The subunit structure of chromatin from Physarum polycephalum.

Nucleosome DNA repeat lengths in Physarum chromatin, determined by nuclease digestion experiments, are shorter than those observed in most mammalian chromatin and longer than those reported for chromatin of certain other lower eukaryotes. After digestion with staphylococcal nuclease for short periods of time an average repeat length of 190 base pairs is measured. After more extensive digestion an average repeat length of 172 base pairs is measured. Upon prolonged digestion DNA is degraded to an average monomer subunit length of 160 base pairs, with only a small amount of DNA found in lengths of 130 base pairs or smaller. Mathematical analysis of the data suggests that the Physarum nucleosome DNA repeat comprises a protected DNA segment of about 159 base pairs with a nuclease-accessible interconnecting segment which ranges from 13 to 31 base pairs. The spacing data are compatible with measurements from electron micrographs of Physarum chromatin.     

Mechanism of exonuclease action of BAL 31 nuclease

Two kinetically and molecularly distinct forms ('fast' (F) and 'slow' (S] of nuclease BAL 31 from Alteromonas espejiana effect the length reduction of linear duplex DNAs through a 3'----5'-directed exonuclease activity in conjunction with an endonuclease activity against the 5'-terminated single-stranded tails generated by the exonuclease activity. No evidence for a 5'----3' mode of exonuclease action was seen. Single-stranded DNA is degraded predominantly by the 3'----5' exonuclease action. There is a pronounced decrease, to roughly constant values, of the average lengths of the tails in partially digested duplexes at a constant extent of digestion with increasing nuclease concentration. This decrease correlates with an increasing extent of ligatability, in the absence of repair, under conditions favoring the joining of fully base-paired ends. The exonuclease action, at least against duplex substrates, is quasi-processive and removes approx. 18 and 28 nucleotides per productive enzyme-substrate encounter for the S and F species, respectively. The dependence on Ca2+ and Mg2+ concentrations of the activities has been determined.     

The structural organization of sperm chromatin

The structure of rabbit, fowl, and Xenopus laevis sperm chromatin was explored by study of the reaction of their decondensed nuclei with DNase 1 and micrococcal nuclease. Those of rabbit and fowl were readily digested by DNase 1, and the polyacrylamide gel electrophoresis profiles of DNAs extracted from the digests were similar, each being polydisperse with a single discrete band of DNA smaller than 72 base pairs. There were differences, however, between the sperm chromatins in the course of their digestion by micrococcal nuclease. A limit digest at about 45% acid solubility was obtained with Xenopus sperm chromatin, while 90% of fowl sperm DNA was rendered acidsoluble by the enzyme. The gel profiles of the limit digests were polydisperse, but only those of rabbit and fowl sperm chromatins possessed a discrete band of DNA smaller than 72 base pairs. Bleomycin did not react with DNA of rabbit, fowl, or Xenopus spermatozoa. Since bleomycin reacts with somatic cell chromatin, and the course of DNase 1 or micrococcal nuclease digestion of sperm chromatin was different from that found for somatic cell chromatin, it would appear that sperm chromatin does not have the repeating nucleosometype structure of somatic cell chromatin. The nuclease digestion studies further suggest that the organization of rabbit and fowl sperm chromatins is similar, and is different from that of Xenopus sperm chromatin. The dependence of the structure of sperm chromatin on the composition of its basic proteins, and a possible structure for a protamine-type sperm chromatin, are discussed.     

AFM characterization of single strand-specific endonuclease activity on linear DNA

The specificity of nucleases for nicked and un-nicked double-stranded DNA has been characterized using atomic force microscopy (AFM). We have found that AFM has advantages over the usual macroscopic analyses, such as sucrose gradient centrifugation or electrophoresis, in characterizing nuclease digestion. In particular, short DNA fragments resulting from non-specific digestion were detected and, thus, the true length distribution of digested DNA was revealed. A simple numerical method is proposed to estimate the number of nicked sites per DNA molecule based on AFM images.     

Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis.

We describe the use of polyacrylamide gel electrophoresis to estimate chain lengths of double- and single-stranded DNA molecules in the size range 20-1000 base pairs (or nucleotides). Double-stranded DNA molecules of known length produced either by organic synthesis or by restriction endonuclease digestion of viral DNAs were used as standards. The relative electrophoretic mobilities of these standards were examined on both nondenaturing (aqueous) polyacrylamide gels and on denaturing gels containing 7 M urea or 98% formamide. Electrophoretic mobility of DNA is a linear function of the log of molecular weight if appropriate conditions are used, although exceptions are noted. Chain lengths can be conveniently estimated by using as standards bacteriophage gamma DNA restriction fragments or commercially available tracking dyes.     

Adenovirus chromatin structure at different stages of infection.

We investigated the structure of adenovirus deoxyribonucleic acid (DNA)-protein complexes in nuclei of infected cells by using micrococcal nuclease. Parental (infecting) DNA was digested into multimers which had a unit fragment size that was indistinguishable from the size of the nucleosomal repeat of cellular chromatin. This pattern was maintained in parenteral DNA throughout infection. Similar repeating units were detected in hamster cells that were nonpermissive for human adenovirus and in cells pretreated with n-butyrate. Late in infection, the pattern of digestion of viral DNA was determined by two different experimental approaches. Nuclear DNA was electrophoresed, blotted, and hybridized with labeled viral sequences; in this procedure all virus-specific DNA was detected. This technique revealed a diffuse protected band of viral DNA that was smaller than 160 base pairs, but no discrete multimers. All regions of the genome were represented in the protected DNA. To examine the nuclease protection of newly replicated viral DNA, infected cells were labeled with [3H]thymidine after blocking of cellular DNA synthesis but not viral DNA synthesis. With this procedure we identified a repeating unit which was distinctly different from the cellular nucleosomal repeat. We found broad bands with midpoints at 200, 400, and 600 base pairs, as well as the limit digest material revealed by blotting. High-resolution acrylamide gel electrophoresis revealed that the viral species comprised a series of closely spaced bands ranging in size from less than 30 to 250 base pairs.     

Chromatin assembly in isolated mammalian nuclei.

Cellular DNA replication was stimulated in confluent monolayers of CV-1 monkey kidney cells following infection with SV40. Nuclei were isolated from CV-1 cells labeled with [3H]thymidine and then incubated in the presence of [alpha-32P]deoxyribonucleoside triphosphates under conditions that support DNA replication. To determine whether or not the cellular DNA synthesized in vitro was assembled into nucleosomes the DNA was digested in situ with either micrococcal nuclease or pancreatic DNase I, and the products were examined by electrophoretic and sedimentation analysis. The distribution of DNA fragment lengths on agarose gels following micrococcal nuclease digestion was more heterogeneous for newly replicated than for the bulk of the DNA. Nonetheless, the state of cellular DNA synthesized in vitro (32P-labeled) was found to be identical with that of the DNA in the bulk of the chromatin (3H-labeled) by the following criteria: (i) The extent of protection against digestion by micrococcal nuclease of DNase I. (ii) The size of the nucleosomes (180 base pairs) and core particles (145 base pairs). (iii) The number and sizes of DNA fragments produced by micrococcal nuclease in a limit digest. (iv) The sedimentation behavior on neutral sucrose gradients of nucleoprotein particles released by micrococcal nuclease. (v) The number and sizes of DNA fragments produced by DNase I digestion. These results demonstrate that cellular DNA replicated in isolated nuclei is organized into typical nucleosomes. Consequently, subcellular systems can be used to study the relationship between DNA replication and the assembly of chromatin under physiological conditions.     

Studies in heterochromatin DNA: accessibility of late replicating heterochromatin DNA in chromatin to micrococcal nuclease digestion

Heterochromatin DNA in cactus mouse (Peromyscus eremicus) replicates in the late S phase of cell cycle. A method of obtaining cells which contain DNA preferentially labeled at heterochromatic areas by a pulse-labeling of late replicating DNA is described. When the nuclei of P. eremicus cells containing radioactively labeled DNA in heterochromatin were digested with micrococcal nuclease and the resultant nucleosomal DNA was separated by gel electrophoresis, it was found that the repeat length of nucleosomal DNA in the heterochromatin DNA is not different from that of the bulk of the genomic DNA. Furthermore, there was no significant difference in the accessibility to digestion by micrococcal nuclease between the late replicating heterochromatin DNA and the total DNA under our digestion conditions. Two dimensional gel electrophoresis patterns of nucleosomal DNAs isolated from micrococcal nuclease digested nuclei from P. eremicus, P. collatus, and P. crinitus cells in culture were very similar. Cytogenetic data showed that these three species are different in heterochromatin but similar in euchromatin.     

Nucleic acid renaturation and restriction endonuclease cleavage analyses show that the DNAs of a transforming and a nontransforming strain of Epstein-Barr virus share approximately 90% of their nucleotide sequences.

Viral DNA molecules were purified from a nontransforming and a transforming strain of Epstein-Barr virus. Each viral DNA was labeled in vitro and renatured in the presence of an excess of either one or the other unlabeled viral DNA. Both viral DNAs were also digested with the Eco R1 restriction endonuclease and subsequently labeled by using avian myeloblastosis virus DNA polymerase to repair either the EcoR1 nuclease-generated single-stranded ends of the DNAs or their single-stranded ends produced by a second digestion with exonuclease III after the first EcoR1 nuclease digestion. The results of these experiments support three general conclusions: (i) the DNAs of these two strains of Epstein-Barr virus share approximately 90% of their nucleotide sequences; (ii) both viral DNA populations are reasonably homogenous; and (iii) both DNAs contain repetitions or inverted repetitions of some of their nucleotide sequences.     

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.     

BAL 31 nuclease as a probe in concentrated salt for the B-Z DNA junction

The BAL 31 nuclease, an extracellular nuclease from A. espejiana, specifically recognizes and cleaves the salt induced conformational junction between B and Z-DNA. Short segments of (dC-dG) left-handed Z-helix, comprising approximately 1% of the total DNA, are specifically detected within two different recombinant plasmids. The BAL 31 enzyme is highly resistant to inactivation by the presence of high concentrations of a variety of electrolytes that stabilize left-handed helices, is active at physiological pH, and can be used to probe both linear and circular DNAs. Additionally, the nuclease cleaves left-handed (dC-dG)n only very poorly, if at all. Thus, the BAL 31 nuclease can be utilized as a probe for helical junctions and consequently for segments of left-handed DNA that might exist within predominantly right-handed naturally occurring genomes.     

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.     

Periodicity and fragment size of DNA from mouse TLT hepatoma chromatin and chromatin fractions using endogenous and exogenous nucleases

Summary The action of micrococcal nuclease, DNase I and DNase II on mouse TLT hepatoma chromatin revealing the periodicity of its structure as visualized by denaturing and nondenaturing gel electrophoresis, was consistent with the action of these enzymes on other chromatins. Micrococcal nuclease showed a complex subnucleosome fragment pattern based on multiples of 10 base pairs with a prominant couplet at 140/160 base pairs and the absence of the 80 base pair fragment. This couplet of the core and minimal nucleosome fragments was conspicuously present in the mononucleosomes found in the 11S fractions of a glycerol gradient centrifugation. DNase I and II produced a fairly even distribution of a 10 base pair increasing series of fragments to about 180 base pairs, a pattern also repeated in the DNA of nucleosome glycerol-gradient fractions. In limited digestions by these nucleases multinucleosomic DNA fragments are pronounced. These fragment lengths are multiples of an estimated average repeat length of nucleosome DNA of 180 base pairs. The action of the endogenous Mg/Ca-stimulated endonuclease produced only limited cuts in the hepatoma chromatin resulting primarily in multi-nucleosommc DNA fragment lengths and only upon lengthy digestion limited subnucleosomic, 10-base-pair multiple fragments are produced. The putative euchromatin-enriched fractions (50–75S) of the glycerol gradient centrifugation of autodigested chromatin, similarly, contained primarily the multinucleosomic DNA fragment lengths. These results are consistent with our previous electron microscopic demonstration that autodigested chromatin as well as the putative euchromatin-enriched fractions were composed of multinucleosomic chromatin segments containing a full complement of histones.     

Evolutionary divergence and length of repetitive sequences in sea urchin DNA

Summary The organization of repetitive and single copy DNA sequences in sea urchin DNA has been examined with the single strand specific nuclease Sl fromAspergillus. Conditions and levels of enzyme were established so that single strand DNA was effectively digested while reassociated divergent repetitive duplexes remained enzyme resistant. About 25% of sea urchin DNA reassociates with repetitive kinetics to form Sl resistant duplexes of two distinct size classes derived from long and short repetitive sequences in the sea urchin genome. Fragments 2,000 nucleotides long were reassociated to Cot 20 and subjected to controlled digestion with Sl nuclease. About half of the resistant duplexes (13% of the DNA) are short, with a mode size of about 300 nucleotide pairs. This class exhibits significant sequence divergence, and principally consists of repetitive sequences which were interspersed with single copy sequences. About one-third of the long duplexes (4% of the DNA) are reduced in size after extensive Sl nuclease digestion to about 300 nucleotide pairs. About two-thirds of the long resistant duplexes (8% of the DNA) remains long after extensive SI nuclease digestion. These long reassociated duplexes are precisely base paired. The short duplexes are imprecisely paired with a melting temperature about 9°C below that of precisely paired duplexes of the same length. The relationship between length of repetitive duplex and precision of repetition is confirmed by an independent method and has been observed in the DNA of a number of species over a wide phylogenetic area.Also Staff Member, Carnegie Institution of Washington     

Structural analysis of hyperperiodic DNA from Caenorhabditis elegans

Several bioinformatics studies have identified an unexpected but remarkably prevalent ~10 bp periodicity of AA/TT dinucleotides (hyperperiodicity) in certain regions of the Caenorhabditis elegans genome. Although the relevant C.elegans DNA segments share certain sequence characteristics with bent DNAs from other sources (e.g. trypanosome mitochondria), the nematode sequences exhibit a much more extensive and defined hyperperiodicity. Given the presence of hyperperiodic structures in a number of critical C.elegans genes, the physical characteristics of hyperperiodic DNA are of considerable interest. In this work, we demonstrate that several hyperperiodic DNA segments from C.elegans exhibit structural anomalies using high-resolution atomic force microscopy (AFM) and gel electrophoresis. Our quantitative analysis of AFM images reveals that hyperperiodic DNA adopts a significantly smaller mean square end-to-end distance, hence a more compact coil structure, compared with non-periodic DNA of similar length. While molecules remain capable of adopting both bent and straight (rod-like) configurations, indicating that their flexibility is still retained, examination of the local curvatures along the DNA contour length reveals that the decreased mean square end-to-end distance can be attributed to the presence of long-scale intrinsic bending in hyperperiodic DNA. Such bending is not detected in non-periodic DNA. Similar studies of shorter, nucleosome-length DNAs that survived micrococcal nuclease digestion show that sequence hyperperiodicity in short segments can likewise induce strong intrinsic bending. It appears, therefore, that regions of the C.elegans genome display a significant correlation between DNA sequence and unusual mechanical properties.     

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.     

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.     

Digestion of insect chromatin with micrococcal nuclease, DNase I and DNase I combined with single-strand specific nuclease S1.

The chromatin of the lepidopteran Ephestia kuehniella was digested by micrococcal nuclease, DNase I and S1-nuclease combined with DNase I pretreatment. The resulting DNA fragments were analyzed by gel electrophoresis and compared with the DNA fragments of rat liver nuclei obtained by the same process. Extensive homology was revealed between insect and mammalian chromatin structure. The combined DNase I- S1-nuclease digestion yields double-stranded DNA fragments of lengths from 30 to 110 base-pairs. These DNA fragments are not obtained from nuclei predigested extensively with micrococcal nuclease. The results are discussed with respect to the internal structure of the chromatin subunit.     

Thermal denaturation of DNA in the chromatin fragments released by mild micrococcal nuclease digestion of HTC cell nuclei

In chromatin a minor fraction melts at a temperature lower than deproteinized DNA, which may be assigned to DNA destabilizing proteins. We attempted to localize the destabilized DNA in the various chromatin fragments separated by electrophoresis after a mild micrococcal nuclease digestion. The small released fragments are enriched in coding sequences. About 20% of the DNA extracted from the released nucleosomes are single-stranded, 60% of the DNA in these fragments are digested by nuclease S₁ after incubation at low temperature, which suggests that the DNA destabilizing proteins are present in the released nucleosomes. Hybridization studies have shown that 25% of the DNA in nucleosomes are specific of this class of fragments. DNA destabilizing proteins could be associated with the specific sequences.