DNA of a circular minichromosome linearized by restriction enzymes or other reagents is resistant to further cleavage: an influence of chromatin topology on the accessibility of DNA

The accessibility of DNA in chromatin is an essential factor in regulating its activities. We studied the accessibility of the DNA in a ∼170 kb circular minichromosome to DNA-cleaving reagents using pulsed-field gel electrophoresis and fibre-fluorescence in situ hybridization on combed DNA molecules. Only one of several potential sites in the minichromosome DNA was accessible to restriction enzymes in permeabilized cells, and in growing cells only a single site at an essentially random position was cut by poisoned topoisomerase II, neocarzinostatin and γ-radiation, which have multiple potential cleavage sites; further sites were then inaccessible in the linearized minichromosomes. Sequential exposure to combinations of these reagents also resulted in cleavage at only a single site. Minichromosome DNA containing single-strand breaks created by a nicking endonuclease to relax any unconstrained superhelicity was also cut at only a single position by a restriction enzyme. Further sites became accessible after ≥95% of histones H2A, H2B and H1, and most non-histone proteins were extracted. These observations suggest that a global rearrangement of the three-dimensional packing and interactions of nucleosomes occurs when a circular minichromosome is linearized and results in its DNA becoming inaccessible to probes.     

Structure of chromatin at deoxyribonucleic acid replication forks: prenucleosomal deoxyribonucleic acid is rapidly excised from replicating simian virus 40 chromosomes by micrococcal nuclease

Replicating simian virus 40 (SV40) chromosomes were found to be similar to other eukaryotic chromosomes in that the rate and extent of micrococcal nuclease (MNase) digestion were greater with replicating than with nonreplicating mature SV40 chromatin. MNase digestion of replicating SV40 chromosomes, pulse labeled in either intact cells or nuclear extracts, resulted in the rapid release of nascent DNA as essentially bare fragments of duplex DNA (3-7S) that had an average length of 120 base pairs and were degraded during the course of the reaction. In addition, nucleosomal monomers, equivalent in size to those from mature chromosomes, were released. On the other hand, MNase digestion of uniformly labeled mature SV40 chromosomes resulted in the release of only nucleosomal monomers and oligomers. The small nascent DNA fragments released from replicating chromosomes represented prenucleosomal DNA (PN-DNA) from the region of replication forks that encompasses the actual sites of DNA synthesis and includes Okazaki fragments. Predigestion of replicating SV40 chromosomes with both Escherichia coli exonuclease III (3'-5') and bacteriophage T7 gene 6 exonuclease (5'-3') resulted in complete degradation of PN-DNA. This result, together with the observation that isolated PN-DNA annealed equally well to both strands of SV40 restriction fragments, demonstrated that PN-DNA originates from both sides of replication forks. Over 90% of isolated Okazaki fragments annealed only to the retrograde DNA template. The characteristics of isolated PN-DNA were assessed by examining its sensitivity to MNase and single strand specific S1 endonuclease, sedimentation behavior before and after deproteinization, buoyant density in CsCl after formaldehyde treatment, and size on agarose gels. In addition, it was observed that MNase digestion of purified SV40 DNA also resulted in the release of a transient intermediate similar in size to PN-DNA, indicating that a DNA-protein complex is not required to account for the appearance of PN-DNA. These and other data provide a model of replicating chromosomes in which DNA synthesis occurs on a region of replication forks that is free of nucleosomes and is designated as prenucleosomal DNA.     

Correlation between premeiotic DNA replication and chromatin transition at yeast recombination initiation sites

The DNA double-strand breaks (DSBs) that initiate meiotic recombination in Saccharomyces cerevisiae are preceded first by DNA replication and then by a chromatin transition at DSB sites. This chromatin transition, detected as a quantitative increase in micrococcal nuclease (MNase) sensitivity, occurs specifically at DSB sites and not at other MNase-sensitive sites. Replication and DSB formation are directly linked: breaks do not form if replication is blocked, and delaying replication of a region also delays DSB formation in that region. We report here experiments that examine the relationship between replication, the DSB-specific chromatin transition and DSB formation. Deleting replication origins (and thus delaying replication) on the left arm of one of the two parental chromosomes III affects DSBs specifically on that replication-delayed arm and not those on the normally replicating arm. Thus, replication timing determines DSB timing in cis. Delaying replication on the left arm of chromosome III also delays the chromatin transition at DSB sites on that arm but not on the normally replicating right arm. Since the chromatin transition precedes DSB formation and requires the function of many genes necessary for DSB formation, these results suggest that initial events for DSB formation in chromatin are coupled with premeiotic DNA replication.     

A sequence of the yeast 2 micron DNA plasmid chromosome near the origin of replication is exposed to restriction endonuclease digestion

The yeast 2 μm DNA plasmid nucleoprotein complex was subjected to restriction endonuclease digestion to ascertain whether all possible sites are equally accessible to hydrolysis. When plasmid nucleoprotein complexes which had been fixed with formaldehyde were exhaustively digested with restriction endonucleases HinfI or CfoI, only a few of the limit digest products were produced. Furthermore, the limited set of restriction endonuclease sites exposed in formaldehyde-treated plasmid chromosomes could be shown to be preferentially hydrolyzed when plasmid chromosomes which had not been treated with formaldehyde were digested with the same restriction endonucleases. Mapping of the preferred sites revealed that they mapped to the region of the plasmid near the replication origin. These results demonstrate that the protection of DNA from nuclease activity is not constant along the plasmid chromatin, and that a region near the replication origin is preferentially exposed to endonuclease hydrolysis.     

Nucleosome positioning as a critical determinant for the DNA cleavage sites of mammalian DNA topoisomerase II in reconstituted simian virus 40 chromatin.

We have assessed the ability of nucleosomes to influence the formation of mammalian topoisomerase II-DNA complexes by mapping the sites of cleavage induced by four unrelated topoisomerase II inhibitors in naked versus nucleosome-reconstituted SV40 DNA. DNA fragments were reconstituted with histone octamers from HeLa cells by the histone exchange method. Nucleosome positions were determined by comparing micrococcal nuclease cleavage patterns of nucleosome-reconstituted and naked DNA. Three types of DNA regions were defined: 1) regions with fixed nucleosome positioning; 2) regions lacking regular nucleosome phasing; and 3) a region around the replication origin (from position 5100 to 600) with no detectable nucleosomes. Topoisomerase II cleavage sites were suppressed in nucleosomes and persisted or were enhanced in linker DNA and in the nucleosome-free region around the replication origin. Incubation of reconstituted chromatin with topoisomerase II protected nucleosome-free regions from micrococcal nuclease cleavage without changing the overall micrococcal nuclease cleavage pattern. Thus, the present results indicate that topoisomerase II binds preferentially to nucleosome-free DNA and that the presence of nucleosomes at preferred DNA sequences influences drug-induced DNA breaks by topoisomerase II inhibitors.     

Is there replication-associated mutational pressure in the Saccharomyces cerevisiae genome?

Compositional bias of yeast chromosomes was analysed using detrended DNA walks. Unlike eubacterial chromosomes, the yeast chromosomes did not show the specific asymmetry correlated with origin and terminus of replication. It is probably a result of a relative excess of autonomously replicating sequences (ARS) and of random choice of these sequences in each replication cycle. Nevertheless, the last ARS from both ends of chromosomes are responsible for unidirectional replication of subtelomeric sequences with pre-established leading/lagging roles of DNA strands. In these sequences a specific asymmetry is observed, resembling the asymmetry introduced by replication-associated mutational pressure into eubacterial chromosomes.     

Restriction endonuclease cleavage of satellite DNA in intact bovine nuclei

We have analyzed the efficiency with which specific nucleotide sequences within nucleosomes are recognized and cleaved by DNA restriction endonucleases. A system amenable to this sort of analysis is the cleavage of the bovine genome with the restriction endonuclease EcoRI. Bovine satellite I comprises 7% of the genome and is tandemly repetitious with an EcoRI site at 1400 base pair (bp) intervals within this sequence. The ease with which this restriction fragment can be measured permits an analysis of the accessibility of this sequence when organized in a nucleosomal array.Initial studies indicated that satellite I sequences are organized in a nucleosomal structure in a manner analogous to that observed for total genomic DNA. We then examined the accessibility of the EcoRI cleavage sites in satellite to endonucleolytic cleavage in intact nuclei. We find that whereas virtually all the satellite I sequences from naked DNA are cleaved into discrete 1400 bp fragments, only 33% of the satellite I DNA is liberated as this fragment from intact nuclei. These data indicate that 57% of the EcoRI sites in nuclei are accessible to cleavage and that cleavage can occur within the core of at least half the nucleosomal subunits. Analysis of the products of digestion suggests a random distribution of nucleosomes about the EcoRI sites of satellite I DNA.Finally, the observation that satellite sequences can be cleaved from nuclei to 1400 bp length fragments with their associated proteins provides a method for the isolation of specific sequences as chromatin. Using sucrose gradient velocity centrifugation, we have isolated a 70% pure fraction of satellite I chromatin. Nuclease digestion of this chromatin fraction reveals the presence of nucleosomal subunits and indicates that specific sequences can be isolated in this manner without gross disorganization of their subunit structure.     

Replication origin region of Bacillus subtilis chromosome contains two rRNA operons.

The first replicating DNA fragment (BamHI-7) of the Bacillus subtilis chromosome contains two promoters for a rRNA operon. A map of restriction enzyme cleavage sites of the region of replication origin suggests the presence of a second rRNA operon in this region. Hybridization of rRNA genes (rDNA) with DNA fragments derived from the origin region by treatment with various enzymes clearly revealed two rRNA operons in this region, one at the B7-B3 junction and the other at the B5-B6 junction. The restriction enzyme cleavage sites surrounding the rRNA operons show that the operon at the B5-B6 junction corresponds to the rrnA operon. A novel operon at the B7-B3 junction was termed rrnO. Transformation by density-labeled fragments of the origin region showed that the first replicating marker, guaA, is located in the B3 fragment. From these results, a map was constructed for the first time to correlate the genetic markers with the physical structure of the replication origin region of the B. subtilis chromosome. The role of the rrnO operon in regulating the initiation of chromosomal replication is discussed, based on the fact that the promoter of the rrnO operon suppresses the replication of the plasmid carrying the promoter.     

Studies on the biological role of DNA methylation: V. The pattern of E.coli DNA methylation.

The distribution of the methylatable sites GATC and CCATGG was studied by analyzing the molecular average size of restriction fragments of E. coli DNA. Both sites were found to be randomly distributed, reflecting a random pattern of methylation. The methylation pattern of specific sequences such as the origin of replication and rRNA genes has been studied in wild type E. coli and a methylation deficient (dam- dcm-) mutant. These sequences were found to be methylated in wild type cells and unmethylated in the mutant indicating that there is no effect of the state of methylation of these sequences on their expression. Analysis of the state of methylation of GATC sites in newly replicating DNA using the restriction enzyme Dpn I (cleaves only when both strands are methylated) revealed no detectable hemimethylated DNA suggesting that methylation occurs at the replication fork. Taking together the results presented here and previously published data (5), we arrive at the conclusion that the most likely function of E. coli DNA methylations is probably in preventing nuclease activity.     

Comparison of the subunit organization of early and late replicating chromatin

A comparison was made of the subunit organization of chromatin from regions of the genome with different metaphase chromosome banding characteristics by analyzing the accessibility of early and late replicating DNA in synchronized Chinese hamster ovary cells to digestion with staphylococcal nuclease. Three measures of nuclease susceptibility were employed: (1) the release of acid-soluble material; (2) a digestion index, P, which corresponds to the proportion of internucleosome segments which experienced at least one cleavage event; and (3) the size distribution of DNA fragments isolated from digested chromatin. Little or no difference was observed in the initial rates with which nuclease converted early and late replicating chromatin to acid-soluble material, although the initial digestion rates varied with time of cell collection in the cycle (metaphase > G1 mid-S > late-S or G2). Measurements of the digestion indices of material isolated from interphase cells suggested that initial cleavage events were more rapid in early replicating chromatin than in late replicating chromatin. In contrast, electrophoretic analysis revealed that oligomer DNA fragments from early labelled metaphase chromatin were slightly larger than corresponding fragments from late labelled metaphase chromatin. The size distribution of DNA in submonomer fragments obtained from extensively digested chromatin appeared to be identical regardless of the timing of replication or cell collection. Those small differences in chromatin digestibility that were observed may reflect subtle variations in the accessibility of internucleosome regions or perhaps in the higher-order arrangement of nucleosomes. However, no gross variation in accessibility to staphylococcal nuclease digestion was observed in chromatin localized to metaphase chromosome regions with vastly different cytological staining properties.     

The majority of minicircle DNA in Crithidia fasciculata strain CF-C1 is of a single class with nearly homogeneous DNA sequence

DNA minicircles found within the kinetoplast of the trypanosomatid Crithidia fasciculata, like those of most other kinetoplastid species, are heterogeneous in sequence. The pattern of minicircle DNA fragments generated by cleavage of kinetoplast DNA with various restriction enzymes has been used to demonstrate this heterogeneity. Here we describe a strain of Crithidia fasciculata in which more than 90% of the DNA minicircles exhibit a common pattern of restriction enzyme cleavage sites. A map of cleavage sites within this major minicircle DNA class is presented for seven restriction enzymes with hexanucleotide recognition sequences. Sequence homogeneity at an even finer level is reflected in minicircle DNA digestion patterns generated by restriction enzymes with tetranucleotide recognition sites. Partial DNA sequence analysis of multiple clones from the major minicircle class shows nearly complete homogeneity at the nucleotide level. The existence of a near homogeneous complement of DNA minicircles in Crithidia should facilitate the study of their replication in this organism.     

Morphological and biochemical effects of endonucleases on isolated mammalian chromosomes in vitro

Endonuclease digestion of isolated and unfixed mammalian metaphase chromosomes in vitro was examined as a means to study the higher-order regional organization of chromosomes related to banding patterns and the mechanisms of endonuclease-induced banding. Isolated mouse LM cell chromosomes, digested with the restriction enzymes AluI, HaeIII, EcoRI, BstNI, AvaII, or Sau96I, demonstrated reproducible G- and/or C-banding at the cytological level depending on the enzyme and digestion conditions. At the molecular level, specific DNA alterations were induced that correlated with the banding patterns produced. The results indicate that: (1) chromatin extraction is intimately involved in the mechanism of endonuclease induced chromosome banding. (2) The extracted DNA fragments are variable in size, ranging from 200 bp to more than 4 kb in length. (3) For HaeIII, there appears to be variation in the rate of restriction site cleavage in G- and R-bands; HaeIII sites appear to be more rapidly cleaved in R-bands than in G-bands. (4) AluI and HaeIII ultimately produce banding patterns that reflect regional differences in the distribution of restriction sites along the chromosome. (5) BstNI restriction sites in the satellite DNA of constitutive heterochromatin are not cleaved intrachromosomally, probably reflecting an inaccessibility of the BstNI sites to enzyme due to the condensed nature of this chromatin or specific DNA-protein interactions. This implies that some enzymes may induce banding related to regional differences in the accessibility of restriction sites along the chromosome. (6) Several specific nonhistone protein differences were noted in the extracted and residual chromatin following an AluI digestion. Of these, some nonhistones were primarily detected in the extracted chromatin while others were apparently resistant to extraction and located principally in the residual chromatin. (7) The chromatin in constitutive heterochromatin is transiently resistant to cleavage by micrococcal nuclease.     

Nucleosome arrangement in alpha-satellite chromatin of African green monkey cells.

By analyzing the accessibility of restriction endonuclease sites in African green monkey alpha-satellite chromatin, we demonstrate the absence of a unique phase relationship between nucleosomes and alpha-satellite DNA. The data indicate a minimum of three different positions for nucleosome cores relative to the alpha-satellite sequence and suggest a random distribution in at least some regions. In addition, while we confirm published reports that staphylococcal nuclease cuts the alpha-satellite sequence in chromatin at a highly preferred site, two-dimensional gel electrophoresis of nuclear digests demonstrates that this site is preferentially cut by staphylococcal nuclease even when it is within the nucleosome core. These data indicate that staphylococcal nuclease is not useful for determining nucleosome positions on alpha-satellite DNA, and perhaps on other specific DNA sequences as well.     

Origin and Direction of Simian Virus 40 Deoxyribonucleic Acid Replication

Double-branched, circular, replicating deoxyribonucleic acid (DNA) molecules of simian virus 40 (SV40) have been cleaved by the R(1) restriction endonuclease from Escherichia coli. This enzyme introduces one double-strand break in SV40 DNA, at a specific site. The site of cleavage in the replicating molecules was used in this study to position the origin and the two branch points. Radioactively labeled molecules fractionated according to their extent of replication were evaluated after cleavage by sedimentation analysis and electron microscopy. The results demonstrate that the R(1) cleavage site is 33% of the genome length from the origin of replication and that both branch points are growing points. These data indicate that SV40 DNA replication is bidirectional and confirm other reports which have shown a unique origin of replication.     

MAMMALIAN CHROMOSOMES IN VITRO : XVIII. DNA Replication Sequence in the Chinese Hamster

The complete DNA replication sequence of the entire complement of chromosomes in the Chinese hamster may be studied by using the method of continuous H³-thymidine labeling and the method of 5-fluorodeoxyuridine block with H³-thymidine pulse labeling as relief. Many chromosomes start DNA synthesis simultaneously at multiple sites, but the sex chromosomes (the Y and the long arm of the X) begin DNA replication approximately 4.5 hours later and are the last members of the complement to finish replication. Generally, chromosomes or segments of chromosomes that begin replication early complete it early, and those which begin late, complete it late. Many chromosomes bear characteristically late replicating regions. During the last hour of the S phase, the entire Y, the long arm of the X, and chromosomes 10 and 11 are heavily labeled. The short arm of chromosome 1, long arm of chromosome 2, distal portion of chromosome 6, and short arms of chromosomes 7, 8, and 9 are moderately labeled. The long arm of chromosome 1 and the short arm of chromosome 2 also have late replicating zones or bands. The centromeres of chromosomes 4 and 5, and occasionally a band on the short arm of the X are lightly labeled.     

Small DNA fragments isolated from human T-cell clones are enriched in sequences involved in DNA bending

IL-2 deprivation induces apoptosis in human IL-2-dependent T-cell clones. This process is characterized by typical cell morphology, changes in the cellular membranes and fragmentation of chromatin into units of single and multiple nucleosomes. We isolated apoptotic DNA of an IL-2-deprived T-cell clone and sequenced randomly selected fragments representing single and multiple nucleosomes.The frequency of phased adenosine tracts was markedly increased in the small apoptotic fragments as compared to oligonucleosomes. Our results thus indicate that chromatin fragmentation in human apoptotic T-cells is not random but preferentially involves DNA sequences with the capability to form bent DNA. Whether this indicates a colocalization of DNase cleavage sites and phased adenosine tracts on the chromosomes or a bias in selecting sites for apoptotic DNA fragmentation is discussed. Analysing the underlying mechanisms will shed new light on DNA degradation in apoptosis.     

Assembly of nascent DNA into nucleosome structures in simian virus 40 chromosomes by HeLa cell extract.

A soluble system was developed that could support DNA replication in simian virus 40 (SV40) chromosomes. DNA synthesis in this system required the presence of purified SV40 large tumor antigen, SV40 chromosomes prepared from virus-infected monkey cells, a crude extract from HeLa cells, and several low-molecular-weight components. In comparison to the replication of purified SV40 form I DNA, the rate of DNA synthesis was 15 to 20% in this system. DNA synthesis started near the replication origin of SV40 and proceeded bidirectionally in a semiconservative manner. Micrococcal nuclease digestion experiments revealed that the replicated DNA produced in this system became organized into a regularly spaced array of nucleosome core particles when an appropriate amount of purified HeLa core histones was added to the reaction mixture. SV40 form I DNA replicating under the same conditions was also assembled into nucleosomes, which were arranged in a rather dispersed manner and formed an aberrant chromatin structure.     

Comparative analysis of the nucleosomal structure of rye,wheat and their relatives

Analysis of the structure of chromatin in cereal species using micrococcal nuclease (MNase) cleavage showed nucleosomal organization and a ladder with typical nucleosomal spacing of 175–185 bp. Probing with a set of DNA probes localized in the authentic telomeres, subtelomeric regions and bulk chromatin revealed that these chromosomal regions have nucleosomal organization but differ in size of nucleosomes and rate of cleavage between both species and regions. Chromatin from Secale and Dasypyrum cleaved more quickly than that from wheat and barley, perhaps because of their higher content of repetitive sequences with hairpin structures accessible to MNase cleavage. In all species, the telomeric chromatin showed more rapid cleavage kinetics and a shorter nucleosome length (160 bp spacing) than bulk chromatin. Rye telomeric repeat arrays were shortest, ranging from 8 kb to 50 kb while those of wheat ranged from 15 kb up to 175 kb. A gradient of sensitivity to MNase was detected along rye chromosomes. The rye-specific subtelomeric sequences pSc200 and pSc250 have nucleosomes of two lengths, those of the telomeric and of bulk nucleosomes, indicating that the telomeric structure may extended into the chromosomes. More proximal sequences common to rye and wheat, the short tandem-repeat pSc119.2 and rDNA sequence pTa71, showed longer nucleosomal sizes characteristic of bulk chromatin in both species. A strictly defined spacing arrangement (phasing) of nucleosomes was demonstrated along arrays of tandem repeats with different monomer lengths (118, 350 and 550 bp) by combining MNase and restriction enzyme digestion.