ATP as an alternative inhibitor of bacterial and endogenous nucleases and its effect on native chromatin compaction

The studies reported here demonstrate that ATP may be used in lieu of EDTA to inhibit nuclease digestion of DNA and chromatin. Because ATP is a milder chelator than EDTA and is a biochemical common to the cellular microenvironment in vivo, critical studies of cellular processes that require native structure to be maintained are more feasible without the presence of strong chelators. During the digestion of chromatin into its components by nuclease treatment, ATP assures the retention of nucleoprotein compaction, particularly for large to intermediate-sized oligosomes (2400bp–1000bp in length). ATP used at a concentration of 3.3 mM appears to be somewhat better than EDTA, 1.0 mM, for minimizing degradation of nuclease-treated chromatin. However, termination of nuclease digestion of chromatin and minimization of further degradation by the addition of ATP to a concentration of 1.0 mM was almost equivalent to the addition of EDTA to a concentration of 1.0 mM. Slightly more degradation was observed for the latter condition. In addition, ATP can be used to inhibit endogenous nuclease activity when specific restriction enzymes are needed. Standard low ionic strength DNP, deoxyribonucleoprotein, and DNA electrophoresis of proteinized and deproteinized chromatin oligomers, respectively, indicated that ATP effectively inhibits staphylococcal nuclease. Low ionic strength nucleoprotein electrophoresis to resolve staphylococcal nuclease-digested chromatin indicates that as little as 10^–4 M EDTA can promote structural unfolding resulting in changes in apparent mobilities for chromatin oligomers 250 and 600 by in length. Comparative digestion of chromatin with staphlococcal nuclease followed by reaction termination by ATP or EDTA showed that this observation was not merely the result of degradation due to inefficiency of ATP enzyme inhibition.     

Nanotribology results show that DNA forms a mechanically resistant 2D network in metaphase chromatin plates

In a previous study, we found that metaphase chromosomes are formed by thin plates, and here we have applied atomic force microscopy (AFM) and friction force measurements at the nanoscale (nanotribology) to analyze the properties of these planar structures in aqueous media at room temperature. Our results show that high concentrations of NaCl and EDTA and extensive digestion with protease and nuclease enzymes cause plate denaturation. Nanotribology studies show that native plates under structuring conditions (5 mM Mg²⁺) have a relatively high friction coefficient (μ ≈ 0.3), which is markedly reduced when high concentrations of NaCl or EDTA are added (μ ≈ 0.1). This lubricant effect can be interpreted considering the electrostatic repulsion between DNA phosphate groups and the AFM tip. Protease digestion increases the friction coefficient (μ ≈ 0.5), but the highest friction is observed when DNA is cleaved by micrococcal nuclease (μ ≈ 0.9), indicating that DNA is the main structural element of plates. Whereas nuclease-digested plates are irreversibly damaged after the friction measurement, native plates can absorb kinetic energy from the AFM tip without suffering any damage. These results suggest that plates are formed by a flexible and mechanically resistant two-dimensional network which allows the safe storage of DNA during mitosis.     

Limited nuclease digestion of heterogeneous nuclear ribonucleoprotein in nuclei.

We have used limited nuclease digestion of nuclei to probe the structure of nuclear ribonucleoprotein (nRNP). Analysis of [³H]uridine-labeled heterogeneous nuclear RNA isolated from nuclease digested nuclei revealed preferential generation of discrete bands of RNA ranging in size from 1.5 × 10⁵ to 6 × 10⁵ daltons. The nuclease digestion pattern of nRNP differed from the nuclease digestion pattern obtained with chromatin in that the RNA bands generated in these experiments were transient, appearing only early in the course of digestion, and no stable nRNP monomer size was evident. Therefore, although nRNP may be organized in a regular configuration, nRNP structure differs considerably from the repeating subunit structure of chromatin.     

Generating Nucleosomal Ladders In Vivo by Releasing Endogenous Endonucleases in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Chromatin maps by enzymatic digestion using micrococcal nuclease (MNase) have served as popular substrates for molecular epigenetic studies. Such analyses have been limited to cell wall minus mutants of Chlamydomonas reinhardtii because of the complications in nucleus isolation due to cell wall removal. Here, we describe an endogenous endonuclease in Chlamydomonas that is specifically released from the cells upon abrasion with beads (glass or zirconia), in the presence of detergents. The resulting in vivo digests obtained from both cell wall containing cc124/cc125 and mutant strains (cc400) are typical of repetitive nucleosomal fragments (MNase-like ladders) ranging from mono- to poly-nucleosomes, indicating that the nuclease acts preferentially on inter-nucleosomal linker DNA. We demonstrate that the endonuclease activity is strictly dependent on divalent cations (Ca⁺²?>?Mg⁺²) and can be inhibited by both Cu⁺² and Zn⁺² (5 mM) or by divalent cation chelators like EDTA. Detailed standardization reveals that the nuclease is released only when the cells are treated with detergents, implying that it might be membrane bound or vesicular and intracellular in nature. This endonuclease seems to accumulate in late log-phase cultures and probably has a role in generation of apoptotic ladders in Chlamydomonas. Further activity-based in-gel DNase analyses of whole cell extracts, using denaturing SDS-PAGE, revealed a single major ~?30-kDa band upon renaturation. We further characterized this endonuclease by partial purification using ammonium sulfate precipitation. Activity-based analysis revealed partial enrichment in the 50% (NH₄)₂SO₄ peak fraction and also confirmed Ca dependence. Finally, adding back the released endonuclease from a detergent-treated supernatant of cc124 cells to intact cc400 cw ^? cells as substrate resulted in a typical ladder formation in the latter, confirming the intracellular release of the nuclease and its activity. In summary, by controlled release of calcium-dependent endogenous endonucleases (altering vortex and detergent conditions), we can generate dose-dependent nucleosomal ladders for epigenetic analyses in Chlamydomonas, as an alternate to MNase-derived substrates.     

Uptake of homologous single-stranded fragments by superhelical DNA. III. The product and its enzymic conversion to a recombinant molecule

When superhelical DNA (RFI)² of phages φX174 or G4 takes up a homologous single-stranded fragment, RF DNA and fragment are linked by as many as 300 base-pairs, and a corresponding length of one strand of the RFI is displaced, forming a displacement loop (D-loop). The length of the base-paired region was estimated from the fraction of the associated ³²P-labeled fragment that was resistant to digestion by exonuclease VII, as well as by electron microscopy. Dissociation of the fragment by heating was characterized by a sharp melting curve. The displaced strand of the RF DNA was digested by two endonucleases that act on single-stranded DNA, the S₁ nuclease of Aspergillus oryzae and the recBC DNAase of Escherichia coli. Acting on complexes, both enzymes converted the form I [³H]DNA into form II DNA, and left some of the associated ³²P-labeled fragment undigested. The remaining ³²P-labeled fragment could no longer be displaced by branch migration, as expected if the displaced strand of the RF DNA were digested. The action of S₁ nuclease also produced the amount of acid-soluble ³H expected from digestion of the D-loop. Treatment of such digested complexes with polynucleotide ligase covalently linked about 35% of the remaining ³²P-labeled fragment to ³H-labeled strands, which proves that S₁ nuclease digested the D-loop.     

Specificity and biological significance of microtubule-associated protein-DNA interactions in chick

The interactions between chick brain microtubule associated proteins (MAPs) and chick DNA have been examined using DNA-cellulose chromatography, cross-blotting, and nitrocellulose filter-binding. Comparison of nitrocellulose filter-binding and cross-blotting results show that while MAPs and a minor, M_r 48 000, protein show significant binding at 50 mM NaCl, only the latter continues to bind a significant amount of DNA at 150 mM NaCl, suggesting an ionic basis for the MAP-DNA interactions. MAP-DNA interactions also show weak preference for AT-rich fractions, and are sensitive to S1 nuclease digestion. We suggest that the MAPs bind preferentially to single-stranded DNA. The binding may involve an interaction between the DNA phosphates and the highly cationic tubulin-binding domain of the MAPs. Repetitive fractions of the chick genome prepared both by hydroxyapatite chromatography and by S1 nuclease digestion show binding to a number of minor proteins present in preparations of microtubule proteins, as well as to the MAPs. We conclude that the MAPs probably do not bind specifically to repetitive DNA, in contrast to earlier reports using mouse DNA. MAP-DNA interactions are therefore unlikely to be involved in the attachment of microtubules to mitotic chromosomes.     

Deoxyribonuclease II as a probe for chromatin structure. I. Location of cleavage sites

DNAase II has been shown to cleave condensed mouse liver chromatin at 100-bp² intervals while chromatin in the extended form is cleaved at 200-bp intervals (Altenburger et al., 1976). Evidence is presented here that DNA digestion patterns of a half-nucleosomal periodicity are also obtained upon DNAase II digestion of chicken erythrocyte nuclei and yeast nuclei, both of which differ in their repeat lengths (210 and 165 bp) from mouse liver chromatin. In the digestion of mouse liver nuclei a shift from the 100-bp to the 200-bp cleavage mode takes place when the concentration of monovalent cations present during digestion is decreased below 1 mM. When soluble chromatin prepared by micrococcal nuclease is digested with DNAase II the same type of shift occurs, albeit at higher ionic strength.In order to map the positions of the DNAase II cleavage sites on the DNA relative to the positions of the nucleosome cores, the susceptibility of DNAase II-derived DNA termini to exonuclease III was investigated. In addition, oligonucleosome fractions from HaeIII and micrococcal nuclease digests were end-labelled with polynucleotide kinase and digested with DNAase II under conditions leading to 100 and 200-bp digestion patterns. Analysis of the chain lengths of the resulting radioactively labelled fragments together with the results of the exonuclease assay allow the following conclusions. In the 200-bp digestion mode, DNAase II cleaves exclusively in the internucleosomal linker region. Also in the 100-bp mode cleavage occurs initially in the linker region. Subsequently, DNAase II cleaves at intranucleosomal locations, which are not, however, in the centre of the nucleosome but instead around positions 20 and 125 of the DNA associated with the nucleosome core. At late stages of digestion intranucleosomal cuts predominate and linkers that are still intact are largely resistant to DNAase II due to interactions between adjacent nucleosomes. These findings offer an explanation for the sensitivity of DNAase II to the higher-order structure of chromatin.     

Characterization and comparative analysis of a second thermonuclease from Staphylococcus aureus

Staphylococcal nuclease (here termed as Nuc1) is considered an important virulence factor and a unique marker widely used in the detection of Staphylococcus aureus. A second functional thermostable nuclease (here termed as Nuc2) in S. aureus was characterized after recombinant expression in Escherichia coli. Sequence alignment and phylogenetic analysis revealed that Nuc2 was a more conserved protein in the staphylococci group compared with Nuc1. Recombinant Nuc2 showed nuclease activity in the zymogram test and was able to degrade various types of nucleic acids. The optimal reaction temperature and pH for Nuc2 were 50 °C and pH 10, respectively. The enzymatic activity of Nuc2 was stimulated in the presence of Ca²⁺ (0.05 mM), Mg²⁺ (0.5 mM), dithiothreitol, β-mecaptoethanol, TritonX-100, Tween-20, and urea; however, activity decreased sharply when exposed to heavy metals such as Zn²⁺ and Mn²⁺, and in the presence of EDTA or SDS. Nuc2 showed weaker activity, lower thermostability and different sensitivity to these chemical agents compared with Nuc1, which was consistent with differences in the sequence pattern and structure predicted. Furthermore, a nuc1 and nuc2 double deletion mutant of S. aureus and respective complementation experiments suggest a major role for nuc1 in terms of thermonuclease activity in S. aureus.     

Letter: Some unusual properties of replicating adenovirus type 2 DNA

Replicating adenovirus type 2 DNA was isolated from KB cells 13 hours after infection. The buoyant density in caesium chloride of the replicating DNA was found to be 5 to 10 mg/cm² greater than that of mature adenovirus type 2 DNA. The single-strand specific nuclease from Neurospora crassa released 25 to 30% of the radioactivity from replicating DNA and the density difference between replicating and mature adenovirus DNA was eliminated after digestion with this enzyme, but not after digestion with RNase or pronase. The results suggest that the complementary strands of adenovirus type 2 DNA are replicated asynchronously.     

Structure of adenovirus chromatin

The structure of adenovirus type 2 chromatin isolated from wild-type and ts1 virions was investigated by micrococcal nuclease digestion and electron microscopy. Partial digestion of wild-type and ts1 chromatin with micrococcal nuclease generated a multimeric DNA smear devoid of the 200 basepair nucleosome repeating pattern characteristic of cellular chromatin. However, 11 S monomer cores of 150 basepairs were detectable. The chromatin of ts1 (39°C) was more resistant to digestion by micrococcal nuclease. Two-dimensional electrophoresis of the monomer core showed that wild-type core contained protein VII while ts1 (39°C) core contained PVI and PVII. Protein V appears to be located on the variable-length intermonomer region. Crosslinking studies suggest that proteins PVII and VII exist in dimeric form within the monomer core. Electron microscopy revealed a 5.5-fold-condensed two-micron-long beaded structure with about 200 monomer particles spaced irregularly. Based on these observations, a model for adenovirus prochromatin and chromatin is proposed that differs in important aspects from the model proposed previously (Corden, J., Engelking, H.M. and Pearson, G.D. (1976) Proc. Natl. Acad. Sci. USA 73, 401–404).     

Isolation of foldback DNA utilizing nuclease S1 digestion in aqueous dioxane.

An improved method for the isolation of the inverted repetitive (foldback) sequences present in mammalian DNAs is described. It makes use of the new observation that nuclease S1 digestion of denatured DNA occurred at a faster rate and was more extensive in medium containing dioxane. The temperature-absorbance characteristics of nuclease S1-resistant DNA were systematically studied as a function of the temperature employed during the step of enzymic hydrolysis. Specimens of human placental and calf thymus DNA which had been denatured and renatured to C₀t ≤ 10^?3 mol s liter^?1 were used as substrates. Foldback DNA was isolated from the enzymic digests by means of hydroxylapatite chromatography. Temperature-absorbance studies showed the enzyme-resistant DNA had a high degree of thermal stability; the hyperchromic rises equaled those obtained in the native speciments. The amount of foldback DNA which could be obtained was not influenced by the fragment size of the starting material, above a certain molecular weight range. Foldback DNA represented about 4% of the human genome and at least 5% of the bovine genome. The size distributions of these strands were studied by means of polyacrylamide gel electrophoresis.     

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.     

E. coli tRNAPhe modified at the 3-(3-amino-3-carboxypropyl)uridine with a photoaffinity label is fully functional for aminoacylation and for ribosomal interaction

E. coli tRNA^Phe was modified at its 3-(3-amino-3-carboxypropyl)uridine residue with the N-hydroxysuccinimide ester of N-4-azido-2-nitrophenyl)glycine. Exclusive modification of this base was shown by two-dimensional TLC analysis of the T₁ oligonucleotide and nucleoside products of nuclease digestion. The fully modified tRNA could be aminoacylated to the same level as control tRNA. The aminoacylated tRNA was as active as control tRNA in non-enzymatic binding to the P site of ribosomes, and in EFTu-dependent binding to the rirobosomal A site. The functional activity of this photolabile modified tRNA allows it to be used to probe the A and P binding sites on ribosomes and on other proteins that interact with tRNA. Crosslinking to the ribosomal P site has been shown.     

Structural organization of ribonucleoproteins containing small nuclear RNAs from HeLa cells. Proteins interact closely with a similar structural domain of U1, U2, U4 and U5 small nuclear RNAs

U₁ snRNP² isolated from HeLa cells and purified by centrifugation in cesium chloride contains a set of proteins that may be resolved into four/five polypeptides by gel electrophoresis. When this particle was submitted to extensive digestion with micrococcal nuclease, RNA fragments of about 25 nucleotides in length were obtained. Sequence analyses showed that these highly protected fragments were derived from the same region of the U₁ molecule, spanning positions 119 to 143. At low concentrations of nuclease, a longer fragment, from nucleotide 119 to the 3′ OH end, was also detected. U₁ core-resistant snRNP, isolated by high performance liquid chromatography, still contains all the protein components of the intact particle.When a less drastically purified U₁ snRNP containing, beside the four/five polypeptides remaining after centrifugation in cesium chloride, a set of at least three polypeptides of larger size, was digested with the nuclease, no other protected RNA fragment was detected.When a mixture of U₁, U₂, U₄, U₅ and U₆ snRNPs, which contains the same four/five polypeptides as U₁ snRNP, was treated with micrococcal nuclease, protected fragments of snRNAs U₂, U₄ and U₅ were found in addition to those derived from U₁. No fragment derived from U₆ was found.In all cases, the region of snRNA shielded from nuclease attack corresponds to a distinctive feature of the molecule. It is a single-stranded region, comprising the sequence A(U)_nG with n ≥ 3, bordered by two double-stranded stems. One of these stems includes the 3′ terminus of the RNA, except in the case of U₂, where there are two stems instead of one on the 3′ side of the single-stranded stretch. Although a comparable structural domain exists also in U₆ snRNA, it does not contain the sequence A(U)_nG which correlates well with the fact that no U₆ snRNA fragment seems to resist micrococcal nuclease digestion.     

Nascent DNA in nucleosome like structures from chromatin

We have used chromatin sensitivity to cleavage by micrococcal nuclease as a probe for differences between chromatin containing nascent DNA and that containing bulk DNA. Micrococcal nuclease digested the nascent DNA in chromatin of swimming blastulae of sea urchins more rapidly to acid-soluble nucleotides than the DNA of bulk chromatin. A part of the nascent DNA occurred in micrococcal nuclease-resistant structures which were either different from, or temporary modifications of, the bulk nucleosomes. This was inferred from the size differences between bulk and nascent DNA fragments in 10% polyacrylamide gels after micrococcal nuclease digestion of nuclei from a mixture of ¹⁴C-thymidine long- and ³H-thymidine pulse-labeled embryos. Bulk monomer and dimer DNA fragments contained about 170 and 410 base pairs (bp), respectively, when 18% of the bulk DNA had been rendered acid-soluble. At this level of digestion, “nascent monomer DNA” fragments of about 150 bp as well as 305 bp “large nascent DNA fragments” were observed. Increasing levels of digestion indicated that the large nascent DNA fragment was derived from a chromatin structure which was more resistant to micrococcal nuclease cleavage than bulk dimer chromatin subunits. Peaks of ³H-thymidine-labeled DNA fragments from embryos which had been pulse-labeled and then chased or labeled for several minutes overlapped those of ¹⁴C-thymidine long-labeled monomer, dimer and trimer fragments. This indicated that the chromatin organization at or near the replication fork which had temporarily changed during replication had returned to the organization of its nonreplicating state.