Specific cleavage of chromatin by restriction nucleases.

Digestion of mouse and rat liver nuclei with a restriction nuclease from Bacillus subtilis (Bsu) is examined in continuation of previous work from this laboratory (Pfeiffer et al., 1975, Nature 258, 450). The finding of more than 95% C in the 5'-termini of the DNA fragments generated during digestion with Bsu shows that the participation of endogenous nucleases in Bsu digestion is extremely small. The restriction nuclease Hae III, an isoschizomer of Bsu, yields identical degradation patterns. The patterns conform to what one expects from statistical calculations based on a nucleosome structure of chromatin with a region preferentially accessible to the nuclease of 40-50 nucleotide pairs per nucleosome. Integrity of the histones is maintained during digestion with restriction nucleases. Digestion of mouse liver nuclei with EcoRII shows that most if not all of the satellite DNA is organized in a nucleosome structure. Also in rat liver, much of the repetitive DNA appears to be present in nucleosomes.     

Stability of DNA-protein interactions in chromatin fractions with different sensitivity to nucleases

It was revealed by means of nucleoprotein-celite-chromatography that DNA-protein interactions in the chromatin fraction sensitive to micrococcal nuclease and DNase II are weaker that in the resistant one. The micrococcal nuclease destroys the DNA-matrix bond resistant to salt-urea, while DNase II does not change the DNA-matrix integrity. Tightness of the DNA-protein interactions is weakened by the increasing chromatin fragmentation, but does not depend on the size of chromatin particles.     

Periodicity and fragment size of DNA from mouse TLT hepatoma chromatin and chromatin fractions using endogenous and exogenous nucleases

Summary The action of micrococcal nuclease, DNase I and DNase II on mouse TLT hepatoma chromatin revealing the periodicity of its structure as visualized by denaturing and nondenaturing gel electrophoresis, was consistent with the action of these enzymes on other chromatins. Micrococcal nuclease showed a complex subnucleosome fragment pattern based on multiples of 10 base pairs with a prominant couplet at 140/160 base pairs and the absence of the 80 base pair fragment. This couplet of the core and minimal nucleosome fragments was conspicuously present in the mononucleosomes found in the 11S fractions of a glycerol gradient centrifugation. DNase I and II produced a fairly even distribution of a 10 base pair increasing series of fragments to about 180 base pairs, a pattern also repeated in the DNA of nucleosome glycerol-gradient fractions. In limited digestions by these nucleases multinucleosomic DNA fragments are pronounced. These fragment lengths are multiples of an estimated average repeat length of nucleosome DNA of 180 base pairs. The action of the endogenous Mg/Ca-stimulated endonuclease produced only limited cuts in the hepatoma chromatin resulting primarily in multi-nucleosommc DNA fragment lengths and only upon lengthy digestion limited subnucleosomic, 10-base-pair multiple fragments are produced. The putative euchromatin-enriched fractions (50–75S) of the glycerol gradient centrifugation of autodigested chromatin, similarly, contained primarily the multinucleosomic DNA fragment lengths. These results are consistent with our previous electron microscopic demonstration that autodigested chromatin as well as the putative euchromatin-enriched fractions were composed of multinucleosomic chromatin segments containing a full complement of histones.     

Differences in chromatin from normal and leukemic human cells as shown by digestion with restriction nucleases

Sequence homology was found by computer analysis between potato spindle tuber viroid (PSTV) RNA and U3B snRNA of Novikoff hepatoma cells. This homology is colinear in arrangement, extends in length to 81% of the entire U3B snRNA molecule and is involved in the PSTV molecule unique sites which, if depicted in terms of the secondary structure of the circular PSTV molecule, reveal a conspicuous regularity in their location. A strong relation in primary structure between PSTV and U3B snRNA is demonstrated by statistical analysis.     

Computer analysis of chromatin fragmentation with nucleases

Computerizing of the densitograms of chromatin hydrolysate DNA is offered. It is based on searching for gaussoids depicting the peaks of individual oligomers so that the resultant curve corresponded to the experimental curve to a high enough accuracy. The method suggested was used to study the time course of mononucleosome and oligomer accumulation during chromatin fragmentation with endogenous nuclease. Comparison of the experimental data to a model calculated for break down of 500-nucleosome chromatin fragments demonstrates that in chromatin, the release of mono-, di- and trinucleosomes is shifted towards nucleosomes during break down induced by endogenous nuclease.     

Cations and the accessibility of chromatin to nucleases.

When rat liver nuclei prepared with polyamines as stabilising cations are digested with DNAase II, release of both inactive chromatin and Mg-soluble, active chromatin is greatly reduced, in comparison to digestion of liver nuclei prepared with Mg2+ as stabilising cation. Chromatin release from polyamine stabilised nuclei is also inhibited relative to Mg-stabilised nuclei following digestion with micrococcal nuclease under two very different cation conditions. Nuclei prepared with polyamines and monovalent ions as stabilising cations exhibit properties intermediate between these two extremes with both nucleases. These effects are due to residual binding of polyamines to chromatin, which is thus maintained in a condensed state, inaccessible to nucleases. Since polyamine binding is not easily reversed, concentrations of polyamines and other cations must be rigidly controlled in experiments on chromatin structure if artefacts are to be avoided. The significance of these findings to the nature and properties of active chromatin within the intact nucleus is considered.     

Creating highly specific nucleases by fusion of active restriction endonucleases and catalytically inactive homing endonucleases

Zinc-finger nucleases and TALE nucleases are produced by combining a specific DNA-binding module and a non-specific DNA-cleavage module, resulting in nucleases able to cleave DNA at a unique sequence. Here a new approach for creating highly specific nucleases was pursued by fusing a catalytically inactive variant of the homing endonuclease I-SceI, as DNA binding-module, to the type IIP restriction enzyme PvuII, as cleavage module. The fusion enzymes were designed to recognize a composite site comprising the recognition site of PvuII flanked by the recognition site of I-SceI. In order to reduce activity on PvuII sites lacking the flanking I-SceI sites, the enzymes were optimized so that the binding of I-SceI to its sites positions PvuII for cleavage of the composite site. This was achieved by optimization of the linker and by introducing amino acid substitutions in PvuII which decrease its activity or disturb its dimer interface. The most specific variant showed a more than 1000-fold preference for the addressed composite site over an unaddressed PvuII site. These results indicate that using a specific restriction enzyme, such as PvuII, as cleavage module, offers an alternative to the otherwise often used catalytic domain of FokI, which by itself does not contribute to the specificity of the engineered nuclease.     

Contractile proteins in the fractions of soluble and insoluble chromatin of liver and thymus

The proteins corresponding in molecular weight and solubility in salt solutions to skeletal muscle actin and myosin were revealed in liver and thymus chromatin fragments. When the ionic strength reached 0.3, about 60% of the myosin-like protein identified by electrophoretic mobility of high chains and the K+-EDTA-ATPase activity was cosedimented with nucleohistones. In the presence of ATP or PPi and Mg2+ the solubility of myosin in such salt solutions increased up to 90%, which was paralleled with significant stimulation of RNA release from the nucleohistones. The conformity in the degree of extraction and sedimentation of RNA and intranuclear myosin was also observed in other solutions used during myosin purification. The supposition that the nuclear system of contractile proteins causes labile, ATP-dependent binding of RNA to chromatin is discussed. No essential differences in the actin or myosin contents in the fractions of soluble and non-soluble chromatin were detected.     

Structure of eukaryotic chromatin. Evaluation of periodicity using endogenous and exogenous nucleases.

DNA isolated from (a) liver chromatin digested in situ with endogenous Ca2+, Mg2+-dependent endonuclease, (b) prostate chromatin digested in situ with micrococcal nuclease or pancreatic DNAase I, and (c) isolated liver chromatin digested with micrococcal nuclease or pancreatic DNAase I has been analyzed electrophoretically on polyacrylamide gels. The electrophoretic patterns of DNA prepared from chromatin digested in situ with either endogenous endonuclease (liver nuclei) or micrococcal nuclease (prostate nuclei) are virtually identical. Each pattern consists of a series of discrete bands representing multiples of the smallest fragment of DNA 200 +/- 20 base pairs in length. The smallest DNA fragment (monomer) accumulates during prolonged digestion of chromatin in situ until it accounts for nearly all of the DNA on the gel; approx. 20% of the DNA of chromatin is rendered acid soluble during this period. Digestion of liver chromatin in situ in the presence of micrococcal nuclease results initially in the reduction of the size of the monomer from 200 to 170 base pairs of DNA and subsequently results in its conversion to as many as eight smaller fragments. The electrophoretic pattern obtained with DNA prepared from micrococcal nuclease digests of isolated liver chromatin is similar, but not identical, to that obtained with liver chromatin in situ. These preparations are more heterogeneous and contain DNA fragments smaller than 200 base pairs in length. These results suggest that not all of the chromatin isolated from liver nuclei retains its native structure. In contrast to endogenous endonuclease and micrococcal nuclease digests of chromatin, pancreatic DNAase I digests of isolated chromatin and of chromatin in situ consist of an extremely heterogeneous population of DNA fragments which migrates as a continuum on gels. A similar electrophoretic pattern is obtained with purified DNA digested by micrococcal nuclease. The presence of spermine (0.15 mM) and spermidine (0.5 mM) in preparative and incubation buffers decreases the rate of digestion of chromatin by endogenous endonuclease in situ approx. 10-fold, without affecting the size of the resulting DNA fragments. The rates of production of the smallest DNA fragments, monomer, dimer, and trimer, are nearly identical when high molecular weight DNA is present in excess, indicating that all of the chromatin multimers are equally susceptible to endogenous endonuclease. These observations points out the effects of various experimental conditions on the digestion of chromatin by nucleases.     

Characterization of plant satellite DNA using restriction nucleases

The structural organization of satellite DNAs of mustard Brassica nigra and lemon Citrus limon has been studied by digestion with restriction nucleases. Analysis of DNA products produced by EcoRI and Bam I shows that two satellite DNAs contain long range periodicities belonging to several repeated sequences. The periodicities in two satellite DNAs differ characteristically, however, they have been found to contain common homologous sequences. Using the restriction nuclease Bsp I, a highly periodical fractions has been found in Citrus satellite DNA, composed of Bsp I fragments ranging from 80 to 1240 basepain. The major repeat units comprise five Bsp I fragments ranging from 80 to 200 bp. These fractions characterized by a high content of 5-methyl-cytosine.     

Effect of irradiation and endogenous nucleases on rat liver chromatin

These assessment of the consequences of irradiation on chromatin is complicated by endogenous nucleases. Isolation and prolonged storage of rat liver nuclei in buffers containing divalent metal ions activates these enzymes and promotes the degradation of chromatin. Irradiation of rat liver nuclei to dose levels of 20,000 rad under conditions in which endogenous nucleases are inhibited and analysis of the irradiated chromatin by sucrose density gradient centrifugation gave no evidence for monosomes or oligosomes. When chromatin from irradiated nuclei was digested with micrococcal nuclease, the levels of monosomes and oligosomes were identical to those of micrococcal nuclease, the levels of monosomes and oligosomes are identical to suggest that irradiation results in neither a direct fragmentation of linkers nor the sensitization of linkers for subsequent cleavage by micrococcal nuclease. Histones isolated from monosomes of irradiated and unirradiated nuclei were intact, showing no fragmentation or loss of residues, as judged by sodium dodecyl sulfate-polyacrylamide electrophoresis.     

Comparative analysis of three guinea pig satellite DNA's by restriction nucleases.

The structures of guinea pig satellite DNAs I, II, and III have been analyzed by digestion with seven restriction nucleases. From the cleavage patterns it is obvious that the long-range periodicities in these three satellites differ rather characteristically Satellite I is fairly resistant to six nucleases and gives only a number of weak discrete bands which do not show a simple regularity. By the restriction nuclease from Arthrobacter luteus, however, it is cleaved extensively and yields very heterogeneous breakdown products. This is consistent with the high extent of divergence previously found for this satellite, e. g. by sequence analysis. Satellite II is almost completely resistant to all nucleases, indicative of a high degree of sequence homogeneity of this satellite. Satellite III is completely broken by the restriction nuclease from Bacillus subtilis into fragments which form a novel, highly regular series of bands in gel electrophoresis. The patterns show that the satellite is composed of tandem repeats ofapproximately 215 nucleotide pairs length, each repeat unit containing two cleavage sites for this nuclease. The data are consistent with the assumption that 30--40% of all cleavage sites have been eliminated by a random process. Satellite III DNA yields weak degradation patterns of the same periodicity with a number of other restriction nucleases. Cleavage sites for these nuclease are clustered on separatesmall segments of the satellite DNA. In this respect, the satellite is similar to others, notably the mouse satellite DNA. The three guinea pig satellites are examples of more general types of satellite structures also found in othe organisms. Similarities and differences to other satellites are discussed with special consideration to theories on the evolution of this class of DNA.     

Characterization of distinct segments in mouse satellite DNA by restriction nucleases.

The long-range periodicity of mouse satellite DNA has been analyzed by digestion with five restriction nucleases. With all nucleases tested, a major repeat unit approximately 245 nucleotide pairs became apparent. Minor registers of shorter length were also detected. The total number of cleavage sites per haploid genome for each restriction enzyme as well as their positions relative to each other were determined. While endo R-EcoRII was known to cleave all of the satellite DNA, the other four restriction enzymes were found to generate only weak degradation patterns. The results taken together with quantitative analyses of codigestion experiments indicate that the recognition sequences for each of these four nucleases are clustered on separate parts of the satellite DNA. It is concluded that the satellite DNA, which appears homogeneous by digestion with endo R-EcRII, contains distinct segments each susceptible to degradation with one of the other nucleases. These results have certain implications for theories on the evolution of mouse satellite DNA. A simple mechanism of multiplication and divergence by mutation is not sufficient to explain the data. Additional and alternative processes which are relevant to the evolutionary considerations are discussed.     

Analysis of the alpha-satellite DNA from African green monkey cells by restriction nucleases.

By the use of restriction endonucleases the organization of the alpha-satellite DNA from African green monkey cells (Cercopithecus aethiops) has been analyzed. With endo R-HindIII, endo R-AluI and with endo R-EcoRI at conditions of low salt and high pH (endo R-EcoRI) all of the satellite was digested while only a part of the satellite was cleaved with endo R-Bsu and endo R-EcoRI under standard conditions. With each of the four nucleases a series of fragments was formed which were multiplies in size of a basic repeat unit linked in tandem arrays in the intact satellite. The quantitative evaluation of the digestion with each nuclease as well as with combinations of two nucleases yielded information about the distribution of the cleavage sites. While the arrangement of the endo R-HindIII cleavage sites conforms to a random distribution across the entire satellite, the results from the endo R-Bsu and endo R-EcoRI cleavage patterns are consistent with a picture where the cleavage sites are clustered in fractions of the satellite. Since endo R-AluI recognizes the central four nucleotide pairs of the endo R-HindIII cleavage site, the redigestion of the endo R-HindIII dimer with endo R-AluI gave information about the distribution of mutations in the satellite. The results of these experiments together with the comparison of the sequence divergence determined from digestion with endo R-HindIII and endo R-EcoRI lend support to the hypothesis that mutations have affected all bases in the satellite evenly. The gamma-satellite, another fraction of the African green monkey DNA, could be separated by Ag+/CsSO4 density gradient centrifugation into two components. With the three restriction nucleases used both components gave a background of fragments of heterogenous length on gel electrophoresis with some faint bands of no apparent regularity in one case.