Increased thermal stability of solubilized chromatin after poly(ADP-ribose) synthesis

A hyperthermic shift in the hyperchromicity curve of thermally denatured swine aortic-smooth-muscle-cell chromatin solubilized by digestion of nuclei with micrococcal nuclease was observed after the chromatin was incubated under conditions to allow poly-(ADP-ribose) synthesis by the endogenous poly(ADP-ribose) polymerase. When the order of solubilization and poly(ADP-ribosyl)ation was reversed, a smaller proportion of the solubilized chromatin exhibited greater thermal stability. Nuclease digestion of nuclei preincubated for poly(ADP-ribose) synthesis revealed no difference in kinetics of digestion or fragment size distribution compared to that of control nuclei. Poly(ADP-ribose) synthesis in these nuclei was proportionately greater in the chromatin fraction most resistant to solubilization by micrococcal nuclease treatment.     

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.     

Histone shuttling by poly(ADP-ribosylation).

We have found that two nuclear enzymes, i.e. poly(ADP-ribose) polymerase (EC 2.4.2.30) and poly(ADP-ribose) glycohydrolase, may cooperate to function as a histone shuttle mechanism on DNA. The mechanism involves four distinct reaction intermediates that were analyzed in a reconstituted in vitro system. In the first step, the enzyme poly(ADP-ribose) polymerase is activated in the presence of histone-DNA complexes and converts itself into a protein carrying multiple ADP-ribose polymers. These polymers attract histones that dissociate from the DNA as a histone-polymer-polymerase complex. The DNA assumes the electrophoretic mobility of free DNA and becomes susceptible to nuclease digestion (second step). In the third step, poly(ADP-ribose) glycohydrolase degrades ADP-ribose polymers and thereby eliminates the binding sites for histones. In the fourth step, histones reassociate with DNA, and the histone-DNA complexes exhibit the electrophoretic mobilities and nuclease susceptibilities of the original complexes prior to dissociation. Our results are compatible with the view that the poly(ADP-ribosylation) system acts as a catalyst of nucleosomal unfolding of chromatin in DNA excision repair.     

Noncovalent binding of poly(ADP-ribose) to nuclear matrix proteins: developmental changes and tissue specificity

Poly(ADP-ribose) is a nuclear polynucleotide involved in the regulation of chromatin functions via covalent and/or noncovalent modification of nuclear proteins. Using a binding assay on protein blots, we searched for poly(ADP-ribose) binding proteins in nuclear matrices from testes of differently aged rats as well as from various adult rat tissues (brain, liver, spleen). We found that nuclear matrix proteins represent a significant subset of the nuclear proteins that can establish noncovalent interactions with poly(ADP-ribose). The profiles of poly(ADP-ribose) binding nuclear matrix proteins appeared to be tissue-specific and changed during postnatal development in the testis. The isolation and analysis of endogenous poly-(ADP-ribose) from rat testes showed that the ADP-ribose polymers that bind nuclear matrix proteins in vitro are also present under physiologic conditions in vivo. These results further substantiate the possibility that poly(ADP-ribose) may affect chromatin functions through noncovalent interaction with specific protein targets, including nuclear matrix components.     

Selective degradation of integrated murine leukemia proviral DNA by deoxyribonucleases

The sensitivity to micrococcal nuclease and DNAase I of the integrated proviral DNA sequences in Swiss mouse cells infected with Moloney murine leukemia virus has been studied. Chromatin was separated into micrococcal nuclease-sensitive and -resistant regions, and the amount of proviral sequences in these DNA preparations was estimated by kinetic hybridization with single-stranded complementary DNA of Moloney murine leukemia virus. At least two thirds of the proviral DNA sequences were found in the open regions of chromatin, and only one third was resistant to nuclease. The proviral DNA sequences are even more sensitive to deoxyribonuclease I. When intact nuclei were treated with limited amounts of enzyme, only 5% of the nuclear DNA was digested, whereas 48% of the proviral DNA was degraded.The proviral DNA sequences in cells which do not produce virus are more resistant to nuclease digestion, as compared to virus producer cells. Thus the endogenous proviral sequences, in normal uninduced Swiss mouse cells, are randomly distributed between resistant and sensitive portions of chromatin when tested with either micrococcal nuclease or pancreatic deoxyribonuclease I. The effect of cell cycle synchronization on the accessibility of the proviral sequences to pancreatic deoxyribonuclease I was investigated with rat cells infected with Moloney murine leukemia virus. The amount of proviral DNA sensitive to pancreatic deoxyribonuclease I is higher in actively dividing cells than in cells arrested at Go phase, which produce only small amounts of virus.     

Structure of rat liver nuclei after depletion and reassociation of histone H1

Chromatin in isolated rat liver nuclei was compared with chromatin in (i) nuclei depleted of H1 by acid extraction; (ii) nuclei treated at pH 3.2 (without removal of H1), and (iii) depleted nuclei following reassociation of H1. Electron microscopy and digestion by DNase I, micrococcal nuclease and endogenous Ca/Mg endonuclease were used for this comparative examination. Electron micrographs of H1-depleted nuclei showed a dispersed and finely granular appearance. The rate of DNA cleavage by micrococcal nuclease or DNase I was increased several-fold after H1 removal. Discretely sized intermediate particles produced by Ca/Mg endonuclease in native nuclei were not observed in digests of depleted nuclei. Digestion by micrococcal nuclease to chromatin particles soluble in 60 mM NaCl buffer appeared not to be affected in depleted nuclei. When nuclei were treated at pH 3.2, neither the appearance of chromatin in electron micrographs nor the mode or rate of nuclease digestion changed appreciably. Following reassociation of H1 to depleted nuclei, electron micrographs demonstrated the reformation of compacted chromatin, but the lower rate of DNA cleavage in native nuclei was not restored. Further, H1 reassociation produced a significant decrease in the solubility of nuclear chromatin cleaved by micrococcal nuclease or Ca/Mg endonuclease. In order to evaluate critically the reconstitution of native chromatin from H1-depleted chromatin we propose the use of digestion by a variety of nucleases in addition to an electron microscopic examination.     

Cholera toxin affects nuclear ADP-ribosylation in GH1 cells

Incubation of GH1 cells with cholera toxin for 24 h inhibits [32P]ADP-ribose incorporation into histones and non-histone nuclear proteins by more than 50%. The toxin produces a generalized decrease of incorporation into all protein acceptors and into the poly(ADP-ribosyl)ated components excised from chromatin after micrococcal nuclease digestion. The cellular levels of NAD were also decreased (40 to 80%) after treatment with cholera toxin. The inhibition of poly(ADP-ribosyl)ation is preceded by an increase of [32P]ADP-ribose incorporation, since incubation with the toxin for 3 h caused an increase instead of a decrease of incorporation. Incubation with dibutyryl cyclic AMP for 24 h also inhibited nuclear poly(ADP-ribosyl)ation, thus showing that the effect of cholera toxin might be mediated by cyclic AMP.     

Fractionation of micrococcal nuclease-digested chromatin solubilized at physiologic ionic strength

When mouse brain nuclei are optimally digested with micrococcal nuclease, most of the chromatin is soluble in a 180 mM salt/1 mM EDTA buffer [1]. At this ionic concentration, chromatin maintains its native structure [2]. In an attempt to selectively extract different fractions of chromatin from digested nuclei, we have examined the differential solubility of chromatin in the 180 mM salt buffer containing concentrations of MgCl2 ranging from 2 to 0 mM. The results suggest that digested chromatin may be fractionated into specific soluble chromatin fractions which correspond to nuclease-sensitive chromatin, bulk chromatin, and heterochromatin. These soluble fractions have a high molecular weight (up to 20 kbp), and contain a full complement of histones as well as a complex assortment of non-histone proteins. The residual insoluble fraction may be equivalent to a native, nuclear matrix-bound chromatin fraction.     

Fractionation of chromatin of liver cell nuclei after mild micrococcal nuclease digestion

Mild micrococcal nuclease treatment of rat and mouse nuclei and fractionation were based on the method of Tata and Baker. Three chromatin fractions, S, P1, P2, were separated, and for each of these fractions the sensitivity to the DNase 1 action was determined. The relative content in these fractions of non-transcribed DNA sequences was established by hydridization with a mouse satellite DNA, and the relative content of transcribed DNA sequences--by hydridization with DNA synthesised on the total poly (A) mRNA. None of the fractions displayed the properties characteristic of active chromatin.     

Poly(ADP-ribosylated) histones in chromatin replication

Poly(ADP-ribosylation) of histones and several other nuclear proteins seem to participate in nuclear processes involving DNA strand breaks like repair, replication, or recombination. This is suggested from the fact that the enzyme poly(ADP-ribose) polymerase responsible for this modification is activated by DNA strand breaks produced in these nuclear processes. In this article I provide three lines of evidence supporting the idea that histone poly(ADP-ribosylation) is involved in chromatin replication. First, cellular lysates from rapidly dividing mouse or human cells in culture synthesize a significant number of oligo- in addition to mono(ADP-ribosylated) histones. Blocking the cells by treatment of cultures with 5 mM butyrate for 24 h or by serum or nutrient depletion results in the synthesis of only mono- but not of oligo(ADP-ribosylated) histones under the same conditions. Thus, the presence of oligo(ADP-ribosylated) histones is related to cell proliferation. Second, cellular lysates or nuclei isolated under mild conditions in the presence of spermine and spermidine and devoid of DNA strand breaks mainly synthesize mono(ADP-ribosylated) histones; introduction of a small number of cuts by DNase I or micrococcal nuclease results in a dramatic increase in the length of poly(ADP-ribose) attached to histones presumably by activation of poly(ADP-ribose) polymerase. Free ends of DNA that could stimulate poly(ADP-ribosylation) of histones are present at the replication fork. Third, putatively acetylated species of histone H4 are more frequently ADP-ribosylated than nonacetylated H4; the number of ADP-ribose groups on histone H4 was found to be equal or exceed by one the number of acetyl groups on this molecule. Since one recognized role of tetraacetylated H4 is its participation in the assembly of new nucleosomes, oligo(ADP-ribosylation) of H4 (and by extension of other histones) may function in new nucleosome formation. Based on these results I propose that poly(ADP-ribosylated) histones are employed for the assembly of histone complexes and their deposition on DNA during replication. Modified histones arise at the replication fork by activation of poly(ADP-ribose) polymerase by unligated Okazaki fragments.     

The extraction by micrococcal nuclease of glucocorticoid receptors and mouse mammary tumor virus DNA sequences is dissociated.

Glucocorticoid receptors (RG) and mammary tumor virus (MM-TV) DNA sequences were extracted by micrococcal nuclease digestion from the nuclei of C3H mouse mammary tumor cells in order to specify their relative distribution in chromatin. RG was labelled and translocated into the nuclei by incubating cells with 3H Dexamethasone (3H Dex). The purified nuclei were then treated at 2 degrees C with micrococcal nuclease. Three chromatin fractions were successively obtained: an isotonic extract (ne3H1), ahypotonic extract (ne2) and the residual pellet (P). The Dex-RG complexes were measured by the hydroxyapatite technique. The MMTV DNA sequences were titrated by molecular hybridization with an excess of MMTV radioactive cDNA probe. Up to 75% of the nuclear 3H Dex and the MMTV radioactive cDNA probe. Up to 75% of the nuclear 3H Dex and MMTV DNA sequences were extracted in a concentration dependent manner while only 10-15% of nucleic acids became soluble in 10% perchloric acid. The extracted 3H Dex-RG complex was found to be partly bound to soluble chromatin and partly free. The free complex displayed similar sedimentation constants (4S, 7S) and DNA binding ability to the cytosol receptor. The 3H Dex-RG complexes were 2 to 8 fold more concentrated in ne1, which is known to be enriched in active chromatin, than in ne2. Conversely, the concentration of MMTV DNA sequences per microgram DNA was the same in the three nuclear fractions. These results suggest that the Dex-RG complexes are concentrated in an active fraction of chromatin. We propose that, among the 20-30 copies of MMTV genes per haploid genome, only a small proportion are transcribed or regulated.     

Differential compartmentalization of plasmid DNA microinjected into Xenopus laevis embryos relates to replication efficiency.

Circular plasmid DNA molecules and linear concatemers formed from the same plasmid exhibit strikingly different fates following microinjection into Xenopus laevis embryos. In this report, we prove quantitatively that only a minority of small, circular DNA molecules were replicated (mean = 14%) from fertilization through the blastula stage of development. At all concentrations tested, very few molecules (approximately 1%) underwent more than one round of DNA synthesis within these multiple cell cycles. In addition, unlike endogenous chromatin, the majority of circular templates became resistant to cleavage by micrococcal nuclease. The extent of nuclease resistance was similar for both replicated and unreplicated templates. Sequestration of circular molecules within a membranous compartment (pseudonucleus), rather than the formation of nucleosomes with abnormal size or spacing, apparently conferred the nuclease resistance. In contrast, most linearly concatenated DNA molecules (derived from end-to-end joining of microinjected monomeric plasmid DNA) underwent at least two rounds of DNA replication during this same period. Linear concatemers also exhibited micrococcal nuclease digestion patterns similar to those seen for endogenous chromatin yet, as judged by their failure to persist in later stages of embryogenesis, were likely to be replicated and maintained extrachromosomally. We propose, therefore, that template size and conformation determine the efficiency of replication of microinjected plasmid DNA by directing DNA to a particular compartment within the cell following injection. Template-dependent compartmentalization may result from differential localization within endogenous nuclei versus extranuclear compartments or from supramolecular assembly processes that depend on template configuration (e.g., association with nuclear matrix or nuclear envelope).     

Assembly of oligonucleosomes into a limit series of multimeric higher-order chromatin structures

Chicken erythrocyte chromatin, obtained after fragmentation with micrococcal nuclease, appears to remain folded in a stable distribution of supranucleosomal structures in buffers containing 80 mM NaCl. These supranucleosomal particles are composed of on average 25 nucleosomes. However, the integrity of the linker DNA within these particles is not required. The supranucleosomal particles have been interpreted by others as superbeads cut out of a preexisting granular nominal 30-nm chromatin fibre. We show that the same distribution of supranucleosomal structures (even those containing internal DNA scissions) can be reconstituted from unfolded nuclear chromatin extracts as present in 10 mM or 600 mM NaCl. Moreover, fractions of oligonucleosomes with mean lengths between 6 and 15 nucleosomes reassemble or aggregate into a limit series of multimeric species. The existence of an assembly barrier could be inferred as we were unable to observe a stable and soluble assembly product containing more than about 25 nucleosomes. We propose an alternative explanation for the generation and observation of a constant distribution of supranucleosomal structures in nuclear extracts, based on the assembly or aggregation property of oligonucleosomes and on the existence of an assembly barrier.     

Two-dimensional gel electrophoretic separation of the proteins present in chromatin of Escherichia coli

The polypeptides present in 35S-labelled chromatin prepared from Escherichia coli cells, and polypeptides present in the DNA and RNA complexes obtained by micrococcal nuclease digestion of the chromatin, were analysed by two-dimensional non-equilibrium polyacrylamide gel electrophoresis. Three hundred and thirty-five 35S-labelled polypeptides were detected in the chromatin whereas the DNA- and RNA-containing fractions of the micrococcal nuclease digest contained 126 and 183 polypeptides respectively. The major basic low-molecular-weight polypeptides were found in the DNA-containing fractions.