A unique DNAse associated with mitogen-induced transformation of rat lymphocytes

A unique DNAse was found, following electrophoretic separation, in rat lymphocytes stimulated with either ConA or pokeweed mitogens. This DNAse was absent or minimal in non-stimulated cells. The enzyme was active on native DNA at neutral pH and was activated by EDTA. A possible biological role is discussed as related to DNA replication.     

Non-random binding of a chemical carcinogen to the DNA in chromatin

The distribution of a liver carcinogen (N-hydroxy-2-aminofluorene) along the DNA of chromatin has been studied using two nucleases as probes for the structure of chromatin. Rats were injected with the carcinogen and killed at various times after the injection. The nuclei of the liver were prepared and digested with Staphylococcal nuclease or pancreatic nuclease DNAse I. We show that the carcinogen is non randomly distributed along the DNA of chromatin since it binds preferentially to the regions of chromatin digested by the Staphylococcal nuclease whereas it is preferentially bound to the DNAse I resistant fraction. Our results also indicate that the two nucleases do not recognize exactly the same region of chromatin.     

Distribution along DNA of the bound carcinogen N-acetoxy-N-2-acetylaminofluorene in chromatin modified in vitro.

Chromatin from duck erythrocytes was modified in vitro by the carcinogen N-acetoxy-N-2-acetylaminofluorene (N-Ac-O-AAF). The distribution of the carcinogen along the DNA molecule was studied using staphylococcal nuclease which allows the fractionation of chromatin DNA into two zones. It was shown that the carcinogen binds preferentially to the regions of chromatin sensitive to the enzyme; however, the regions of DNA tightly bound to histones and resistant to the enzyme react comparatively well. The single-strand specific nuclease S1 which digests DNA modified by the carcinogen in vitro did not digest chromatin under the conditions used. Some possible mechanisms for the interaction of the carcinogen with chromatin are discussed.     

DNA topoisomerase I cleavage sites in DNA and in nucleoprotein complexes.

The intracellular substrate for eukaryotic DNA topoisomerases is chromatin rather than protein-free DNA. Yet, little is known about the action of topoisomerases on chromatin-associated DNA. We have analyzed to what extent the organization of DNA in chromatin influences the accessibility of DNA molecules for topoisomerase I cleavage in vitro. Using potassium dodecyl sulfate precipitation (Trask et al., 1984), we found that DNA in chromatin is cleaved by the enzyme with somewhat reduced efficiency compared to protein-free DNA. Furthermore, using native SV40 chromatin and mononucleosomes assembled in vitro, we show that DNA bound to histone octamer complexes is cleaved by topoisomerase I and that the cleavage sites as well as their overall distribution are identical in histone-bound and in protein-free DNA molecules.     

Effect of liver chromatin DNAse on closed circular DNA of PM-2 phage and simian virus 40]

Comparative effect of the DNAse from rat liver chromatin and Neurospora crassa endonuclease S1 on closed circular superhelical DNA of PM-2 phage and Simian Virus 40 is studied. It is shown that both of them--the DNAse from chromatin proteins and endonuclease S1--are specific to single-stranded regions in DNA molecular. It is suggested that chromatin protein DNAse participates in reparation processes.     

Association of DNA with the nuclear lamina in Ehrlich ascites tumor cells

We have studied in vitro binding of DNA to nuclear lamina structures isolated from Ehrlich ascites tumor cells. At low ionic strength in the presence of Mg++, they bind considerable amounts of mouse and bacterial DNA, forming complexes stable in 2 M NaCl. Single-stranded DNA and pulse-labeled DNA show higher binding efficiencies than native uniformly labeled DNA. When mixing occurs in 2 M NaCl, complex formation is inhibited. When nuclei are digested with DNAse I under conditions that favor chromatin condensation, DNA associated with matrices subsequently prepared from such nuclei is markedly enriched in satellite DNA. If digestion is carried out with DNAse II while nuclei are decondensed in EDTA, no enrichment in satellite DNA is observed. Preparations of purified, high-molecular weight, double-stranded DNA contain variable amounts of fast-sedimenting aggregates, which are insoluble in 2 M NaCl but are dispersed by DNA fragmentation or denaturation. These results point at some artifacts inherent in studies of DNA bound to residual nuclear structures in vivo and suggest conditions expected to avoid these artifacts. Further, using controlled digestion with DNAse II, we have studied the in vivo association of DNA with nuclear lamina isolated from Ehrlich ascites tumor cells. In the course of DNA fragmentation from above 50 kbp to about 20 kbp average size, the following events were observed. The DNA of high molecular weight (much longer than 50 kbp) behaved as if tightly bound to the nuclear lamina, as judged by sedimentation in sucrose and metrizamide density gradients, electron microscopy, and retention on glass fiber filters. As the size of DNA decreased, it was progressively detached from the nuclear lamina, and at about 20 kbp average length practically all DNA was released. The last 1-4% of DNA, although cosedimenting with the nuclear lamina in sucrose gradients, behaved as free DNA, banding at 1.14 g/cm3 in metrizamide density gradients and showing less than 4% retention on filters. At no stage of digestion did the DNA cosedimenting with nuclear lamina show changes in satellite DNA content relative to that of total DNA or enrichment in newly replicated DNA. It was shown, however, that digestion of nuclear lamina-DNA complex with EcoRI or Hae III led to the formation of DNA-protein aggregates, which banded at 1.35 g/cm3 in high salt containing metrizamide density gradients and which were strongly enriched in satellite DNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Digestion of chromatin with deoxyribonuclease II

The action of DNAse II on DNA in chromatin was studied. The formation of acid-soluble products followed a two-phase kinetic curve. At the end of the first more rapid phase about 25% of DNA was degraded. Early in the degradation process DNA was converted into double stranded fragments, whose sizes were multiples of about 180 base pairs. As the degradation proceeded these fragments were reduced in size. After denaturation DNA from digested chromatin was resolved into discrete single stranded fractions, exact multiples of a ten-nucleotide length, forming a pattern very similar to that observed with DNAse I.     

Removal of histone H1 exposes linker DNA in chromatin to DNAse I

After removal of histone H1 about 40% of DNA in chromatin acquires the sensitivity of naked DNA to DNAse I. Digestion of H1-depleted chromatin with DNAse I leads to a qualitative change in the digestion pattern, generating DNA fragments of approx. 200 b.p. and multiples, similar to those obtained with micrococcal nuclease. Both effects are reversed upon reconstitution of purified H1 to H1-depleted chromatin.     

Formation of native-like mammalian sperm cell chromatin with folded bull protamine

The DNA of most vertebrate sperm cells is packaged by protamines. The primary structure of mammalian protamine I can be divided into three domains, a central DNA binding domain that is arginine-rich and amino- and carboxyl-terminal domains that are rich in cysteine residues. In native bull sperm chromatin, intramolecular disulfide bonds hold the terminal domains of bull protamine folded back onto the central DNA binding domain, whereas intermolecular disulfide bonds between DNA-bound protamines help stabilize the chromatin of mature mammalian sperm cells. Folded bull protamine was used to condense DNA in vitro under various solution conditions. Using transmission electron microscopy and light scattering, we show that bull protamine forms particles with DNA that are morphologically similar to the subunits of native bull sperm chromatin. In addition, the stability provided by intermolecular disulfide bonds formed between bull protamine molecules within in vitro DNA condensates is comparable with that observed for native bull sperm chromatin. The importance of the bull protamine terminal domains in controlling the bull sperm chromatin morphology is indicated by our observation that DNA condensates formed under identical conditions with a fish protamine, which lacks cysteine-rich terminal domains, do not produce as uniform structures as bull protamine. A model is also presented for the bull protamine.DNA complex in native sperm cell chromatin that provides an explanation for the positions of the cysteine residues in bull protamine that form intermolecular disulfide bonds.     

DNase I hypersensitivity in the gamma globin gene locus of K562 cells.

We have probed the chromatin conformation of the G gamma-A gamma-delta-beta globin gene locus of K562 cells, a human hematopoietic cell line, with the enzyme pancreatic DNAse I. This enzyme preferentially digests genes in an active configuration. We have found that in K562 cells, which produce embryonic and fetal but not adult hemoglobins, both the active gamma and inactive beta genes are DNAse I sensitive. However, only the active gamma genes have DNAse I hypersensitive regions. The hypersensitive regions have been mapped to an area approximately 100 base pairs 5' to the G gamma and A gamma genes.     

Increased susceptibility of activated rat liver chromatin to DNAse I

Rat liver chromatin activated by partial hepatectomy is more susceptible to the action of DNAse I than control chromatin isolated from intact liver. The study on the transfer of chromatin material to the acid-soluble fraction reveals a higher rate of activated chromatin degradation. Activated chromatin shows also an increased capacity for ethidium bromide (EB) binding as estimated from the isotherms of adsorption. The difference in EB binding between activated and control chromatin is abolished after DNAse I treatment. Conditions of mild digestion with DNAse I have been found under which the number of binding sites for EB per nucleotide decreases to almost the same level in activated and non-activated chromatin. The results suggest a preferential degradation of those DNA sequences in activated chromatin that are responsible for the increase in the ligand binding.     

Complementation by intraspecific protoplast fusion of auxotrophic cell lines of Datura innoxia P. Mill.

It was determined using electrophoresis in polyacrylamide gels containing native DNA or RNA that sugar non-specific nuclease active at pH 5.2 was expressed in tobacco callus. The nuclease had a relative molecular mass of about 34.6 kDaltons and degraded substrates in the following order of decreasing rate: denaturated DNA>poly dA>UV-irradiated native DNA>native DNA>alkylated native DNA>apurinated native DNA>poly dGpoly dC. The nuclease activity changed during callus growth and plant regeneration, but no developmental changes in electrophoretic patterns were detected. The increase in specific DNAse activity of nuclease was maximal in the exponential phase of callus growth on both growth and regeneration media, except for activity in the cytokinin-independent cell strain grown on growth medium. The specific DNAse activity of nuclease decreased during the bud formation period, while total DNAse activity calculated per mg of dry weight was slightly higher in vegetative buds (9.1U) than in undifferentiated tissue of callus (8.5U). Specific DNAse activity was, on the average, several hundred-fold lower in the vegetative tissues of flowering tobacco plants than in calluses in the exponential phase of growth.     

Analysis of chromatin reconstitutiion.

The ability of high molecular weight chicken erythrocyte chromatin to spontaneously self-assemble into native-like material, after dissociation by high ionic strength and reassociation by salt gradient dialysis, was critically examined. The native conformational state of the reassembled nucleoprotein complex was regenerated to the extent reflected by circular dichroism spectra and thermally induced helix--coil transition of the nucleoprotein DNA. However, internucleosomal packing of approximately 205 base pairs of DNA per repeating unit, as probed by digestion with micrococcal nuclease, was not regenerated upon reassembly and was replaced by a packing of approximately 160 base pairs per repeating unit. Thus, high molecular weight chromatin containing only lysine-rich histones (H1 and H5) and core histones (H2A, H2B, H3, and H4) is not a true self-assembling system in vitro using the salt gradient dialysis system used herein. Circular dichroism and thermal denaturation studies on core chromatin (lysine-rich histones removed) showed that core histones alone are not capable of reassembling high molecular weight DNA into native-like core particles at low temperature (4 degree C). Reassembly at 21 degree C restored the circular dichroism but not the thermal denaturation properties to those characteristic of undissociated core chromatin. Nonetheless, micrococcal nuclease digestions of both reassembled core chromatin products were identical with undissociated native core chromatin. Ressembly in the presence of the complete complement of histones, followed by removal of the lysine-rich histones, did regenerate the thermal denaturation properties of undissociated native core particles. These results indicated multiple functions of the lysine-rich histones in the in vitro assembly of high molecular weight chromatin.     

A nucleosome assembly factor is a constituent of simian virus 40 minichromosomes.

Using in vitro replication assays, we compared native with salt-treated simian virus 40 minichromosomes isolated from infected cell nuclei. Minichromosomes from both preparations contain the full complement of nucleosomes, but salt treatment removes histone H1 and a fraction of nonhistone chromatin proteins. Both types of minichromosomes served well as templates for in vitro replication, but the structures of the replication products were strikingly different. Replicated salt-treated minichromosomes contained, on average, about half the normal number of nucleosomes as previously shown (T. Krude and R. Knippers, Mol. Cell. Biol. 11:6257-6267, 1991). In contrast, the replicated untreated minichromosomes were found to be densely packed with nucleosomes, indicating that an assembly of new nucleosomes occurred during in vitro replication. Biochemical and immunological data showed that the fraction of nonhistone chromatin proteins associated with native minichromosomes includes a nucleosome assembly activity that appears to be closely related to chromatin assembly factor I (S. Smith and B. W. Stillman, Cell 58:15-25, 1989). Furthermore, this minichromosome-bound nucleosome assembly factor is able to exert its activity in trans to replicating protein-free competitor DNA. Thus, native chromatin itself contains the activities required for an ordered assembly of nucleosomes during the replication process.     

Methylation of chromatin DNA.

E. coli DNA methylase has been used to methylate chromatin DNA in vitro. At saturation only 50% of the chromatin DNA becomes methylated. The methylated regions of chromatin correspond to that fraction of the chromatin which is sensitive to staphylococcal nuclease. Using in vitro methylated chromatin followed by nuclease digestion movement of chromatin proteins along the DNA can be detected. By this criterion, sonication of chromatin or precipitation with MnCl2 causes 10% of the previously uncovered methylated regions to become covered by protein. Reconstitution of methylated chromatin results in the randomization of the chromatin proteins. Using nuclei which were methylated in vitro we have demonstrated that a small degree of protein sliding does occur during the preparation of chromatin from nuclei. Finally, we have prepared open region DNA by polylysine titration. This procedure does not cause displacement of chromatin proteins.     

Nuclease Digestion of Chromatin from the Eukaryotic Algae Olisthodiscus luteus,Peridinium balticum,and Crypthecodinium cohnii 1, 2

Chromatin from a uninucleate dinoflagellate, Crypthecodinium cohnii, a binucleate dinoflagellate, Peridinium balticum, and a chromophyte, Olisthodiscus luteus, was examined by nuclease digestion and the results were compared to those from vertebrates. Gel analysis of the products of staphylococcal (micrococcal) nuclease digestion revealed a DNA repeat unit of 220(±5) base pairs for O. luteus and 215(±5) for P. balticum. Limit digestion gave a core particle of 140 base pairs, revealing that these longer repeat sizes are due to longer linker regions. No repeating subunit structure was found upon electrophoresis of digests of C. cohnii nuclei. Examination of the DNA fragments produced by DNAse I digestion of nuclei isolated from P. balticum and O. luteus showed the same ladder of ten base multiples as seen in chromatin from other eukaryotes. Examination of the kinetics of digestion by DNAse II of Peridinium chromatin revealed less susceptibility when compared to DNAse I digestions while 70% of Olisthodiscus chromatin and 35% of C. cohnii chromatin was sensitive to DNAse II. These data, taken together with previous results from Euglena, indicate that while algal chromatin is similar to that of higher eukaryotes in regard to DNAse I and II action, it differs in that the linker DNA is longer. In addition, the Hl-like histone from O. luteus and P. balticum is located in the linker DNA as in higher eukaryotes.