No abstract

Staphylococcal nuclease digestion of purified chromatin from duck reticulocytes or calf thymus results in the production of a series of double-stranded DNA fragments of discrete molecular size, ranging from about 130 to 45 base pairs, which can be detected by polyacrylamide gel electrophoresis. Similar patterns of protected DNA fragments are obtained from limit digests of chromatin "reconstituted" from purified DNA and chromatin proteins. The results obtained with reconstituted material do not depend upon the origin of the DNA, which may be derived from a bacterial, viral, or homologous source. The specificity of the protective mechanism, therefore, resides in the structure of the bound histones, and probably not in any special nucleotide sequences present in the DNA. Removal of lysine-rich histones from chromatin before digestion results principally in disappearance from the digest of a DNA fragment about 130 base pairs long. Our preliminary results suggest that other elements of the digest pattern can be assigned uniquely to the remaining histone components. These results indicate that the binding of histones to DNA in chromatin involves a limited number of specific and very well defined contacts between protein and nucleic acid, which arise from structural properties of the histones.Many studies of the binding of histones to the DNA of chromatin have suggested that the histones interact with DNA through well defined sites on the proteins (1). Other evidence indicates that the histones may be bound to DNA in the form of specific complexes involving more than one histone species (2-4). It has been proposed (4) that such complexes are arranged in a regular repeating sequence along the DNA, giving rise to the tertiary DNA structure present in chromatin. Evidence suggesting that histone complexes may cover the DNA in a regular fashion is provided by studies of the products of nuclear autodigestion (5, 6). These studies reveal that autodigestion of nuclei results in release of DNA segments that are multiples of a subunit about 200 base pairs in length. Incubation of nuclei with added nuclease results in release of fragments of similar size (7).In our laboratory we have made use of the enzyme staphylococcal nuclease as a probe of the structure of purified chromatin, and we have isolated those regions of the DNA that are sufficiently tightly covered by protein to be protected from digestion (8, 9). About half the DNA is susceptible to digestion. The rest is protected, and is reduced to double-stranded segments with a weight average length of about 110 to 130 base pairs regardless of the amount of enzyme used (9). In this paper we examine in more detail the nature of the reaction product of this digestion, and show that it consists of a well-defined set of DNA segments. We believe that these segments correspond to the points of intimate contact between histones and DNA, and reveal a regular and highly specific mode of interaction between them. MATERIALS AND METHODSChromatin was prepared from washed nuclei isol...

Several small alkylammonium ions can eliminate, or even reverse, the usual dependence of the DNA transition temperature on base composition. For example, in 3 M tetramethylammonium chloride, or 2.4 M tetraethylammonium chloride, DNAs of different base compositions all melt at a common temperature, and with a greatly decreased breadth of transition reflecting only the sequence-independent components of melting cooperativity. At still higher concentrations of such additives, dG-dC-rich DNAs melt at lower temperatures than dA dT-rich molecules. Circular dichroism spectra show that these additives alter the structure of the DNA double helix very little at room temperature. This differential (base-specific) effect on helix stability is investigated with several small additives related to the tetraalkylammonium ions. Additives larger than tetraethylammonium ion have little differential effect on helix stability. Preferential binding of ions to dA dT base pairs, requiring fit into a "groove" of DNA, is consistent with these data and with equilibrium binding studies. These differential effects can be distinguished from general destabilizing effects, which are independent of specific features of macromolecular conformation or chemistry. Possible experimental uses of this ability to alter the base-composition-dependent components of the stability of the DNA helix are discussed, as well as the insight this phenomenon provides into the molecular basis for the differential stability of dA * dT and dG * dC base pairs.The differential stability of dA-dT and dG-dC base pairs and, thus, of segments of native DNA differing in base composition, may be critical to several of its biological functions, i.e., DNA-protein recognition mechanisms in processes such as transcription, recombination, etc. (for a recent review see ref. 1). These stability differences have also had practical consequences for biochemists and molecular biologists, who have used differences in melting temperature (Tm) as a measure of DNA base composition (2). They have used preferential melting of dA dT-rich sequences to physically map bacteriophage and bacterial chromosomes (3) and to fractionate DNA molecules by thermal elution chromatography (4). On the other hand, base sequence heterogeneity has complicated the theoretical interpretation of melting transitions of nucleic acids and the analysis of other aspects of nucleic acid structure in solution (5), in particular making it very difficult to separate sequence-dependent effects from intrinsic (sequence-independent) structural contributions to the cooperativity of melting of the DNA helix.For the design of experiments in which differential stability of base sequences can be manipulated as an experimental variable, it is necessary to establish aqueous solvent systems in which the differential stability of dA-dT and dG-dC base pairs can be altered with a minimum of disturbance of the other properties of the native DNA helix. Certain solvent additives, such as methanol (6) and several inorganic salts (7)...

A 276 bp region from the tetracycline resistance gene of the plasmid pBR322 was modified with 2-acetylaminofluorene (AAF), 2-aminofluorene (AF), 4-aminobiphenyl (ABP), N'-acetylbenzidine or 1-aminopyrene (AP) in order to determine the effect of adduct structure upon mutation induction. Each modification reaction gave one major adduct and these adducts had chromatographic properties, as determined by 32P-postlabeling, identical to those in which substitution had occurred at C8 of deoxyguanosine through the amine or amide nitrogen. The types and distribution of mutations were then characterized following introduction of the modified plasmids into SOS-induced Escherichia coli using Hanahan et al.'s procedure (Methods Enzymol., 204, 63-113, 1991). With AAF-modified plasmid, 60% of the mutations were deletions or additions, and these were detected primarily at NarI sites or in repetitive G sequences. Modification with AF gave -G deletions, primarily in runs of Gs, and base substitution mutations, which were mainly G to T transversions. Substitution with ABP or N'-acetylbenzidine resulted in G to T and G to C transversions, the latter being a mutation not detected with AF; in addition, -G deletions were detected at only very low frequency. AP modification gave both -G frameshift and base substitution mutations, of which G to T transversions predominated. A comparison of the mutation frequencies per adduct indicated that the mutagenic efficiencies of the adducts decreased in the order AP > AF > AAF approximately ABP approximately N'-acetylbenzidine. AAF- and ABP-modified pBR322 were also introduced with a CaCl2 method. The mutation frequency per adduct increased with this transformation procedure, and this appeared to be a reflection of a greater percentage of frameshift mutations. These data indicate that a series of structurally related aromatic amines will induce both base substitution and frameshift mutations when incorporated into pBR322, but that frameshift mutations occur almost exclusively with the planar derivatives. Furthermore, the ability to induce frameshift mutations increases the mutagenic efficiency of an adduct.