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.     

Changes in chromatin structure at the replication fork. II The DNPs containing nascent DNA and a transient chromatin modification detected by DNAase I.

Discrete deoxyribonucleoproteins (DNPs) containing nascent and/or bulk DNA, were obtained by fractionating micrococcal nuclease digests of nuclei form 3H-thymidine pulse (15-20 sec) and 14C-thymidine long (16 h) labeled sea urchin embryos in polyacrylamide gels. One of these DNPs was shown to contain the micrococcal nuclease resistant 300 bp "large nascent DNA" described in Cell 14, 259-267, 1978. The bulk and nascent mononucleosome fractions provided evidence for the preferential digestion by micrococcal nuclease of nascent over bulk linker regions to yield mononucleosome cores with nascent DNA. DNAase I was used to probe whether any nascent DNA is in nucleosomes. Nascent as well as bulk single-stranded DNA fragments occurred in multiples of 10.4 bases with higher than random frequencies of certain fragment sizes (for instance 83 bases) as expected from a nucleosome structure. However, a striking background of nascent DNA between nascent DNA peaks was observed. This was eliminated by a pulse-chase treatment or by digestion of pulse-labeled nuclei with micrococcal nuclease together with DNAase I. One of several possible interpretations of these results suggests that a transient change in nucleosome structure may have created additional sites for the nicking of nascent DNA by DNAase I; the micrococcal nuclease sensitivity of the interpeak radioactivity suggest its origin from the linker region. Endogenous nuclease of sea urchin embryos cleaves chromatin DNA in a manner similar to that of DNAase I.     

Assembly of newly replicated chromatin.

Mild staphylococcal nuclease digestions under isotonic conditions release fragments of a 200 Å diameter fiber from nuclei of Drosophila melanogaster tissue culture cells. These soluble fragments have high sedimentation coefficients (30–100S) and show tightly packed nucleosomes in the electron microscope. Under the same conditions, newly replicated chromatin is released as more slowly sedimenting fragments (14S). Within 20 min after DNA replication, the nascent chromatin gradually matures into compact supranucleosomal structures which are indistinguishable from bulk chromatin on the isokinetic sucrose gradients.We have used this fractionation technique to examine the question of the fate and assembly of the new histones. After short pulses with either ³⁵S-methionine or ³H-lysine, the radioactive histones do not co-sediment with the bulk chromatin but appear instead in the fractions where the newly replicated DNA is found. Furthermore, the various nascent histones appear in different fractions on the gradient: histones H3 and H4 in 10–15S structures, histones H2A and H2B in 15–50S structures and histone H1 in 30–100S structures. These results, together with the analysis of pulse and pulse-chase experiments of both nascent DNA and histones, strongly suggest that histones H3 and H4 are deposited first on the nascent DNA (during or slightly after the DNA is replicated), histones H2A and H2B are deposited next (2–10 min later) and histone H1 is deposited last (10–20 min after DNA replication). A high turnover 20,000 dalton protein is also associated with the newly replicated chromatin.     

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.     

Structure of chromatin at deoxyribonucleic acid replication forks: location of the first nucleosomes on newly synthesized simian virus 40 deoxyribonucleic acid

Exonucleases specific for either 3' ends (Escherichia coli exonuclease III) or 5' ends (bacteriophage T7 gene 6 exonuclease) of nascent DNA chains have been used to determine the number of nucleotides from the actual sites of DNA synthesis to the first nucleosome on each arm of replication forks in simian virus 40 (SV40) chromosomes labeled with [3H]thymidine in whole cells. Whereas each enzyme excised all of the nascent [3H]DNA from purified replicating SV40 DNA, only a fraction of the [3H]DNA was excised from purified replicating SV40 chromosomes. The latter result was attributable to the inability of either exonuclease to digest nucleosomal DNA in native replicating SV40 chromosomes, as demonstrated by the following observations: (i) digestion with either exonuclease did not reduce the amount of newly synthesized nucleosomal DNA released by micrococcal nuclease during a subsequent digestion period; (ii) in briefly labeled molecules, as much as 40% of the [3H]DNA was excised from long nascent DNA chains; (iii) the fraction of [3H]DNA excised by exonuclease III was reduced in proportion to the actual length of the radiolabeled DNA; (iv) the effects of the two exonucleases were additive, consistent with each enzyme trimming only the 3' or 5' ends of nascent DNA chains without continued excision through to the opposite end. When the fraction of nascent [3H]DNA excised from replicating SV40 DNA by exonuclease III was compared with the fraction of [32P]DNA simultaneously excised from an SV40 DNA restriction fragment, the actual length of nascent [3H]DNA was calculated. From this number, the fraction of [3H]DNA excised from replicating SV40 chromosomes was converted into the number of nucleotides. Accordingly, the average distance from either 3' or 5' ends of long nascent DNA chains to the first nucleosome on either arm of replication forks was found to be 125 nucleotides. Furthermore, each exonuclease excised about 80% of the radiolabel in Okazaki fragments, suggesting that less than one-fifth of the Okazaki fragments were contained in nucleosomes. On the basis of these and other results, a model for eukaryotic replication forks is presented in which nucleosomes appear rapidly on both the forward and retrograde arms, about 125 and 300 nucleotides, respectively, from the actual site of DNA synthesis. In addition, it is proposed that Okazaki fragments are initiated on nonnucleosomal DNA and then assembled into nucleosomes, generally after ligation to the 5' ends of long nascent DNA chains is completed.     

Changes in chromatin structure at the replication fork. DNase I and trypsin-micrococcal nuclease effects on approximately 300- and 150-base pair nascent DNAs

DNase I, trypsin, and micrococcal nuclease are used to further probe the structure of nascent deoxyribonucleoprotein (DNP) fractions which appear after in vivo 20-s pulse labeling of sea urchin embryos with [3H]thymidine. We present evidence that the large nascent DNP which protects the approximately 300-base pair large nascent DNA consists of at least one nucleosome core. This is based on fractionation in denaturing polyacrylamide gels of DNA extracted from large nascent DNP fractions of a micrococcal nuclease + DNase I digest of nuclei. The data also suggest the existence of a DNase I-hypersensitive site(s) within the large nascent DNP; this is consistent with the hypothesis that the latter consists of closely packed dinucleosome cores. Histone H1 and non-histone proteins do not account for the previously reported unusual hyperresistance of the large nascent DNA against micrococcal nuclease. The protection offered this approximately 300-base pair nascent DNA was not eliminated by an 0.2-microgram/ml trypsin pretreatment which removes the above proteins from the chromatin. However, 5-10 micrograms/ml of trypsin, which remove a portion of the NH2 termini of the four core histones of nucleosomes, eliminate the hyperresistance of the large nascent DNA to subsequent micrococcal nuclease digestion, while nascent and bulk monomer DNAs remain unaffected. This indicates histone-histone and/or histone-DNA interactions within the large nascent DNP which differ from those of nascent and bulk mononucleosome cores.     

Assembly of an active chromatin structure during replication.

MSB cells were pulse labeled with 3H-thymidine and the isolated nuclei digested with either staphylococcal nuclease (to about 40% acid solubility) or DNase I (to 15% acid solubility). The purified, nuclease resistant single-copy DNA was then hybridized to nuclear RNA (nRNA). The results of these experiments show that actively transcribed genes are assembled into nucleosome-like structures within 5-10 nucleosomes of the replication fork and that they also acquire a conformation characteristic of actively transcribed nucleosomes (ie, a DNase I sensitive structure) within 20 nucleosomes of the fork. Assuming DNA sequence specific interactions are required for establishing a DNase I sensitive conformation on active genes during each round of replication, our results indicate that a specific recognition event can occur very rapidly and very specifically in eukaryotic cells. The results are discussed in terms of the possible mechanisms responsible for propagating active, chromosomal conformations from mother cells to daughter cells.     

Two-stage maturation process for newly replicated chromatin

HTC cells have been labeled by short exposures to [3H]thymidine in order to identify newly synthesized DNA. By either isolating nuclei directly or isolating them after an extensive fixation with formaldehyde, we have been able to identify two phases in the maturation process of newly replicated chromatin. The first phase which is relatively brief (less than 5 min) is reflected in a diffuse, irregular organization of nucleosomes on new DNA immediately postreplicatively . The second phase which lasts from 5 to 30 min postreplication is characterized by a normal repeat length for the nucleosomes which are nonetheless more weakly bound than bulk nucleosomes. This is reflected in increased sliding during nuclease digestion as well as increased nuclease sensitivity and the presence of easily dissociated histones which has been described by other workers.     

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.     

Eight different highly specific nucleosome phases on alpha-satellite DNA in the African green monkey.

The question of nucleosome phasing on African Green Monkey (AGM) alpha-satellite DNA has been addressed by employing a new approach. Nucleosome cores were prepared from AGM nuclei with micrococcal nuclease, exonuclease III and nuclease S1. The core DNA population derived from alpha-satellite DNA containing chromatin was purified from total core DNA by denaturation of the DNA, reassociation to a low Cot value, and hydroxyapatite chromatography to separate the renatured satellite fraction. After end-labeling the termini of the alpha-satellite containing core DNA fragments were mapped by high resolution gel electrophoresis relative to known restriction sites along the 172 bp repeat unit of the satellite DNA. The results show that nucleosomes occupy eight strictly defined positions on the alpha-satellite DNA which could be determined with an accuracy of +/- 1 base pair. Approximately 35% of all nucleosomes are organized in one of these frames while the other seven registers contribute about 10% each.     

Histone deacetylation is required for the maturation of newly replicated chromatin

The effects of inhibiting histone deacetylation on the maturation of newly replicated chromatin have been examined. HeLa cells were labeled with [3H]thymidine in the presence or absence of sodium butyrate; control experiments demonstrated that butyrate did not significantly inhibit DNA replication for at least 70 min. Like normal nascent chromatin, chromatin labeled for brief periods (0.5-1 min) in the presence of butyrate was more sensitive to digestion with DNase I and micrococcal nuclease than control bulk chromatin. However, chromatin replicated in butyrate did not mature as in normal replication, but instead retained approximately 50% of its heightened sensitivity to DNase I. Incubation of mature chromatin in butyrate for 1 h did not induce DNase I sensitivity: therefore, the presence of sodium butyrate was required during replication to preserve the increased digestibility of nascent chromatin DNA. In contrast, sodium butyrate did not inhibit or retard the maturation of newly replicated chromatin when assayed by micrococcal nuclease digestion, as determined by the following criteria: 1) digestion to acid solubility, 2) rate of conversion to mononucleosomes, 3) repeat length, and 4) presence of non-nucleosomal DNA. Consistent with the properties of chromatin replicated in butyrate, micrococcal nuclease also did not preferentially attack the internucleosomal linkers of chromatin regions acetylated in vivo. The observation of a novel chromatin replication intermediate, which is highly sensitive to DNase I but possesses normal resistance to micrococcal nuclease, suggests that nucleosome assembly and histone deacetylation are not obligatorily coordinated. Thus, while deacetylation is required for chromatin maturation, histone acetylation apparently affects chromatin organization at a level distinct from that of core particle or linker, possibly by altering higher order structure.     

The sites of deposition of newly synthesized histone.

The chromosomal fragments produced by nuclease digestion of freshly replicated chromatin migrate more rapidly relative to bulk chromatin when analyzed in nucleoprotein gels. The cause of the anomalous migration has been studied and the evidence indicates that rather than reflecting a shorter nucleosomal repeat in vivo that it may be a consequence of nucleosome sliding during the digestion itself. The distinct electrophoretic characteristics of nucleosomal material containing newly replicated DNA have enabled us to examine their histone composition by two dimensional electrophoresis. We find that nucleosomes containing new DNA also contain newly synthesized histones H3 and H4. In contrast more than 50% of newly synthesized H2A and H2B, and essentially all of new H1, are deposited at sites on the bulk chromatin distinct from that material containing newly replicated DNA. In addition we show that newly synthesized histones H3 and H4 are bound unusually weakly when they first become associated with the chromatin.     

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.     

Reduced repeat length of nascent nucleosomal DNA is generated by replicating chromatin in vivo

Micrococcal nuclease digestion of nuclei from sea urchin embryos revealed transient changes in chromatin structure which resulted in a reduction in the repeat length of nascent chromatin DNA as compared with bulk DNA. This was considered to be entirely the consequence of in vivo events at the replication fork (Cell 14, 259, 1978). However, a micrococcal nuclease-generated sliding of nucleosome cores relative to nascent DNA, which might account for the smaller DNA fragments, was not excluded. In vivo [3H]thymidine pulse-labeled nuclei were fixed with a formaldehyde prior to micrococcal nuclease digestion. This linked chromatin proteins to DNA and thus prevented any in vitro sliding of histone cores. All the nascent DNAs exhibiting shorter repeat lengths after micrococcal nuclease digestion, were resolved at identical mobilities in polyacrylamide gels of DNA from fixed and unfixed nuclei. We conclude that these differences in repeat lengths between nascent and bulk DNA was generated in vivo by changes in chromatin structure during replication, rather than by micrococcal nuclease-induced sliding of histone cores in vitro.     

Enrichment of transcribed and newly replicated DNA in soluble chromatin released from nuclei by mild micrococcal nuclease digestion

A chromatin fraction solubilized from mouse myeloma nuclei under near-physiological ionic conditions by very mild micrococcal nuclease digestion at 0°C is enriched at least 7-fold in DNA complementary to total myeloma polyadenylated mRNA, and 15-fold in DNA originating near the replication fork (labeled within 30 s). Newly replicated DNA recovered in solubilized chromatin after brief labeling was incorporated mainly into particles sedimenting with, or faster than, mononucleosomes. A rapid decrease in enrichment of newly replicated DNA in readily released, soluble chromatin with increasing labeling times indicated that newly replicated chromatin matured within 90 s to a form that was partitioned similarly to bulk chromatin by this fractionation method. Previous studies showed that chromatin readily solubilized from myeloma nuclei is enriched in high-mobility-group (HMG) and other non-histone proteins, RNA and single-stranded DNA; and depleted in H1 and 5-methylcytosine, relative to bulk chromatin (Jackson, J.B., Pollock, J.M., Jr., and Rill, R.L. (1979) Biochemistry 18, 3739–3748). Mild digestion of chicken erythrocyte nuclei with micrococcal nuclease yielded a soluble chromatin fraction (1–2% of the total DNA) with similar properties. This fraction was enriched at least 6-fold in DNA complementary to chicken globin mRNA, relative to total erythrocyte DNA.     

Assembly kinetics of replicating chromatin: Isolation and characterization of prenucleosomal and nucleosomal DNA

Replicating chromatin is known to be more sensitive to micrococcal nuclease than bulk chromatin. We have used this property and a fractionation procedure based on the specific release of replicating material under mild micrococcal nuclease digestion, in order to analyse both the kinetics of maturation of newly replicated DNA into nucleosomes and the structure of the replicating material. As other authors, we initially observed that repetitive unit of newly replicated chromatin was shorter than that of bulk chromatin, however this result appears to be due to sliding of nucleosomes along the chromatin fibers close to the replicating fork. Replicative chromatin was fractionated and analysed. A prenucleosomal peak was observed and preliminary characterized.     

Organization of 5-methylcytosine in chromosomal DNA

The 5-methylcytosine residues of L-cells have been labeled with [methyl-3H]-L-methionine and their chromatin localization studied using deoxyribonucleases. The kinetics of micrococcal nuclease digestion showed that the methylated cytosine residues are concentrated within regions resistant to nuclease digestion and preferentially missing from those regions between nucleosomes which are nuclease sensitive. Using DNA hybridization kinetic analysis, it is shown that 5-methylcytosine is abundant in highly repeated sequences but is also present in middle repetitive and unique sequence DNA.     

Accessibility of some regions of DNA in chromatin (chicken erythrocytes) to single strand-specific nucleases.

The susceptibility of the DNA in chromatin to single strand-specific nucleases was examined using nuclease P1, mung bean nuclease, and venom phosphodiesterase. A stage in the reaction exists where the size range of the solubilized products is similar for each of the three nucleases and is nearly independent of incubation time. During this stage, the chromatin fragments sediment in the range of 30 to 100 S and contain duplex DNA ranging from 1 to 10 million daltons. Starting with chromatin depleted of histones H1 and H5 similar fragments are generated. In both cases these nucleoprotein fragments are reduced to nucleosomes and their multimers by micrococcal nuclease. Thus, chromatin contains a limited number of DNA sites which are susceptible to single strand-specific nucleases. These sites occur at intervals of 8 to 80 nucleosomes and are distributed throughout the chromatin. Nucleosome monomers, dimers, or trimers were not observed at any stage of single strand-specific nuclease digestion of nuclei, H1- and H5-depleted chromatin, or micrococcal nuclease-generated oligonucleosomes. Each of the three nucleases converted mononucleosomes (approximately 160 base pairs) to nucleosome cores (approximately 140 base pairs) probably by exonucleolytic action that was facilitated by the prior removal of H1 and H5. The minichromosome of SV40 is highly resistant to digestion by nuclease P1.     

Dispersive segregation of nucleosomes during replication of simian virus 40 chromosomes

The distribution of preformed ("old") histone octamers between the two arms of DNA replication forks was analyzed in simian virus 40(SV40)-infected cells following treatment with cycloheximide to prevent nucleosome assembly from nascent histones. Viral chromatin synthesized in the presence of cycloheximide was shown to be deficient in nucleosomes. Replicating SV40 DNA (wild-type 800 and capsid assembly mutant, tsB11) was radiolabeled in either intact cells or nuclear extracts supplemented with cytosol. Nascent nucleosomal monomers were then released by extensive digestion of isolated nuclei, nuclear extracts or isolated viral chromosomes with micrococcal nuclease. The labeled nucleosomal DNA was purified and found to hybridize to both strands of SV40 DNA restriction fragments taken from each side of the origin of DNA replication, whereas Okazaki fragments hybridized only to the strand representing the retrograde DNA template. In addition, isolated, replicating SV40 chromosomes were digested with two strand-specific exonucleases that excised nascent DNA from either the forward or the retrograde side of replication forks. Pretreatment of cells with cycloheximide did not result in an excess of prenucleosomal DNA on either side of replication forks, but did increase the amount of internucleosomal DNA. These data are consistent with a dispersive model for nucleosome segregation in which "old" histone octamers are distributed to both arms of DNA replication forks.     

Maturation of newly replicated chromatin of simian virus 40 and its host cell

The DNA in replicating simian virus 40 chromatin and cellular chromatin was labeled with short pulses of [³H]thymidine. The structure of pulse-labeled nucleoprotein complexes was studied by micrococcal nuclease digestion. It was found that in both newly replicated viral and cellular chromatin, a structural state appears which is characterized by an increased sensitivity to nuclease and a faster than usual rate of cleavage to DNA fragments of monomeric nucleosome size and smaller. Pulse-chase experiments show that each of these effects requires a characteristic time to disappear in both systems, suggesting the existence of different sub-processes of chromatin maturation. One of these processes, detectable by the reversion of the unusually fast production of subnucleosomal fragments, is delayed in SV40 chromatin replication.