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.     

Nucleosome cores reconstituted from poly (dA-dT) and the octamer of histones.

In this paper we describe a detailed investigation of the reconstitution of nucleosome cores from poly (dA-dT) and the octamer of histones. We also attempted the reconstitution from the copolymers poly dA.poly dT, poly dG.poly dC and poly (dG-dC). The repeat of the reconstituted chromatin fibre is discussed. The micrococcal nuclease released poly (dA-dT) core particle is found to contain a considerably narrower DNA size distribution that of the native random DNA nucleosome core (12). In addition we have succeeded in obtaining small crystals of the poly (dA-dT) nucleosome core. The DNAase I digestion pattern of the poly (dA-dT) containing nucleosome core is presented. The periodicity of DNAase I cutting sites is found to be about 10.5 bases and is similar to that of the native nucleosome core (12, 13).     

Homopolymer tract organization in the human malarial parasite Plasmodium falciparum and related Apicomplexan parasites

Background

Homopolymeric tracts, particularly poly dA.dT, are enriched within the intergenic sequences of eukaryotic genomes where they appear to act as intrinsic regulators of nucleosome positioning. A previous study of the incomplete genome of the human malarial parasite Plasmodium falciparum reports a higher than expected enrichment of poly dA.dT tracts, far above that anticipated even in this highly AT rich genome. Here we report an analysis of the relative frequency, length and spatial arrangement of homopolymer tracts for the complete P. falciparum genome, extending this analysis to twelve additional genomes of Apicomplexan parasites important to human and animal health. In addition, using nucleosome-positioning data available for P. falciparum, we explore the correlation of poly dA.dT tracts with nucleosome-positioning data over key expression landmarks within intergenic regions.

Results

We describe three apparent lineage-specific patterns of homopolymeric tract organization within the intergenic regions of these Apicomplexan parasites. Moreover, a striking pattern of enrichment of overly long poly dA.dT tracts in the intergenic regions of Plasmodium spp. uniquely extends into protein coding sequences. There is a conserved spatial arrangement of poly dA.dT immediately flanking open reading frames and over predicted core promoter sites. These key landmarks are all relatively depleted in nucleosomes in P. falciparum, as would be expected for poly dA.dT acting as nucleosome exclusion sequences.

Conclusions

Previous comparative studies of homopolymer tract organization emphasize evolutionary diversity; this is the first report of such an analysis within a single phylum. Our data provide insights into the evolution of homopolymeric tracts and the selective pressures at play in their maintenance and expansion.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-848) contains supplementary material, which is available to authorized users.     

Unique poly(dA).poly(dT) B'-conformation in cellular and synthetic DNAs

Poly(dA).poly(dT), but not B-form DNA, is specifically recognized by experimentally induced anti-kinetoplast or anti-poly(dA).poly(dT) immunoglobulins. Antibody binding is completely competed by poly(dA).poly(dT) and poly(dA).poly(dU) but not by other single- or double-stranded DNA sequences in a right-handed B-form. Antibody interaction with poly(dA).poly(dT) depends on immunoglobulin concentration, incubation time and temperature, and is sensitive to elevated ionic strengths. Similar conformations, for example, (dA)4-6 X (dT)4-6, in the kinetoplast DNA of the parasite Leishmania tarentolae are also immunogenic and induce specific anti-poly(dA).poly(dT) antibodies. These antibody probes specifically recognize nuclear and kinetoplast DNA in fixed flagellated kinetoplastid cells as evidenced by immunofluorescence microscopy. Anti-poly(dA).poly(dT) immunofluorescence is DNase-sensitive and competed by poly(dA).poly(dT), but not other classical double-stranded B-DNAs. Thus, these unique cellular B'-DNA helices are immunogenic and structurally similar to synthetic poly(dA).poly(dT) helices in solution.     

A correlation between nucleosome spacer region susceptibility to DNase I and histone acetylation.

Hepatoma tissue culture (HTC) cell nuclei were digested with either DNase I or micrococcal nuclease and the nucleohistone digestion products fractionated by gel electrophoresis or exclusion chromatography. Under appropriate conditions, gel electrophoresis demonstrates that for both nucleases, only cleavages within the nucleosome spacer regions and not within the nucleosome core lead to freely migrating nucleohistone particles. These particles consist of nucleosome cores, nucleosomes and nucleosome oligomers. Following DNase I digestion and fractionation by exclusion chromatography, analysis of the histones indicates a direct relationship between increased spacer region susceptibility to nuclease and increased nucleosomal histone acetylation. Evidently digestion sites outside the regions of DNA protected by core histones can reflect the degree of acetylation of core histones. Such a relationship is not found when micrococcal nuclease is used to digest the samples.     

Complete disproportionation of duplex poly(dT)*poly(dA) into triplex poly(dT)*poly(dA)*poly(dT) and poly(dA) by coralyne

Coralyne is a small crescent-shaped molecule known to intercalate duplex and triplex DNA. We report that coralyne can cause the complete and irreversible disproportionation of duplex poly(dT)·poly(dA). That is, coralyne causes the strands of duplex poly(dT)·poly(dA) to repartition into equal molar equivalents of triplex poly(dT)·poly(dA)·poly(dT) and poly(dA). Poly(dT)·poly(dA) will remain as a duplex for months after the addition of coralyne, if the sample is maintained at 4°C. However, disproportionation readily occurs upon heating above 35°C and is not reversed by subsequent cooling. A titration of poly(dT)·poly(dA) with coralyne reveals that disproportionation is favored by as little as one molar equivalent of coralyne per eight base pairs of initial duplex. We have also found that poly(dA) forms a self-structure in the presence of coralyne with a melting temperature of 47°C, for the conditions of our study. This poly(dA) self-structure binds coralyne with an affinity that is comparable with that of triplex poly(dT)·poly(dA)·poly(dT). A Job plot analysis reveals that the maximum level of poly(dA) self-structure intercalation is 0.25 coralyne molecules per adenine base. This conforms to the nearest neighbor exclusion principle for a poly(dA) duplex structure with A·A base pairs. We propose that duplex disproportionation by coralyne is promoted by both the triplex and the poly(dA) self-structure having binding constants for coralyne that are greater than that of duplex poly(dT)·poly(dA).     

The Flexibility of Alternating dA—dT Sequences

Abstract

The flexibility of alternating poly (dA—dT) has been investigated by the technique of transient electric dichroism. Rotational relaxation times, which are very sensitive to changes in the end-to-end length of flexible polymers, are determined from the field free dichroism decay curves of four, well defined fragments of poly (dA—dT) ranging in size from 136 to 270 base pairs. Persistence lengths, calculated from the results of Hagerman and Zimm (Biopolymers (1981) 29, 1481–1502), are in the range 200–250 A. This makes alternating dA—dT sequences about twice as flexible as naturally occurring, “random” sequence DNA. Considering a bend around a nucleosome, for example, this difference in persistence length translates to an energy difference between poly (dA—dT) and random sequence DNA of 0. 17 kT/base pair or 1 kcal per 10 base pair stretch. This energy difference is sufficiently large to suggest that dA—dT sequences could serve as markers in DNA packaging, for example, at sites where DNA must tightly bend to accommodate structures.     

Two nuclear DNA binding proteins of Dictyostelium discoideum with a high affinity for poly(dA)-poly(dT).

Intergenic regions of the Dictyostelium genome contain an extremely high proportion of AT base pairs. Those intergenic regions which have been subjected to nucleotide sequence analysis are predominantly composed of alternating runs of poly(dA) and poly(dT) and there is evidence to suggest that nucleosomes do not form on such sequences. We have identified two nuclear proteins, of molecular weight 70,000 and 74,000 daltons, which bind only to intergenic regions of a cloned Dictyostelium gene. Binding is specifically inhibited in the presence of synthetic poly(dA) - poly (dT) as competitor. These proteins may play some role in the chromosomal organization of intergenic regions in Dictyostelium discoideum.     

Localization of curved DNA and its association with nucleosome phasing in the promoter region of the human estrogen receptor alpha gene

We determined DNA bend sites in the promoter region of the human estrogen receptor (ER) gene by the circular permutation assay. A total of five sites (ERB-4 to -1, and ERB+1) mapped in the 3 kb region showed an average distance of 688 bp. Most of the sites were accompanied by short poly(dA) x poly(dT) tracts including the potential bend core sequence A2N8A2N8A2 (A/A/A). Fine mapping of the ERB-2 site indicated that this A/A/A and the 20 bp immediate flanking sequence containing one half of the estrogen response element were the sites of DNA curvature. All of the experimentally mapped bend sites corresponded to the positions of DNA curvature as well as to nucleosomes predicted by computer analysis. In vitro nucleosome mapping at ERB-2 revealed that the bend center was located 10-30 bp from the experimental and predicted nucleosome dyad axes.     

Inhibition of DNA replication by berenil in plasmids containing Poly(dA)poly(dT) sequences

The effect of berenil on plasmid DNA replication was studied on pBR322-derived plasmids containing poly(dA)poly(dT) sequences. In comparison to the parental plasmid pBR322, plasmid pKH47 harboring 100 bp of poly(dA)poly(dT) at the PvuII site showed a decrease in plasmid yield in the presence of berenil. This effect was also observed in pVL26, a related plasmid in which the location of the poly(dA)poly(dT) region had been shifted to the EcoRV site in pBR322. [(3)H]Thymidine incorporation experiments indicated that DNA synthesis may be affected in these plasmids in the presence of the drug. Bromodeoxyuridine incorporation experiments coupled to Cs(2)SO(4) equilibrium density gradient centrifugation indicated that the lower plasmid yield was due to an inhibition of DNA replication by berenil. We have also found that berenil induces DNA degradation in plasmids containing the homopolymer. Our studies strongly suggest that the effect of berenil on plasmid replication and DNA stability results from its binding to the poly(dA)poly(dT) region present in these plasmids. Moreover, we have found a correlation between the position of the poly(dA)poly(dT) region and this inhibitory effect. Thus, plasmid pKH47, containing the poly(dA)poly(dT) region most proximal to the origin of pBR322 replication, was most severely affected.     

Evidence for long poly(dA).poly(dT) tracts in D. discoideum DNA at high frequencies and their preferential avoidance of nucleosomal DNA core regions

The eukaryote, Dictyostelium discoideum, has one of the most (A+T) rich genomes studied to date. Isolated nuclear D. discoideum DNA (AX3 strain) was used to qualitatively determine the frequency and length distribution of long (dA).(dT) homopolymer tracts in this genome, in comparison to the less (A+T) rich calf thymus and Schistosoma mansoni DNAs that had few observable long tracts. These experimental data accurately reflect the significantly elevated frequencies of long tracts found computationally within the D. discoideum intron and flanking sequences, but not exons. PCR amplification of long (dA).(dT) homopolymer tract containing sequences was carried out. Then experimental biotinylated (dT)18 probe hybridization to the PCR amplified DNA showed that the long (dA).(dT) homopolymer tracts were enriched in D. discoideum sequences only hundreds of base pair in length, under conditions where no equivalent hybridization was observed to S. mansoni DNA or calf DNA sequences. Similar probe hybridization to DNA isolated following micrococcal nuclease digestion of D. discoideum chromatin demonstrated that long (dA).(dT) homopolymer tracts were more highly enriched in nucleosomal DNA lengths that included the internucleosomal linker as compared to shorter linker free mononucleosomal lengths. This observation is in agreement with the frequency of tract spacing results calculated from GenBank sequence data. These frequency data indicate that adjacent long tracts plus the intervening spacer DNA are found at peak lengths (average 42 bp), exactly characteristic of the internucleosomal spacer region of D. discoideum chromatin and are in sufficient number to be found in nearly half of all nucleosomes. Compared to shuffled tract sequence controls, these lengths of adjacent long tracts plus the intervening spacer DNA were found to be significantly enriched. Lesser enrichments are observed at lengths corresponding to adjacent tracts being separated by nucleosomal core length DNA sequences (145-185 bp). These data strongly suggest that adjacent long tracts occur spaced at selected lengths so as to avoid the central core regions of nucleosomes and instead are found localized within internucleosomal DNA linker and core edge regions in D. discoideum chromatin.