Alkaline lysis of mammalian cells for sedimentation analysis of nuclear DNA. Conformation of released DNA as monitored by physical, electron microscopic and enzymological techniques.

The degree of single strandedness of the DNA released from rat liver nuclei by various alkaline lysing solutions (including some with sodium dodecyl sulfate) was determined both before and after sedimentation in alkaline sucrose gradients employing electron microscopy, melting profiles, circular dichroism measurements, and digestibility by S1 nuclease. Regardless of the technique employed, the results obtained following alkaline sucrose gradient centrifugation of the DNA are consistent. The DNA was completely single stranded as judged by electron microscopy, circular dichroism spectra, and digestibility by S1 nuclease, an enzyme that specifically hydrolyzes single-stranded DNA. This was not true if the DNA was analyzed following alkaline lysis of the nuclei but before centrifugation. Under conditions which gave a complete transition to the single-stranded state, as judged by melting profiles and circular dichroism spectra, only 10-15% of the DNA was hydrolyzed by S1 nuclease. An increase in the susceptibility of the released DNA to S1 nuclease was observed with increases in the pH of the lysing solution. In order to release DNA which was single stranded as judged by both physical and enzymological techniques, the rat liver nuclei were lysed for 30 min with a 0.3 M NaOH lysing solution containing 0.5% dodecyl sulfate, 0.3 M NaCl and 0.03 M EDTA.     

Divalent metal ion effect on helix-coil transition of high molecular weight DNA in neutral and alkaline solutions

Effect of Mg(2+), Ca(2+), Ni(2+) and Cd(2+) ions on parameters of DNA helix-coil transition in sodium cacodylate (pH 6.5), Tris (pH 8.5) and sodium tetraborate (pH 9.0) buffers have been studied by differential UV-visible spectroscopy and by thermal denaturation. Anomalous behavior of the melting temperature T(m) and the melting interval ΔT in the presence of MgCl(2) was observed in Tris, but not in cacodylate or tetraborate buffers. Changes in the buffer type and pH did not influence T(m) and ΔT dependence on Ca(2+) and Cd(2+) concentrations. Decrease of the T(m) and ΔT of DNA in the presence of Ni(2+) and Cd(2+) was caused by preferential ion interaction with N7 of guanine. This type of interaction was also found for Mg(2+) in Tris buffer. The anomalous decrease in the T(m) and ΔT values was connected to formation of complexes between metal ions and Tris molecules. Transition of DNA single-stranded regions into a compact form with the effective radius of the particles of 300±100 ? was induced by Mg(2+) ions in Tris buffer.     

Analysis of rat repetitive DNA sequences.

Parameters of repetitive sequence organization have been measured in the rat genome. Experiments using melting, hydroxylapatite binding, and single strand specific nuclease digestion have been used to measure the number, length, and arrangement of repeated DNA sequences. Renaturation and melting or S1 nuclease digestion of 1.0 kbp DNA fragment show about 20% of rat DNA sequences are 3000-fold repeated. Renatured duplexes from 4.0 kbp DNA fragments display two repetitive size fractions after nuclease digestion. About 60% of the repeated sequences are 0.2-0.4 kbp long while the remainder are longer than 1.5 kbp. The arrangement of the repeated sequences has been measured by hydroxylapatite fractionation of DNA fragments of varying lengths bearing a repeated sequence. Repeated DNA sequences are interspersed among 2.5 kbp long nonrepeated sequences throughout more than 70% of the rat genome. There are approximately 350 different 3000-fold short repeated sequences in the rat interspersed among 600,000 nonrepeated DNA sequences.     

Degradation of DNA by silicic acid

S1 nuclease hydrolysis and hydroxyapatite chromatography were used to study the effect of silicic acid on DNA. Native calf thymus DNA was incubated with increasing concentrations of silicic acid (DNA nucleotide/silicic acid molar ratios of 1:0.25, 1:0.5 and 1:1) and subjected to S1 nuclease hydrolysis. An increasing degree of DNA degradation was seen suggesting a destabilization of the secondary structure. A decrease in melting temperature was also observed. Hydroxyapatite chromatography indicated that incubation at the molar ratio of 1:1 resulted in denaturation and degradation of DNA.     

Evidence for a subunit structure of chromatin in mouse myeloma cells

If micrococcal nuclease is allowed to digest chromatin as it exists inside intact nuclei isolated from mouse myeloma tissue culture cells, more than 60% of the DNA can be isolated as a homogeneous fragment on a sucrose gradient. Analytical ultracentrifugation indicates that the protected DNA is native, unnicked, and about 140 +/- 10 base pairs long. After less extensive nuclease digestion, the protected DNA migrates in gels in lengths which are integral multiples of this 140 base pair "monomer" band. A submonomer band, 105 "/- 10 base pairs long, can also be detected. Similar digestion patterns were obtained by two different nuclear isolation procedures and even when intact cells were gently lysed directly in the digestion medium. These results confirm and extend the chromatin digestion studies of previous investigators and provide support for a subunit model for eukaryotic chromatin. The single strand specific S1 nuclease did not digest intranuclear chromatin under the conditions used.     

A comparison of the digestion of nuclei and chromatin by staphylococcal nuclease.

We have followed the kinetics of staphylococcal nuclease digestion of duck reticulocyte nuclei and chromatin from early stages to the digestion limit. We confirm that partial digestion of nuclei produces discrete DNA bands which are multiples of a monomer, 185 base pairs in length. The multimers are shown to be precursors of the monomer, which is next digested to a homogeneous, 140 base pair fragment. This fragment in turn gives rise to an array of nuclear limit digest DNA bands, which is almost identical with the limit digest pattern of isolated chromatin. As in the case of chromatin, half the DNA of nuclei is acid soluble at this limit. While the DNA limit digest patterns of nuclei and chromatin are similar, the large multimeric structures present as intermediates in nuclear digestion are absent in chromatin digestion. Alternate methods of chromatin gel preparation appear to leave more of the higher order structure intact, as measured by the production of these multimeric bands. Our results are consistent with the "beads on a string" model of chromatin proposed by others.     

A Nuclease from Neurospora Crassa Conidia Specific for Single-Stranded Nucleic Acids

This report describes the purification from sonicates of Neurospora crassa conidia of a nuclease with extremely high specificity for single-stranded nucleic acids. The enzyme was purified 510-fold from streptomycin-treated sonicates in successive steps by (NH₄)₂SO₄ fractionation, acetone fractionation, by chromatography on phosphocellulose, DEAE-cellulose, Sephadex G-200 and hydroxy apatite and, finally, by preparative polyacrylamide gel electrophoresis. The yield of purified enzyme was 7%. Only one protein component was detected by analytical polyacrylamide gel electrophoresis at pH8.9, but, in the presence of 1% sodium dodecyl sulfate and 1% mercaptoethanol at pH7.0, one minor component (approximately 10% of the total protein, mol. wt. approximately 77,000) and one major component (mol. wt. approximately 72,000) were detected. The enzyme degraded denatured DNA rapidly but did not release any acid-soluble material from native DNA. It also did not alter the sedimentation properties of native bacteriophage T7 DNA. The only action on native DNA that was detected was a slow conversion of the superhelical form of bacteriophage S13 DNA to the open circle form. The products of a 10% digest (10% acid-soluble material) of denatured DNA contained 5′-mono-nucleotides and oligonucleotides (di- to decanucleotides) in a ratio of 3 to 1, indicating that the digestion was predominantly exonucleolytic in character.     

Parameters influencing the flow cytometric analysis of DNA sensitivity to nuclease S1

Some parameters that influence the analysis in situ of DNA sensitivity to digestion with nuclease S1 have been studied in isolated HeLa nuclei with flow cytometry. DNA staining with the intercalating fluorochrome propidium iodide allowed the nucleolytic activity on double-stranded (ds) DNA to be determined by monitoring the relative reduction in nuclear fluorescence intensity. Nuclei isolated in buffer at low ionic strength in order to decondense chromatin fibres, showed a lower fluorescence intensity than nuclei with native chromatin, after digestion with nuclease S1 under identical conditions. Nuclei prepared with dispersed chromatin and digested with increasing amounts of enzyme showed a decrease in fluorescence intensity that reached a limit value at about 50% of the value of undigested control samples. On the other hand, in nuclei with native chromatin, fluorescence intensity decreased only about 18%. The NaCl concentration in the reaction buffer strongly influenced the DNA sensitivity to S1 nuclease. By increasing salt molarity from 5 mM to 200 mM, the digestion of dsDNA was significantly reduced as also shown by the amount of released nucleotides from purified calf thymus DNA. The detection of DNA sensitivity to nuclease S1, as assessed by the cytometric method, was shown to be more sensitive than a biochemical technique involving hydrolysis of purines. These results indicate that both the procedure for nuclei isolation and the digestion conditions have to be carefully controlled when evaluating in situ the presence of S1-sensitive sites.     

A correlation between nucleosome spacer region susceptibility to DNase I and histone acetylation.

Hepatoma tissue culture (HTC) cell nuclei were digested with either DNase I or micrococcal nuclease and the nucleohistone digestion products fractionated by gel electrophoresis or exclusion chromatography. Under appropriate conditions, gel electrophoresis demonstrates that for both nucleases, only cleavages within the nucleosome spacer regions and not within the nucleosome core lead to freely migrating nucleohistone particles. These particles consist of nucleosome cores, nucleosomes and nucleosome oligomers. Following DNase I digestion and fractionation by exclusion chromatography, analysis of the histones indicates a direct relationship between increased spacer region susceptibility to nuclease and increased nucleosomal histone acetylation. Evidently digestion sites outside the regions of DNA protected by core histones can reflect the degree of acetylation of core histones. Such a relationship is not found when micrococcal nuclease is used to digest the samples.     

Chromatin core particle unfolding induced by tryptic cleavage of histones.

Chromatin 'core particles' have been digested with trypsin to varying extents. The resulting particles are homogeneous by the criterion of ultracentrifuge boundary analysis. Sedimentation coefficients are lowered as cleavages are introduced into the histones, showing that an unfolding of the core particle occurs. This unfolding is further characterised by a lower melting temperature together with a premelting phase, higher molar ellipticity in the circular dichroism spectra at 280 nm and increased kinetics of digestion by both micrococcal nuclease and DNase I. Differences are also observed in the products of nuclease digestion. The most consistent interpretation of the data involves an unfolding process whereby free rods of DNA are released to extend from a nucleoprotein core.     

Physical-chemical properties of the estrogen receptor solubilized by micrococcal nuclease

The physical-chemical properties of the nuclear estrogen receptor from MCF-7 cells were determined. The receptor was solubilized by micrococcal nuclease. Nuclei were isolated from cells previously exposed to 10 nM [3H]estradiol. The amount of receptor released was parallel to the extent of chromatin solubilized, which suggested that the receptor is homogeneously distributed on the chromatin. Following mild nuclease digestion the excised receptor sedimented as an abundant 6-7 S form and as a less abundant approximately 12 S species. The 6-7 S form represented the receptor excised in association with linker DNA, while the approximately 12 S may represent receptor bound to linker DNA which remained associated with the nucleosome. Increasing the extensiveness of digestion resulted in one receptor form sedimenting at 5.6 S. Additional digestion with DNase I did not affect the sedimentation coefficient of the receptor. Sedimentation of the micrococcal nuclease hydrolysate in a 0.4 M KCl sucrose gradient resulted in a 4.2 S receptor form. The same receptor form was extracted from undigested nuclei with 0.4 M KCl. Using Sephadex G-200 column chromatography we have determined the Stokes radii (Rs), molecular weight (Mr) and frictional ratio (f/fo) for the 5.6 S and 4.2 S receptor forms. For the 5.6 S form: Rs = 7.04 nm, Mr = 163,000 and (f/fo) = 1.80. For the 4.2 S receptor, Rs = 4.45 nm, Mr = 77,000 and (f/fo) = 1.46. The ability of the nuclease solubilized 5.6 S receptor to bind DNA was tested using DNA-cellulose column and highly polymerized DNA. About 40% of the applied receptor bound to the column and could be eluted by high salt concentrated buffer. The 5.6 S receptor form was sedimented on sucrose gradient by the highly polymerized DNA. These results suggested that the receptor is bound in chromatin as a dimer or as a monomer in association with other protein(s) which complexed it with DNA.     

Parameters influencing the flow cytometric analysis of DNA sensitivity to nuclease S1

Summary Some parameters that influence the analysis in situ of DNA sensitivity to digestion with nuclease S1 have been studied in isolated HeLa nuclei with flow cytometry. DNA staining with the intercalating fluorochrome propidium iodide allowed the nucleolytic activity on double-stranded (ds) DNA to be determined by monitoring the relative reduction in nuclear fluorescence intensity. Nuclei isolated in buffer at low ionic strength in order to decondense chromatin fibres, showed a lower fluorescence intensity than nuclei with native chromatin, after digestion with nuclease S1 under identical conditions. Nuclei prepared with dispersed chromatin and digested with increasing amounts of enzyme showed a decrease in fluorescence intensity that reached a limit value at about 50% of the value of undigested control samples. On the other hand, in nuclei with native chromatin, fluorescence intensity decreased only about 18%. The NaCl concentration in the reaction buffer strongly influenced the DNA sensitivity to S1 nuclease. By increasing salt molarity from 5 mM to 200 mM, the digestion of dsDNA was significantly reduced as also shown by the amount of released nucleotides from purified calf thymus DNA. The detection of DNA sensitivity to nuclease S1, as assessed by the cytometric method, was shown to be more sensitive than a biochemical technique involving hydrolysis of purines. These results indicate that both the procedure for nuclei isolation and the digestion conditions have to be carefully controlled when evaluating in situ the presence of S1-sensitive sites.     

Ribosomal DNA sequences attached to the nuclear matrix

The organization of rat liver ribosomal DNA (rDNA) as matrix-attached DNA loops was examined using a protocol which fractionates chromatin from discrete regions of DNA loops. Southern blot analysis of matrix-attached and solubilized chromatin DNA fragments demonstrated that rDNA is associated with the matrix via its 5' and 3' nontranscribed spacer sequences (NTS). Although the 45 S rRNA coding sequences were approximately threefold enriched in matrix preparations, the recovery of this DNA (unlike the NTS) was dependent on the extent of nuclease digest and proportional to the length of the matrix-attached DNA fragments. The data suggest that rDNA is organized as matrix-attached DNA loops and only the NTS are directly involved in matrix binding. Further, we demonstrated that while the kinetics and extent of nuclease digestion were similar in all regions of the DNA loops, the nuclease digestion pattern of bulk nuclear and matrix DNA showed a typical nucleosome organization, but the rDNA fragments retained with the nuclear matrix did not.     

Small repeated sequences in the chloroplast genome of Chlamydomonas reinhardi

Summary Chloroplast DNA of Chlamydomonas reinhardi contains many inverted repeated sequences. Analysis by hydroxyapatite binding, S1 nuclease digestion, and electron microscopy indicates that these sequences are 0.1–0.3 kilobase pairs in length, are widely distributed in the chloroplast genome, and make up 4–7% of the chloroplast DNA.Abbreviations RNA ribonucleic acid - rRNA ribosomal RNA - RNA complementary RNA - DNA deoxyribonucleic acid - chl DNA chloroplast DNA - HAP hydroxypatite - SSC 0.15 M NaCl, 0.015 M sodium citrate - 0.1xSSC, 2xSSC, 4.67xSSC 0.1, 2, and 4.67 times the concentration of SSC, respectively - TCA trichloroacetic acid - PB NaPO₄ buffer, pH 6.8 - Kb Kilobase - KbP Kilobase pair     

In vitro studies on mild alkali induced lesions in DNA.

S1 nuclease hydrolysis, benzoylated naphthoylated DEAE cellulose (BND-cellulose) chromatography as well as certain immunological and genetic techniques have been used to evaluate the effect of mild alkali (pH10.0) on the DNA molecule. Native calf thymus DNA after exposure to alkaline pH10.0 when subjected to S1 nuclease hydrolysis released significant amount of acid soluble nucleotides as compared to the untreated control. With pBR322 DNA, the population of linear DNA species increased on S1 digestion with concomitant reduction in the supercoiled form. BND cellulose chromatographic studies also suggested the formation of single strandedness and/or distortions in the alkali treated DNA molecule. Antisera raised against the alkali treated DNA exhibited high cross-reactivity with both single stranded and Z DNA. Moreover, a significant reduction in transformation frequency of the treated DNA molecule compared with the untreated control further ascertained the structural alterations in DNA as a result of exposure to mild alkali.     

Single-strand-specific degradation of DNA during isolation of rat liver nuclei

We have investigated structural change in rat liver DNA produced by different isolation procedures and specifically compared the integrity of DNA derived by phenol extraction from isolated and purified nuclei with preparations extracted immediately from a crude liver homogenate containing intact nuclei. As indicated by stepwise elution from benzoylated DEAE-cellulose, most structural change in DNA was evident following nuclei isolation. Damage principally involved generation of single-stranded regions in otherwise double-stranded DNA fragments; totally single-stranded DNA was not detected by hydroxylapatite chromatography. Caffeine gradient elution suggested formation of single-stranded regions extending for up to several kilobases. In neutral sucrose gradients, differences in sedimentation rates of respective DNA samples consequent upon S1 nuclease digestion could be detected after isolation of nuclei, though not in other circumstances. The observed single-strand-specific nuclease digestion of DNA could apparently be reduced if steps were taken to reduce autodigestion during nuclei isolation by reduction of temperature and covalent cation concentration. The results are discussed in terms of the use of exogenous and endogenous nucleases in chromatin fractionation studies involving isolated nuclei and possible artifactual findings that may be generated by single-strand-specific autodigestion.     

The assembly of an H2A2,H2B2,H3,H4 hexamer onto DNA under conditions of physiological ionic strength

A novel nucleohistone particle is generated in high yield when a complex of DNA with the four core histones formed under conditions that are close to physiological (0.15 M NaCl, pH 8) is treated with micrococcal nuclease. The particle was found to contain 102 base pairs of DNA in association with six molecules of histones in the ratio 2H2A:2H2B:1H3:1H4 after relatively brief nuclease treatment. Prolonged nuclease digestion resulted in a reduction in the DNA length to a sharply defined 92-base pair fragment that was resistant to further degradation. Apparently normal nucleosome core particles containing two molecules each of the four core histones in association with 145 base pairs of DNA and a particle containing one molecule each of histones H2A and H2B in association with approximately 40 base pairs of DNA were also generated during nuclease treatment of the histone-DNA complexes formed under physiological ionic strength conditions. Kinetic studies have shown that the hexamer particle is not a subnucleosomal fragment produced by the degradation of nucleosome core particles. Furthermore, the hexamer particle was not found among the products of nuclease digestion when histones and DNA were previously assembled in 0.6 M NaCl. The high sedimentation coefficient of the hexameric complex (8 S) suggests that the DNA component of the particle has a folded conformation.     

Ordered replication of DNA sequences: synthesis of mouse satellite and adjacent main band sequences.

The replication of mouse satellite DNA was delayed when synchronized 3T3 cells were exposed to low concentrations of hydroxyurea during S phase, It appears that the onset of satellite replication is not a time dependent event, but instead requires that a certain amount of main band DNA be synthesized first. Using hydroxyapatite chromatography and S1 nuclease digestion, a procedure was developed to quantitate the synthesis of both satellite and neighboring main band sequences. The replication kinetics of satellite determined by this method agree with previous estimates. Main band sequences adjacent to satellite appear to replicate in concert with satellite DNA. The results are discussed and related to the limitations of the techniques utilized.     

Transformation in Bacillus subtilis: characterization of an intermediate in recombination observed during transformation.

Summary From recombination-proficient competent cells of Bacillus subtilis in which the donor DNA entered at 17°, and which were kept at the same temperature, a complex of donor DNA and the recipient chromosome can be obtained which has a relatively high buoyant density in CsCl gradients. Exposure of the isolated complex to nuclease S1 liberates donor radioactivity. The limited biological activity of DNA re-extracted from cells attempting to recombine at 17° is decreased upon incubation with nuclease S1. If recombination is allowed to proceed at 30°, the high buoyant density of the donor-recipient complex decreases to normal values and less radioactivity can be liberated from the complex by nuclease S1. Concomitantly the biological activity of re-extracted DNA becomes less vulnerable to nuclease S1 under these conditions. On the basis of these observations we assume that the intermediate complex partly consists of unpaired single-stranded donor DNA.Support for the correctness of this assumption is derived from experiments with a mutant, which is delayed in the processing of high buoyant density donor-recipient complex to normal buoyant density donor-recipient complex. This delay is reflected in the time of acquisition of resistance to nuclease S1 digestion of the isolated complex.