A nucleosome assembly factor is a constituent of simian virus 40 minichromosomes.

Using in vitro replication assays, we compared native with salt-treated simian virus 40 minichromosomes isolated from infected cell nuclei. Minichromosomes from both preparations contain the full complement of nucleosomes, but salt treatment removes histone H1 and a fraction of nonhistone chromatin proteins. Both types of minichromosomes served well as templates for in vitro replication, but the structures of the replication products were strikingly different. Replicated salt-treated minichromosomes contained, on average, about half the normal number of nucleosomes as previously shown (T. Krude and R. Knippers, Mol. Cell. Biol. 11:6257-6267, 1991). In contrast, the replicated untreated minichromosomes were found to be densely packed with nucleosomes, indicating that an assembly of new nucleosomes occurred during in vitro replication. Biochemical and immunological data showed that the fraction of nonhistone chromatin proteins associated with native minichromosomes includes a nucleosome assembly activity that appears to be closely related to chromatin assembly factor I (S. Smith and B. W. Stillman, Cell 58:15-25, 1989). Furthermore, this minichromosome-bound nucleosome assembly factor is able to exert its activity in trans to replicating protein-free competitor DNA. Thus, native chromatin itself contains the activities required for an ordered assembly of nucleosomes during the replication process.     

Accessibility to topoisomerases I and II regulates the replication efficiency of simian virus 40 minichromosomes.

We determined the effects of chromatin structure on template accessibility to replication factors and used three different templates as substrates for simian virus 40 (SV40) DNA replication in vitro: native and salt-treated SV40 minichromosomes and protein-free SV40 DNA. Native minichromosomes contain histone H1 and numerous nonhistone proteins in addition to the core histones, whereas salt-treated minichromosomes carry essentially only core histones. We reasoned that the less densely packed salt-treated minichromosomes should be more effective replication templates due to their more extended configuration. However, contrary to this expectation, we found that native minichromosomes replicated with significantly higher efficiency than salt-treated minichromosomes, while protein-free DNA was most active as a replication template. The higher replication efficiency of native minichromosomes was due to two activities bound to the chromatin, which were identified as DNA topoisomerases I and II. By using chromatin substrates of different general configurations, we also showed that the overall chromatin structure determines accessibility to topoisomerases I and II and thereby the efficiency of replicative chain elongation.     

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.     

Structure of replicating simian virus 40 minichromosomes. The replication fork, core histone segregation and terminal structures

The structure of replicating simian virus 40 (SV40) minichromosomes was studied by DNA crosslinking with trimethyl-psoralen. The procedure was used both in vitro with extracted SV40 minichromosomes as well as in vivo with SV40-infected cells. Both procedures gave essentially the same results. Mature SV40 minichromosomes are estimated to contain about 27 nucleosomes (error +/- 2), except for those molecules with a nucleosome-free gap, which are interpreted to contain 25 nucleosomes (error +/- 2). In replicative intermediates, nucleosomes are present in the unreplicated parental stem with the replication fork possibly penetrating into the nucleosomal DNA before the histone octamer is removed. Nucleosomes reassociate on the newly replicated DNA branches at distances from the branch point of 225 ( +/- 145) nucleotides on the leading strand and of 285( +/- 120) nucleotides on the lagging strand. In the presence of cycloheximide, daughter duplexes contained unequal numbers of nucleosomes, supporting dispersive and random segregation of parental nucleosomes. These were arranged in clusters with normal nucleosome spacing. We detected a novel type of interlocked dimer comprising two fully replicated molecules connected by a single-stranded DNA bridge. We cannot decide whether these dimers represent hemicatenanes or whether the two circles are joined by a Holliday-type structure. The joining site maps within the replication terminus. We propose that these dimers represent molecules engaged in strand segregation.     

Preferential association of newly synthesized histones with replicating SV40 DNA.

The assembly of newly synthesized histones into nucleosomes during replication of SV40 minichromosomes in vivo was studied. Infected cells were labeled with 35S-methionine for a time shorter than that required to complete a round of viral DNA replication. Mature and replicating SV40 minichromosomes were extracted and separated by zonal sedimentation, and their histone content was analyzed by polyacrylamide gel electrophoresis (SDS and acidic urea). We show that the pulse-labeled histones associate preferentially with the replicating DNA.     

Effects of cell cycle dependent histone H1 phosphorylation on chromatin structure and chromatin replication.

We have reconstituted salt-treated SV40 minichromosomes with differentially phosphorylated forms of histone H1 extracted from either G0-, S- or M-phase cells. Sedimentation studies revealed a clear difference between minichromosomes reconstituted with S-phase histone H1 compared with histone H1 from G0- or M-phase cells, indicating that the phosphorylation state of histone H1 has a direct effect on chromatin structure. Using reconstituted minichromosomes as substrate in the SV40 in vitro replication system, we measured a higher replication efficiency for SV40 minichromosomes reconstituted with S-phase histone H1 compared with G0- or M-phase histone H1. These data indicate that the chromatin structure induced by the phosphorylation of histone H1 influences the replication efficiency of SV40 minichromosomes in vitro.     

SV40 viral minichromosome: preferential exposure of the origin of replication as probed by restriction endonucleases.

Isolated SV40 minichromosomes [1-3] were treated with different single-cut restriction endonucleases to probe the arrangement of nucleosomes in relation to the SV70 DNA sequence. While Eco RI and Bam HI each cut 22-27% of the SV40 minichromosomes under limit-digest conditions, Bgl I, which cuts SV40 DNA at or very near the origin of replication [4,5], cleaves 90-95% of the minichromosomes in a preparation. Similar results were obtained with minichromosomes which had been fixed with formaldehyde before endonuclease treatment. One possible interpretation of these findings is that the arrangement of nucleosomes in the compact SV40 minichromosomes is nonrandom at least with regard to sequences near the origin of DNA replication.     

Re-replication of SV40 minichromosomes is inhibited at the stage of chain elongation.

The template activities of protein-free SV40 DNA and SV40 minichromosomes for DNA re-replication are compared in in vitro replication assays. Density substitution experiments and two-dimensional gel electrophoresis show that protein-free DNA can replicate for at least two cycles whereas salt-treated minichromosomes replicate only once. Re-replication of minichromosomes is blocked at the stage of replicative chain elongation suggesting that replicatively assembled chromatin has structural features that prevent a second round of replication.     

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.     

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.     

Role of amino-terminal histone domains in chromatin replication.

Simian virus 40 minichromosomes were treated with trypsin to specifically remove the amino-terminal histone domains (tails). Trypsin treatment does not affect the spacing and the number of nucleosomes on minichromosomes but indices a more extended conformation, as shown by the reduced sedimentation coefficient of trypsinized minichromosomes compared with the untreated controls. Trypsinized minichromosomes replicate more efficiently than control minichromosomes in in vitro replication assays. The increased template efficiency appears to be due to higher rates of replicative fork movement. In vitro replication in the presence of protein-free competitor DNA shows that replicating trypsinized minichromosomes do not lose nucleosomes and replicating competitor DNA does not gain nucleosomes. This finding suggests that tailless nucleosomes are transferred from the unreplicated prefork stem to replicated DNA branches and excludes a participation of the basic histone domains in nucleosome transfer.     

A large-tumor-antigen-specific monoclonal antibody inhibits DNA replication of simian virus 40 minichromosomes in an in vitro elongation system.

In productively infected cells, a fraction of large-tumor antigen (T antigen) is tightly bound to replicating simian virus 40 (SV40) minichromosomes and does not dissociate at salt concentrations of greater than 1 M NaCl. We present electronmicrograms demonstrating the presence of T antigen on the replicated sections of replicating SV40 minichromosomes. We also show that the fraction of tightly bound T antigen is recognized by antibodies from mouse tumor serum and, more specifically, by a particular T-antigen-specific monoclonal antibody, PAb 1630. A second T-antigen-specific monoclonal antibody, PAb 101, does not react with the T-antigen fraction remaining on replicating SV40 chromatin at high salt concentrations. We used an in vitro replication system which allows, via semiconservative DNA replication, the completion of in vivo-initiated replicative intermediate DNA molecules. We show that monoclonal antibody PAb 1630, but not monoclonal antibody PAb 101, inhibits viral DNA replication. We discuss the possibility that SV40 T antigen may play a role in chain elongation during SV40 chromatin replication.     

Conditions for sliding of nucleosomes along DNA: SV 40 minichromosomes

'Sliding' of nucleosomes along DNA under nearly physiological conditions was studied using treatment of SV 40 minichromosomes with the single-cut restriction endonucleases EcoRI and BamHI. Each enzyme can convert no more than 20-25% of the circular DNA molecules of minichromosomes into the linear form irrespective of the presence of histone H1. This suggests absence of the nucleosomes lateral migration (sliding) along DNa at least in the vicinity of the restriction endonucleases cleavage sites during several hours of incubation. The sites available for EcoRI and BamHI in minichromosomes seem to be located predominantly in the spacer DNA regions of nucleosomes. Introduction of only one double-strand (but not single-strand) break into the DNA of minichromosomes stripped of histone H1 is sufficient to induce redistribution of the nucleosome core particles due to their sliding along DNA. Thus, sliding of the nucleosome core particles can be induced under physiological conditions by rather low energy expenditures.     

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.     

Influence of core histone acetylation on SV40 minichromosome replication in vitro

We have used the SV40 in vitro replication system to analyze the replication efficiencies of SV40 minichromosomes associated with normal or hyperacetylated histones. We found that elongation of replication occurs with higher efficiency in hyperacetylated minichromosomes in comparison with normal minichromosomes. Our results indicate that the movement of the replication machinery through nucleosomal DNA is facilitated by charge neutralization due to acetylation of the histone tails. Edited by: A. Wolffe