as well as or better than eukaryotic DNAs.     

Effect of DNA length on the nucleosome low salt transition.

The effect of DNA length on the low salt unfolding transition of nucleosomes has been studied by the use of fluorescently labeled histones. Nucleosomes were formed by the reconstitution of bulk DNA fragments averaging 173 and 250 base pairs in length. These nucleosomes exhibited a conformational change in a transition centered at about 7 mM ionic strength, very different from that observed for the standard 145 bp nucleosomes (1-3mM). In addition, the conformational change of the 173 and 250 bp nucleosomes involves twice as many ions as that of the 145 bp nucleosomes.     

Limitations of the poly(glutamic acid) reconstitution method in the reassembly of mono- and dinucleosomes

Reconstitution of mononucleosomes and dinucleosomes at physiological ionic strength by means of poly(glutamic acid) is not efficient at physiological histone octamer:DNA ratios, unlike that with the salt dialysis method. The shorter the DNA is, the less transfer of octamers from poly(glutamic acid) to DNA occurs. By increasing the octamer:DNA ratio it is possible to involve all the DNA in the assembly, but for DNA longer than core particle length, nucleoprotein particles containing extra histones are concomitantly generated. Except for core particle and chromatosome lengths of DNA reassembled at 0.6:1 or 1:1 octamer:DNA ratio (and thus with low yield), reconstituted nucleoprotein particles proved to be different from native nucleosomes by their insolubility upon isolation. In the aggregates, DNA ends seemed to be sufficiently loose to allow exonuclease III digestion up to a certain limit. This resulted in patterns that for some cloned DNA fragments could give the impression, without knowledge of the above, of resulting from a unique octamer position. In view of the small range of length of DNA and the low yield of faithful reconstitution, the assembly method using poly(glutamic acid) is only of limited use in mono- or dinucleosome reconstitution experiments, at least in our hands.     

Effects of DNA sequence and histone-histone interactions on nucleosome placement

Using competitive reconstitution, we have refined the parameters for the binding of histone octamers to artificial nucleosome-positioning sequences of the form: (A/T3nn(G/C)3nn. We find that the optimal period between flexible segments is approximately 10.1 base-pairs, supporting the view that the DNA on the nucleosome surface is overwound. The strongest requirement for flexible DNA is near the protein dyad. However, we see no indication of changes in DNA helical repeat in this region. Using a series of repetitive sequences, we confirm that neither all A/T-rich nor all G/C-rich regions are identical in promoting nucleosome formation. Surprisingly, A/T-rich segments containing the TpA step, subject to purine-purine clash in the minor groove, favor nucleosome formation over sequences lacking this step. Short tracts of adenine residues are found to position on the histone surface like other A/T-rich regions, in the manner predicted by the direction of their sequence-directed bends as determined by electrophoretic methods. Tracts containing five adenine residues are extremely aniostropic in their flexibility and are strongly detrimental to nucleosome formation when positioned for major groove compression. Longer adenine tracts are found to position near the ends of the nucleosomal DNA. However, other positions may be occupied by an A12 tract, with only a minor penalty in the free energy of nucleosome formation. Overall, reconstituted nucleosome positions are translationally degenerate, suggesting a weak dependence on DNA flexibility for nucleosome positioning. Dinucleosomal reconstitutions on tandem dimers of the 5 S RNA gene of Lytechinus variegatus demonstrate a weak phasing dependence for the interaction between nucleosomes. This interaction is maximal for the 202 base-pair repeat and suggests a co-operative mechanism for the formation of ordered nucleosomal arrays based on a combination of DNA flexibility and nucleosome-nucleosome interactions.     

Oligopurine.oligopyrimidine tracts do not have the same conformation as analogous polypurine.polypyrimidines

The ability of tracts of synthetic oligopurine.oligopyrimidines containing both adenosine and guanosine residues to approach the conformation of analogous polypurine.polypyrimidines has been examined as a function of tract length by CD spectroscopy. Tracts of up to 19 contiguous, alternating dA and dG residues yield CD spectra that are distinctly different from that of the analogous alternating polymer. Thus the structural changes reflected in the unusual CD spectrum of poly[d(AG)].poly[d(CT)] must require even longer tract lengths. Tracts of contiguous adenosines flanked by guanosine residues were seen to approach the CD spectrum of poly[dA].poly[dT] quite slowly as a function of tract length, requiring more than 24 contiguous adenosines to give CD spectra similar to the homopolymer. These results lead us to the conclusion that oligopurine tracts in vivo are not well modeled by synthetic polypurine.polypyrimidines with one or two base pair repeating units.     

Nucleosomes structure and dynamics: effect of CHAPS

Dynamics of nucleosomes and spontaneous unwrapping of DNA are fundamental property of the chromatin enabling access to nucleosomal DNA for regulatory proteins. Probing of such dynamics of nucleosomes performed by single molecule techniques revealed a large scale dynamics of nucleosomes including their spontaneous unwrapping. Dissociation of nucleosomes at low concentrations is a complicating issue for studies with single molecule techniques. In this paper, we tested the ability of 3-[(3-Cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS) to prevent dissociation of nucleosomes. The study was performed with mononucleosome system assembled with human histones H2A, H2B, H3 and H4 on the DNA substrate containing sequence 601 that provides the sequencespecific assembly of nucleosomes. We used Atomic Force Microscopy (AFM) to directly identify nucleosomes and analyze their structure at the nanometer level. These studies showed that in the presence of CHAPS at millimolar concentrations, nucleosomes, even at sub-nanomolar concentrations, remain intact over days compared to a complete dissociation of the same nucleosome sample over 10 min in the absence of CHAPS. Importantly, CHAPS does not change the conformation of nucleosomes as confirmed by the AFM analysis. Moreover, 16 µM CHAPS stabilizes nucleosomes in over one hour incubation in the solution containing as low as 0.4 nM in nucleosomes. The stability of nucleosomes is slightly reduced at physiological conditions (150 mM NaCl), although the nucleosomes dissociate rapidly at 300 mM NaCl. The sequence specificity of the nucleosome in the presence of CHAPS decreased suggesting that the histone core translocates along the DNA substrate utilizing sliding mechanism.     

Recombination between poly[d(GT).d(CA)] sequences in simian virus 40-infected cultured cells.

CVI cells were transfected with oversized simian virus 40 (SV40) genomes that could be reduced to packageable size by alternative homologous recombination pathways involving either two polydeoxyguanylic-thymidylic acid X polydeoxycytidylic-adenylic acid (poly[d(GT).d(CA)]; abbreviated hereafter as poly(GT)] tracts or two tracts of homologous SV40 sequence. Plaque-forming viruses rescued by this procedure were found to contain genomes formed by homologous and nonhomologous recombination events. Half of the viable viral DNA molecules recovered were the result of recombination between two tracts of poly(GT). Approximately 20% of the rescued viral genomes were produced by homologous recombination between tracts of SV40 DNA. Nonhomologous recombination involving SV40 sequences was also a major pathway of deletion, producing ca. 30% of the viral plaques. Tracts of poly(GT) generated by recombination were variable in length, suggesting that recombination between poly(GT) tracts was usually unequal. On a per-nucleotide basis, poly(GT) recombination occurred eight times more frequently than did recombination between homologous SV40 DNA. This eightfold difference is the maximum recombinatory enhancement attributable to poly(GT) sequences. Although DNA sequence analysis showed that tracts of poly(GT) generated by recombination retained the alternating G-T repeat motif throughout their length, the contribution of the nonhomologous pathway to poly(GT) recombination cannot be ruled out, and the relative proclivity of a given length of d(GT).d(CA) sequence to undergo homologous recombination is probably less than eight times greater than that of an SV40 sequence of the same length.     

Assembly and characterization of nucleosomal cores on B- vs. Z-form DNA

The ability of right- vs. left-handed alternating purine/pyrimidine copolymers to support the formation of nucleosomes has been examined by using a trout testis assembly factor. The protein, which is thermostable, has a molecular weight of 29000 and will assemble nucleosomes onto both SV40 and calf thymus DNA. This assembly factor has been used to assemble nucleosomes onto the B and Z conformations of poly[d(Gm5C)] and the B conformation of poly[d(GC)]. The isolated B-form particles, which sediment at approximately 11 S in a sucrose density gradient, contain DNA of 140-200 bases in length and the four core histones. The isolated Z-form particles, which also sediment at approximately 11 S, contain the four core histones and DNA of 170-250 bases in length. Physical analysis of the particles by absorbance and circular dichroic spectroscopy indicates that the DNA remains in the original conformation throughout the isolation procedure. Further, the particles reconstituted onto left-handed DNA compete effectively for an anti-Z DNA antibody, while the corresponding right-handed particles do not. Analytical sedimentation velocity determinations indicate that the B-form poly[d(Gm5C)] and poly[d(GC)] particles sediment at 11.2 and 11.1 S, respectively. In contrast, the poly[d(Gm5C)] Z-form particles have an S20,w of 10.6 S. The differences in the sedimentation velocity and the density of the cores, and in the lengths of DNA associated with the particles, suggest that the conformation of the DNA affects the manner in which it associates with the histone octamer.