Sites in simian virus 40 chromatin which are preferentially cleaved by endonucleases.

Simian virus 40 (SV40) chromatin isolated from infected BSC-1 cell nuclei was incubated with deoxyribonuclease I, staphylococcal nuclease or an endonuclease endogenous to BSC-1 cells under conditions selected to introduce one doublestrand break into the viral DNA. Full-length linear DNA was isolated, and the distribution of sites of initial cleavage by each endonuclease was determined by restriction enzyme mapping. Initial cleavage of SV40 chromatin by deoxyribonuclease I or by endogenous nuclease reduced the recovery of Hind III fragment C by comparison with the other Hind III fragments. Similarly, Hpa I fragment B recovery was reduced by comparison with the other Hpa I fragments. When isolated SV40 DNA rather than SV40 chromatin was the substrate for an initial cut by deoxyribonuclease I or endogenous nuclease, the recovery of all Hind III or Hpa I fragments was approximately that expected for random cleavage. Initial cleavage by staphylococcal nuclease of either SV40 DNA or SV40 chromatin occurred randomly as judged by recovery of Hind III or Hpa I fragments. These results suggest that, in at least a portion of the SV40 chromatin population, a region located in Hind III fragment C and Hpa I fragment B is preferentially cleaved by deoxyribonuclease I or by endogenous nuclease but not by staphylococcal nuclease.Complementary information about this nuclease-sensitive region was provided by the appearance of clusters of new DNA fragments after restriction enzyme digestion of DNA from viral chromatin initially cleaved by endogenous nuclease. From the sizes of new fragments produced by different restriction enzymes, preferential endonucleolytic cleavage of SV40 chromatin has been located between map positions 0.67 and 0.73 on the viral genome.     

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.     

Methylation and degradation of chromatin DNA in isolated rat liver cell nuclei]

Methylation of chromatin DNA in rat liver cell nuclei incubated in a medium with [3H]CH3-S-adenosyl methionine was studied. It was shown that under the given experimental conditions DNA methylation and chromatin degradation by endogenous nuclear nuclease (nucleases) with a formation of chromatin structural subunits occur simultaneously. An analysis of methylated chromatin DNA degradation products based on a number of approaches demonstrated a predominant methylation of extra-nucleosomal DNA. The data obtained suggest that chromatin of isolated nuclei contain sites with supermethylated DNA fragments incorporating not less than 400 nucleotide pairs. These sites possess an increased sensitivity to endogenous nuclease.     

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.     

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.     

Androgen treatment protects mouse liver chromatin from cleavage by endogenous nucleases during aging

We have examined the endogenous nuclease activity of the liver of intact, castrated and testosterone-treated mice of different ages. Both Mg²⁺- and Ca²⁺-dependent endogenous nuclease activities decline in old age. Withdrawal of the hormone increases nuclease activity in the immature and young. However, testosterone administration prevents the digestion of nuclei to different extents in all ages. These findings suggest a possible protective role of testosterone in the cleavage of liver chromatin by endogenous nucleases during the aging of mice.     

The histone chaperone Asf1p mediates global chromatin disassembly in vivo

The packaging of the eukaryotic genome into chromatin is likely to be mediated by chromatin assembly factors, including histone chaperones. We investigated the function of the histone H3/H4 chaperones anti-silencing function 1 (Asf1p) and chromatin assembly factor 1 (CAF-1) in vivo. Analysis of chromatin structure by accessibility to micrococcal nuclease and DNase I digestion demonstrated that the chromatin from CAF-1 mutant yeast has increased accessibility to these enzymes. In agreement, the supercoiling of the endogenous 2mu plasmid is reduced in yeast lacking CAF-1. These results indicate that CAF-1 mutant yeast globally under-assemble their genome into chromatin, consistent with a role for CAF-1 in chromatin assembly in vivo. By contrast, asf1 mutants globally over-assemble their genome into chromatin, as suggested by decreased accessibility of their chromatin to micrococcal nuclease and DNase I digestion and increased supercoiling of the endogenous 2mu plasmid. Deletion of ASF1 causes a striking loss of acetylation on histone H3 lysine 9, but this is not responsible for the altered chromatin structure in asf1 mutants. These data indicate that Asf1p may have a global role in chromatin disassembly and an unexpected role in histone acetylation in vivo.     

Site and stage specific action of endogenous nuclease and micrococcal nuclease on histone genes of sea urchin embryos

The early histone genes of sea urchin embryos are expressed exclusively during cleavage stages of embryogenesis. The chromatin containing these genes was examined by nuclease sensitivity. An endogenous nuclease active during cleavage, produces 1300-bp segments containing early histone genes. The cutting sites have been mapped; there are very sensitive sites close to the cap site for H1, H2A, H2B, and H4. Chromatin obtained from embryos of later stages, when the genes are not expressed, do not display this pattern of nuclease sensitivity. Micrococcal nuclease produces nucleosomes that contain histone genes when used with nuclei from later stages, but not with nuclei from cleavage stages.     

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.     

Novobiocin induces the in vivo cleavage of active gene sequences in intact cells

When the topoisomerase II inhibitor, novobiocin, is administered to embryonic chicken red blood cells, it induces the in vivo release of an endogenous nuclease which cleaves specifically within internucleosomal spacer DNA and within nuclease-hypersensitive sites in the active chromatin of intact cells. This in vivo released nuclease activity is induced by novobiocin only in metabolically active immature red blood cells. Little induction occurs in mature erythrocytes and no induction occurs in cells previously treated with 2,4-dinitrophenol. Although novobiocin is required to induce release and/or activation of the nuclease, the activity of the nuclease, once activated, is independent of novobiocin. Analysis of the cleaved DNA in drug-treated immature cells demonstrates that the novobiocin-induced nuclease has an unusual blunt-ended double-stranded mode of cleavage. Because of its special properties and apparent chromatin related function in vivo, the novobiocin-induced nuclease activity offers a novel and useful in vivo and in vitro probe of chromatin structure.     

Transient conformation changes in chromatin during excision repair of ultraviolet damage to DNA.

DNA labeled for 15 minutes during UV induced repair synthesis is two-fold more sensitive to micrococcal nuclease than the bulk nuclear DNA. As the length of the labeling period increases from 15 minutes to 4 hours the nuclease sensitivity of repair labeled DNA approaches that of bulk chromatin. Pulse-chase experiments indicate that the nuclease sensitivity of the repaired DNA labeled during a brief pulse decreases with a half-life of about 15 minutes. In contrast to previous interpretations, we consider these results to mean that immediately after synthesis, chromatin labeled during repair has a conformation which renders it more susceptible to nuclease digestion than the bulk chromatin. With time these repaired regions are assembled into a nucleosome structure with normal nuclease sensitivity.     

DNase I hypersensitive regions correlate with a site-specific endogenous nuclease activity on the r-chromatin of Tetrahymena

A novel nuclease activity have been detected at three specific sites in the chromatin of the spacer region flanking the 5'-end of the ribosomal RNA gene from Tetrahymena. The endogenous nuclease does not function catalytically in vitro, but is in analogy with the DNA topoisomerases activated by strong denaturants to cleave DNA at specific sites. The endogenous cleavages have been mapped at positions +50, -650 and -1100 relative to the 5'-end of the pre-35S rRNA. The endogenous cleavage sites are associated with micrococcal nuclease hypersensitive sites and DNase I hypersensitive regions. Thus, a single well-defined micrococcal nuclease hypersensitive site is found approximately 130 bp upstream from each of the endogenous cleavages. Clusters of defined sites, the majority of which fall within the 130 bp regions defined by vicinal micrococcal nuclease and endogenous cleavages, constitute the DNase I hypersensitive regions.     

Distinct roles of two separable in vitro activities of yeast Mre11 in mitotic and meiotic recombination.

In Saccharomyces cerevisiae, Mre11 protein is involved in both double-strand DNA break (DSB) repair and meiotic DSB formation. Here, we report the correlation of nuclease and DNA-binding activities of Mre11 with its functions in DNA repair and meiotic DSB formation. Purified Mre11 bound to DNA efficiently and was shown to have Mn2+-dependent nuclease activities. A point mutation in the N-terminal phosphoesterase motif (Mre11D16A) resulted in the abolition of nuclease activities but had no significant effect on DNA binding. The wild-type level of nuclease activity was detected in a C-terminal truncated protein (Mre11DeltaC49), although it had reduced DNA-binding activity. Phenotypes of the corresponding mutations were also analyzed. The mre11D16A mutation conferred methyl methanesulfonate-sensitivity to mitotic cells and caused the accumulation of unprocessed meiotic DSBs. The mre11DeltaC49 mutant exhibited almost wild-type phenotypes in mitosis. However, in meiosis, no DSB formation could be detected and an aberrant chromatin configuration was observed at DSB sites in the mre11DeltaC49 mutant. These results indicate that Mre11 has two separable functional domains: the N-terminal nuclease domain required for DSB repair, and the C-terminal dsDNA-binding domain essential to its meiotic functions such as chromatin modification and DSB formation. Keywords: DNA binding/double-strand break repair/DSB formation/Mre11/nuclease     

Different repeat lengths in rat satellite I DNA containing chromatin and bulk chromatin.

The nucleosome repeat structure of a rat liver chromatin component containing the satellite I DNA (repeat length 370 bp) was investigated. Digestion experiments with micrococcal nuclease, DNAase II, and the Ca2+/Mg2+-dependent endogenous nuclease of rat liver nuclei revealed a repeat unit of 185 nucleotide pairs which is shorter by approximately 10 bp than the repeat unit of the bulk chromatin of this cell type. The difference seems not to be related to the histone composition which was found to be similar in the two types of chromatin.     

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).     

ChIC and ChEC; genomic mapping of chromatin proteins

To map the genomic interaction sites of chromatin proteins, two related methods were developed and experimentally explored in Saccharomyces cerevisiae. The ChIC method (chromatin immunocleavage) consists of tethering a fusion protein (pA-MN) consisting of micrococcal nuclease (MN) and staphylococcal protein A to specifically bound antibodies. The nuclease is kept inactive during the tethering process (no Ca2+). The ChEC method (chromatin endogenous cleavage) consists of expressing fusion proteins in vivo, where MN is C-terminally fused to the proteins of interest. The specifically tethered nucleases are activated with Ca2+ ions to locally introduce double-stranded DNA breaks. We demonstrate that ChIC and ChEC map proteins with a 100-200 bp resolution and excellent specificity. One version of the method is applicable to formaldehyde-fixed nuclei, another to native cells with comparable results. Among various model experiments, these methods were used to address the conformation of yeast telomeres.     

Internucleosome interaction: detection of dinucleosome fragmentation of chromatin by micrococcal nuclease. Analysis of the products of cleavage of chromatin from rat liver nuclei and L cells by micrococcal nuclease

In murine L-cell nuclei micrococcal nuclease causes chromatin fragmentation with predominant liberation of dinucleosomes. Analysis of dynamics of rat liver nuclear chromatin cleavage by micrococcal nuclease revealed that the "dinucleosomal" mode of fragmentation is due to the pretreatment of nuclei with the non-ionic detergent Triton X-100 in the course of the isolation procedure. The set of particles detected in nuclease hydrolysates of nuclear chromatin pretreated with Triton X-100 and those isolated by the standard procedure was shown to be significantly different. In Triton X-100 treated nuclei the dichromatosome is the main hydrolysate component under various experimental conditions of nuclease hydrolysis and the sole component under "mild" conditions, whereas sucrose-treated nuclei contain three types of dinucleosomes. In Triton-treated nuclei prolongation of hydrolysis results in the liberation of the chromatosome which is absent in chromatin hydrolysates of sucrose-treated nuclei. Hydrolysis of Triton-treated nuclear chromatin by micrococcal nuclease is unaccompanied by the liberation (up to the stage of "deep" hydrolysis) of the core particle, the major component of the "sucrose" nuclear hydrolysate under the conditions used. The sharp differences in the accessibility of various types of dinucleosomes observed during pretreatment of nuclei with Triton X-100 are interpreted in terms of the localization of histone H1. The non-random type of the histone H1 molecule orientation along the nucleosome fibril is postulated.