Disruption of the nucleosomes at the replication fork.

The fate of parental nucleosomes during chromatin replication was studied in vitro using in vitro assembled chromatin containing the whole SV40 genome as well as salt-treated and native SV40 minichromosomes. In vitro assembled minichromosomes were able to replicate efficiently in vitro, when the DNA was preincubated with T-antigen, a cytosolic S100 extract and three deoxynucleoside triphosphates prior to chromatin assembly, indicating that the origin has to be free of nucleosomes for replication initiation. The chromatin structure of the newly synthesized daughter strands in replicating molecules was analysed by psoralen cross-linking of the DNA and by micrococcal nuclease digestion. A 5- and 10-fold excess of protein-free competitor DNA present during minichromosome replication traps the segregating histones. In opposition to published data this suggests that the parental histones remain only loosely or not attached to the DNA in the region of the replication fork. Replication in the putative absence of free histones shows that a subnucleosomal particle is randomly assembled on the daughter strands. The data are compatible with the formation of a H3/H4 tetramer complex under these conditions, supporting the notion that under physiological conditions nucleosome core assembly on the newly synthesized daughter strands occurs by the binding of H2A/H2B dimers to a H3/H4 tetramer complex.     

Nucleosome assembly in mammalian cell extracts before and after DNA replication.

Protein-free DNA in a cytosolic extract supplemented with SV40 large T-antigen (T-Ag), is assembled into chromatin structure when nuclear extract is added. This assembly was monitored by topoisomer formation, micrococcal nuclease digestion and psoralen crosslinking of the DNA. Plasmids containing SV40 sequences (ori- and ori+) were assembled into chromatin with similar efficiencies whether T-Ag was present or not. Approximately 50-80% of the number of nucleosomes in vivo could be assembled in vitro; however, the kinetics of assembly differed on replicated and unreplicated molecules. In replicative intermediates, nucleosomes were observed on both the pre-replicated and post-replicated portions. We conclude that the extent of nucleosome assembly in mammalian cell extracts is not dependent upon DNA replication, in contrast to previous suggestions. However, the highly sensitive psoralen assay revealed that DNA replication appears to facilitate precise folding of DNA in the nucleosome.     

Chromatin assembly during DNA replication in somatic cells.

Newly replicated DNA is assembled into chromatin through two principle pathways. Firstly, parental nucleosomes segregate to replicated DNA, and are transferred directly to one of the two daughter strands during replication fork passage. Secondly, chromatin assembly factors mediate de-novo assembly of nucleosomes on replicating DNA using newly synthesized and acetylated histone proteins. In somatic cells, chromatin assembly factor 1 (CAF-1) appears to be a key player in assembling new nucleosomes during DNA replication. It provides a molecular connection between newly synthesized histones and components of the DNA replication machinery during the S phase of the cell division cycle.     

Transfer of nucleosomes from parental to replicated chromatin.

Simian virus 40 (SV40) minichromosomes were used as the substrate for in vitro replication. Protein-free SV40 DNA or plasmids, carrying the SV40 origin of replication, served as controls. Replicated minichromosomal DNA possessed constrained negative superhelicity indicative of the presence of nucleosomes. The topological state of replicated minichromosomal DNA was precisely determined by two-dimensional gel electrophoresis. We show that most or all nucleosomes, present on the replicated minichromosomal DNA, were derived from the parental minichromosome substrate. The mode and the rate of nucleosome transfer from parental to minichromosomal daughter DNA were not influenced by high concentrations of competing replicating and nonreplicating protein-free DNA, indicating that nucleosomes remain associated with DNA during the replication process. The data also show that parental nucleosomes were segregated to the replicated daughter DNA strands in a dispersive manner.     

Xenopus egg extracts: a model system for chromatin replication

A cell-free system derived from Xenopus eggs enables in vitro reproduction of the steps occurring during eukaryotic DNA replication. With a circular single-stranded DNA template, extracts obtained from high-speed centrifugation perform complementary DNA strand synthesis coupled to chromatin assembly. Nucleosomes are formed on the newly replicated DNA and the overall reaction mimics the events occurring during chromosomal replication on the lagging strand at the replication fork. ATP is necessary at all steps examined individually, including RNA priming, elongation of DNA strands and chromatin assembly. Although not required for nucleosome formation, ATP is involved in the correct spacing of nucleosomes and the stability of the assembled chromatin. Replication of double-stranded DNA was observed only with extracts obtained from low-speed centrifugation using demembraned sperm nuclei as substrate. Nuclei are reconstituted around the DNA and then undergo a series of events characteristic of a cell cycle. In contrast, neither DNA elongation or chromatin assembly require formation of the nucleus, and both are independent of the cell cycle.     

Xenopus egg extracts: A model system for chromatin replication

A cell-free system derived from Xenopus eggs enables in vitro reproduction of the steps occurring during eukaryotic DNA replication. With a circular single-stranded DNA template, extracts obtained from high-speed centrifugation perform complementary DNA strand synthesis coupled to chromatin assembly. Nucleosomes are formed on the newly replicated DNA and the overall reaction mimics the events occuring during chromosomal replication on the lagging strand at the replication fork. ATP is necessary at all steps examined individually, including RNA priming, elongation of DNA strands and chromatin assembly. Although not required for nucleosome formation, ATP is involved in the correct spacing of nucleosomes and the stability of the assembled chromatin. Replication of double-stranded DNA was observed only with extracts obtained from low-speed centrifugation using demembraned sperm nuclei as substrate. Nuclei are reconstituted around the DNA and then undergo a series of events characteristic of a cell cycle. In contrast, neither DNA elongation or chromatin assembly require formation of the nucleus, and both are independent of the cell cycle.     

The fate of parental nucleosomes during SV40 DNA replication.

The fate of parental nucleosomes during the replication of chromatin templates was studied using a modification of the cell-free SV40 DNA replication system. Plasmid DNA molecules containing the SV40 origin were assembled into chromatin with purified core histones and fractionated assembly factors derived from HeLa cells. When these templates were replicated in vitro, the resulting progeny retained a nucleosomal organization. To determine whether the nucleosomes associated with the progeny molecules resulted from displacement of parental histones during replication followed by reassembly, the replication reactions were performed in the presence of control templates. It was observed that the progeny genomes resulting from the replication of chromatin templates retained a nucleosomal structure, whereas the progeny of the control DNA molecules were not assembled into chromatin. Additional experiments, involving direct addition of histones to the replication reaction mixtures, confirmed that the control templates were not sequestered in some form which made them unavailable for nucleosome assembly. Thus, our data demonstrate that parental nucleosomes remain associated with the replicating molecules and are transferred to the progeny molecules without displacement into solution. We propose a simple model in which nucleosomes ahead of the fork are transferred intact to the newly synthesized daughter duplexes.     

Chromatin replication.

Just as the faithful replication of DNA is an essential process for the cell, chromatin structures of active and inactive genes have to be copied accurately. Under certain circumstances, however, the activity pattern has to be changed in specific ways. Although analysis of specific aspects of these complex processes, by means of model systems, has led to their further elucidation, the mechanisms of chromatin replication in vivo are still controversial and far from being understood completely. Progress has been achieved in understanding: 1. The initiation of chromatin replication, indicating that a nucleosome-free origin is necessary for the initiation of replication; 2. The segregation of the parental nucleosomes, where convincing data support the model of random distribution of the parental nucleosomes to the daughter strands; and 3. The assembly of histones on the newly synthesized strands, where growing evidence is emerging for a two-step mechanism of nucleosome assembly, starting with the deposition of H3/H4 tetramers onto the DNA, followed by H2A/H2B dimers.     

Histone octamer dissociation is not required for in vitro replication of simian virus 40 minichromosomes

Replication of chromosomal templates requires the passage of the replication machinery through nucleosomally organized DNA. To gain further insights into these processes we have used chromatin that was reconstituted with dimethyl suberimidate-cross-linked histone octamers as template in the SV40 in vitro replication system. By supercoiling analysis we found that cross-linked histone octamers were reconstituted with the same kinetic and efficiency as control octamers. Minichromosomes with cross-linked nucleosomes were completely replicated, although the efficiency of replication was lower compared with control chromatin. Analysis of the chromatin structure of the replicated DNA revealed that the cross-linked octamer is transferred to the daughter strands. Thus, our data imply that histone octamer dissociation is not a prerequisite for the passage of the replication machinery and the transfer of the parental nucleosomes.     

Site-specific DNA repair at the nucleosome level in a yeast minichromosome

The rate of excision repair of UV-induced pyrimidine dimers (PDs) was measured at specific sites in each strand of a yeast minichromosome containing an active gene (URA3), a replication origin (ARS1), and positioned nucleosomes. All six PD sites analyzed in the transcribed URA3 strand were repaired more rapidly (greater than 5-fold on average) than any of the nine PD sites analyzed in the nontranscribed strand. Efficient repair also occurred in both strands of a disrupted TRP1 gene (ten PD sites), containing four unstable nucleosomes, and in a nucleosome gap at the 5' end of URA3 (two PD sites). Conversely, slow repair occurred in both strands immediately downstream of the URA3 gene (12 of 14 PD sites). This region contains the ARS1 consensus sequence, a nucleosome gap, and two stable nucleosomes. Thus, modulation of DNA repair occurs in a simple yeast minichromosome and correlates with gene expression, nucleosome stability, and (possibly) control of replication.     

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.     

Purification and characterization of CAF-I, a human cell factor required for chromatin assembly during DNA replication in vitro

The purification and characterization of a replication-dependent chromatin assembly factor (CAF-I) from the nuclei of human cells is described. CAF-I is a multisubunit protein that, when added to a crude cytosol replication extract, promotes chromatin assembly on replicating SV40 DNA. Chromatin assembly by CAF-I requires and is coupled with DNA replication. The minichromosomes assembled de novo by CAF-I consist of correctly spaced nucleosomes containing the four core histones H2A, H2B, H3, and H4, which are supplied in a soluble form by the cytosol replication extract. Thus, by several criteria, the CAF-I-dependent chromatin assembly reaction described herein reflects the process of chromatin formation during DNA replication in vivo.