Negative constrained DNA supercoiling in archaeal nucleosomes

Archaeal histones have significant sequence and structural similarity to their eukaryal counterparts. However, whereas DNA is wrapped in negatively constrained supercoils in eukaryal nucleosomes, it has been reported that DNA is positively supercoiled by archaeal nucleosomes. This was inferred from experiments performed at low temperature and low salt concentrations, conditions markedly different from those expected for many archaea in vivo. Here, we report that the archaeal histones HMf and HTz wrap DNA in negatively constrained supercoils in buffers containing potassium glutamate (K-Glu) above 300 mM, either at 37 degrees C or at 70 degrees C. This suggests that high salt concentrations allow an alternate archaeal nucleosome topology: a left-handed tetramer rather than the right-handed tetramer seen in low salt conditions. In contrast, the archaeal histone MkaH produces DNA negative supercoiling at all salt concentrations, suggesting that this duality of structure is not possible for this atypical protein, which is formed by the association of two histone folds in a single polypeptide. These results extend the already remarkable similarity between archaeal and eukaryal nucleosomes, as it has been recently shown that DNA can be wrapped into either positive or negative supercoils around the H3/H4 tetramer. Negative supercoiling could correspond to the predominant physiological mode of DNA supercoiling in archaeal nucleosomes.     

DNA associated with nucleosomes in plants.

50 to 55% of tobacco and barley nuclear DNA is accessible to micrococcal endonuclease digestion. The DNA fragments resulting from a mild endonuclease treatment are multiples of a basic unit of 194 +/- 6 base pairs in tobacco and 195 +/- 6 base pairs in barley. After extensive digestion, a DNA fragment of approximately 140 base pairs is predominant. Hence the "extra-core" or "linker"-DNA is 55 base pairs long. Other fragments having 158 and less than 140 base pairs are present as well. Treatment with DNase I results in multiples of 10 bases when analysed under denaturating conditions. These results show that the general organization of the DNA within the nucleosomes is about the same in higher plants as in other higher eukaryotes.     

Polyamine-induced hydrolysis of apurinic sites in DNA and nucleosomes.

The ability of different polyamines to catalyze hydrolysis of phosphodiester linkages in apurinic and apyrimidinic (AP) sites has been investigated in supercoiled, relaxed and denatured DNA, and also in core and chromatosome particles. The rate constants for the hydrolysis in the DNAs have been determined. In general the order of effectiveness of the polyamines were: spermine greater than spermidine greater than putrescine greater than cadaverine. A 9 fold difference in rate constants was found between spermine and cadaverine. No difference in the rate of hydrolysis was seen between AP-sites in supercoiled and relaxed DNAs, whereas the rate for the single-stranded DNA and DNA in core and chromatosome particles was only half of that in the double-stranded DNA. All AP-sites in both free DNA and DNA-histone particles were hydrolyzed in the presence of polyamines. For all polyamines, with the exception of spermine, increasing concentration of both Mg++ and salts such as KCl both led to a large decrease in the rate of polyamine-induced hydrolysis of AP-sites. The rate of hydrolysis increased markedly with increasing pH in the pH range pH 6 - pH 11.     

Photochemical cross-linking of histones to DNA nucleosomes.

Ultraviolet (UV)-induced cross-linking was utilized in order to identify histone-DNA interacting regions in the chromatin repeating unit. Fractionated mononucleosomes which contained 185 base pairs of DNA and a full complement of the histones, including histone H1, were irradiated with light of lambda greater than 290nm in the presence of a photosensitizer. Equimolar amounts of histones H2A and H2B were found, by two independent labeling experiments, to be cross-linked to the DNA. Based on previous finding that the UV irradiation specifically cross-links residues which are in close proximity, irrespective of the nature of the amino acid side chain or the nucleotide involved, our results indicate that the four core histones are not positioned equivalently with respect to the DNA. This arrangement allows histones H2A and H2B to preferentially cross-link to the DNA. A water soluble covalent complex of DNA and histones was isolated. This complex was partially resistant to mild nuclease digestion, it exhibited a CD spectrum similar to that of chromatin, and was found to contain histone H1. These results are compatible with a model which suggests that histone H1, though anchored to the linker, is bound to the DNA at additional sites. By doing so it spans the whole length of the nucleosome and clamps together the DNA fold around the histone core.     

DNA wrapping in nucleosomes. The linking number problem re-examined.

Chromatin was assembled in vitro from relaxed closed circular DNA (SV40) and core histones at histone to DNA ratios of 0.2 to 0.3 (g/g) and incubated with topoisomerase I to relax supercoils in DNA regions not constrained by protein. Addition of histones H1 + H5 to the chromatin at an ionic strength of 0.1 M, in the presence of the solubilizing agent, polyglutamic acid, and topoisomerase I, increased the magnitude of the DNA linking number change, relative to protein-free DNA. No change in the linking number distribution occurred for relaxed protein-free DNA under these conditions. Control experiments indicated that the increase in the absolute value of the DNA linking number change in the chromatin could not be attributed to an increase in the number of nucleosomes per DNA molecule. These data suggest a solution to the linking number problem associated with models of chromatin structure.     

Anthracycline antibiotics. Interaction with DNA and nucleosomes and inhibition of DNA synthesis

We report studies of the interaction of four anthracycline antibiotics, iremycin (IM), daunomycin (DM), aclacinomycin A (AM), and violamycin B1 (VM), with naked DNA, nucleosomal core particles, and 175 base pair (bp) nucleosomes lacking histone H1. In all cases the binding strength increases in the order IM less than DM approximately AM less than VM. The binding substrates increased in affinity for the drugs in the following order: core particles less than 175-bp nucleosomes less than DNA. The apparent DNA length increment per drug bound decreases in the progression IM greater than DM greater than AM greater than VM, the same serial order as is characterized by increasing binding affinity. Dichroism amplitude measurements show that for all drugs the long-wavelength absorbance transition moment is tilted by 26-29 degrees relative to the plane perpendicular to the helix axis; this angle probably corresponds to the long axis tilt of the intercalated chromophore. Finally, it was found that the ability of the drugs to inhibit DNA synthesis by Escherichia coli DNA polymerase I increases in the same order as their binding affinity.     

Yeast nucleosomes allow thermal untwisting of DNA.

Thermal untwisting of DNA is suppressed in vitro in nucleosomes formed with chicken or monkey histones. In contrast, results obtained for the 2 micron plasmid in Saccharomyces cerevisiae are consistent with only 30% of the DNA being constrained from thermal untwisting in vivo. In this paper, we examine thermal untwisting of several plasmids in yeast cells, nuclei, and nuclear extracts. All show the same quantitative degree of thermal untwisting, indicating that this phenomenon is independent of DNA sequence. Highly purified yeast plasmid chromatin also shows a large degree of thermal untwisting, whereas circular chromatin reconstituted using chicken histones is restrained from thermal untwisting in yeast nuclear extracts. Thus, the difference in thermal untwisting between yeast chromatin and that assembled with chicken histones is most likely due to differences in the constituent histone proteins.     

Ion-induced DNA structure change in nucleosomes

Physical methods have been used to study calcium binding to the nucleosome core particle. Equilibrium dialysis of Ca2+ and spectroscopic analysis of a Ca2+ analogue show that the ion binds tightly to the particles, resulting in a significant change of DNA circular dichroism. This suggests that base stacking may be altered as a result of Ca2+ binding. In the presence of Ca2+, the absorbance and fluorescence properties of methylene blue (MB), a DNA-specific intercalator, confirm that the dye binds tightly to nucleosomes by intercalation. However, secondary changes occur which suggest that the MB binding site is altered as a result of Ca2+ binding. Triplet state anisotropy decay and triplet lifetime quenching both show that in the Ca2+-nucleosome complex, methylene blue is capable of wobbling over a substantial angular range at its binding site. To explain these data, it is proposed that Ca2+ binding to nucleosomes causes DNA to fold by means of a series of sharp bends (kinks). The properties of bound MB are best explained if it is presumed that the intercalator binds tightly to such kinked sites in the nucleosome. On the basis of these observations, we discuss the possibility that multivalent ion concentration in the nucleus is high enough that the smooth to kinked helix equilibrium may be near to its midpoint. Near such a midpoint, the secondary structure of DNA in the nucleosome might prove to be sensitive to effector molecule binding and to site-specific variation of DNA or histone composition within genes.     

Cooperativity in nucleosomes assembly on supercoiled pBR322 DNA.

Many studies have shown that in reconstituted chromatin model systems, containing only purified DNA and histone octamer, nucleosomes can adopt well defined locations with respect to DNA nucleotide sequence. Recently, nucleosome-nucleosome interactions were suggested as one of the factors underlying preferential nucleosomes positioning. In the present paper this aspect has been studied by topological analysis and electron microscopy visualization of minichromosomes reconstituted at different histone/DNA ratios. Both methods suggest that cooperativity plays a role in nucleosomes formation. A linear cooperative model in which nucleosomes are formed on discrete sites with cooperative interactions occurring only between nearest neighbours allows to calculate the cooperative constant. The reported results show that basic interactions, which are of relevance in the process of chromatin folding, are present also in very simple model system.     

Molecular machines in archaeal DNA replication

The archaeal DNA replication apparatus is a simplified version of that of eukaryotes and has attracted attention as a tractable model system for the orthologous, but significantly more complex eukaryal machinery. A variety of archaeal model organisms have been investigated with strong emphasis on structural and biochemical analyses of replication-associated proteins. In this review we will describe recent advances in understanding the properties of the replicative helicase, the MCM complex, and the role of the sliding clamp, PCNA, in mediating a range of protein-DNA transactions. Although both complexes form ring shaped assemblies, they play very distinct roles at the leading and trailing edges of the replication fork machinery respectively.     

DNA sequence patterns in precisely positioned nucleosomes

Several investigators have recognized the importance of non-periodic DNA sequence information in determining the translational position of precisely positioned nucleosomes. The purpose of this study is to determine the extent of such information, in addition to the character of periodic information present. This is accomplished by examining the half-nucleosome DNA sequences of a considerable number of precisely positioned nucleosomes, and determining the probability of occurrence of each dinucleotide type as a function of position from the nucleosome center to the terminus (positions 0 to 72). By the nature of this procedure, no assumptions of periodicity are made. The results show the importance of several DNA sequence periodicities including 6-7, 10, and 21 base pairs, in addition to significant nonperiodic information. The results demonstrate that each dinucleotide type is unique in terms of its positional preference in precisely positioned nucleosomes (for example AA not equal to TT). The probabilities of occurrence for the dinucleotide types can be used to predict the translational positions of a number of observed nucleosomes.     

Enhanced DNA repair synthesis in hyperacetylated nucleosomes

We have investigated the level of "early" DNA repair synthesis in nucleosome subpopulations, varying in histone acetylation, from normal human fibroblasts treated with sodium butyrate. We find that repair synthesis occurring during the first 30 min after UV irradiation is significantly enhanced in hyperacetylated mononucleosomes. Nucleosomes with an average of 2.3 acetyl residues/H4 molecule contained approximately 1.8-fold more repair synthesis than nucleosomes with an average of 1.5 or 1.0 acetyl residues/H4 molecule. Fractionation of highly acetylated nucleosomes by two-dimensional gel electrophoresis yielded an additional 2.0-fold enrichment of repair synthesis in nucleosomes containing 2.7 acetyl residues/H4 molecule as compared to nucleosomes containing 1.9 acetyl residues/H4 molecule. This enhanced repair synthesis is associated primarily with nucleosome core regions and does not appear to result from increased UV damage in hyperacetylated chromatin. In addition, the distribution of repair synthesis within nucleosome core DNA from hyperacetylated chromatin is nonrandom, showing a bias toward the 5' end which is similar to that obtained for bulk (unfractionated) chromatin. These results provide strong evidence that enhanced repair occurs within nucleosome cores of hyperacetylated chromatin in butyrate-treated human cells. Finally, pulse-chase experiments demonstrate that the association of enhanced repair synthesis with hyperacetylated nucleosomes is transient, lasting only about 12 h after UV damage.