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.     

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.     

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.     

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.     

Replication of SV40 minichromosomes in vitro

We operationally define two forms of SV40 minichromosomes, a 75S-form, prepared at low salt concentration, referred to as native minichromosomes, and a 50S-form, obtained after treatment with 0.5M potassium acetate, the salt-treated minichromosomes. Both preparations of minichromosomes serve well as templates for replication in vitro. Their respective replication products are strikingly different: replicated native minichromosomes contain a densely packed array of the maximal number of nucleosomes whereas replicated salt-treated minichromosomes carry, on average, half of the maximal number. We conclude that in both cases parental nucleosomes are transferred to progeny DNA, and, in addition, that an assembly of new nucleosomes occurs during the replication of native minichromosomes. This is apparently due to the presence of a nucleosome assembly factor as a constituent of native minichromosomes that dissociates upon treatment with salt. We further show that preparations of minichromosomes usually contain significant amounts of copurifying hnRNP particles and SV40 virion precursor particles. However, these structures do not detectably affect the replication and the chromatin assembly reactions.     

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.     

Replicative intermediates in UV-irradiated simian virus 40

We have used Simian virus 40 (SV40) as a probe to study the replication of UV-damaged DNA in mammalian cells. Viral DNA replication in infected monkey kidney cells was synchronized by incubating a mutant of SV40 (tsA58) temperature-sensitive for the initiation of DNA synthesis at the restrictive temperature and then adding aphidicolin to temporarily inhibit DNA synthesis at the permissive temperature while permitting pre-replicative events to occur. After removal of the drug, the infected cells were irradiated at 100 J/m2 (254 nm) to produce 6-7 pyrimidine dimers per SV40 genome, and returned to the restrictive temperature to prevent reinitiation of replication from the SV40 origin. Replicative intermediates (RI) were labeled with [3H]thymidine, and isolated by centrifugation in CsCl/ethidium bromide gradients followed by BND-cellulose chromatography. The size distribution of daughter DNA strands in RI isolated shortly after irradiation was skewed towards lengths less than the interdimer spacing in parental DNA; this bias persisted for at least 1 h after irradiation, but disappeared within 3 h, by which time the size of the newly-synthesized DNA exceeded the interdimer distance. No significant excision of dimers from parental strands in either replicative intermediates or Form I (closed circular) DNA molecules was detected. These data are consistent with the hypothesis that replication forks are temporarily blocked by dimers encountered on the leading strand side of the fork, but that daughter strand continuity opposite dimers is eventually established. Evidence was obtained for the generation at late times after irradiation, of Form I molecules in which the daughter DNA strands contain dimers. Thus DNA strand exchange as well as trans-dimer synthesis may be involved in the generation of supercoiled Form I DNA from UV-damaged SV40 replicative intermediates.     

Replication of the simian virus 40 chromosome with purified proteins.

SV40 chromosomes prepared from infected CV-1 cells were replicated with the purified proteins of SV40 T antigen, HeLa DNA polymerase alpha-primase complex, single-stranded DNA-binding protein, and topoisomerases I and II, all of which have been shown to be essential for SV40 DNA replication in vitro. Replication started near the origin and proceeded bidirectionally. The maximum speed of replication fork movement was 200-300 nucleotides/min, which was similar to the rate of SV40 DNA replication with the same set of proteins. When replication products were digested with micrococcal nuclease, DNA fragments of 160-180 base pairs, which is the typical size of mononucleosomal DNA, were protected. This result indicates that replicated DNA was reconstructed into the nucleosome structure, complexed with parental histones.     

Structure of Replicating Simian Virus 40 Deoxyribonucleic Acid Molecules

Properties of replicating simian virus 40 (SV40) deoxyribonucleic acid (DNA) have been examined by sedimentation analysis and by direct observation during a lytic cycle of infection of African green monkey kidney cells. Two types of replicating DNA molecules were observed in the electron microscope. One was an open structure containing two branch points, three branches, and no free ends whose length measurements were consistent with those expected for replicating SV40 DNA molecules. A second species had the same features as the open structure, but in addition it contained a superhelix in the unreplicated portion of the molecule. Eighty to ninety per cent of the replicative intermediates (RI) were in this latter configuration, and length measurements of these molecules also were consistent with replicating SV40 DNA. Replicating DNA molecules with this configuration have not been described previously. RI, when examined in ethidium bromide-cesium chloride (EB-CsCl) isopycnic gradients, banded in a heterogeneous manner. A fraction of the RI banded at the same density as circular SV40 DNA containing one or more single-strand nicks (component II). The remaining radioactive RI banded at densities higher than that of component II, and material was present at all densities between that of supercoiled double-stranded DNA (component I) and component II. When RI that banded at different densities in EB-CsCl were examined in alkaline gradients, cosedimentation of parental DNA and newly replicated DNA did not occur. All newly replicated DNA sedimented more slowly than did intact single-stranded SV40 DNA, a finding that is inconsistent with the rolling circle model of DNA replication. An inverse correlation exists between the extent of replication of the SV40 DNA and the banding density in EB-CsCl. Under alkaline conditions, the parental DNA strands that were contained in the RI sedimented as covalently closed structures. The sedimentation rates in alkali of the covalently closed parental DNA decreased as replication progressed. Based on these observations, some possible models for replication of SV40 DNA are proposed.     

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.     

Chromatin assembly during SV40 DNA replication in vitro

A cytosol extract from human 293 cells supports efficient replication of SV40 origin-containing plasmid DNA in the presence of the SV40 T antigen. Addition of a nuclear extract from the same cells promotes negative supercoiling of the replicated DNA but not the bulk of the unreplicated DNA. The level of superhelicity is affected by the concentrations of T antigen and nuclear extract factors and by the time of addition of the nuclear extract. The replicated DNA in isolated DNA-protein complexes resists relaxation by purified HeLa cell topoisomerase I. Micrococcal nuclease digestion, sucrose gradient sedimentation, and electron microscopy demonstrate that the negative supercoils result from assembly of the replicating DNA into a chromatin structure. These results suggest that, during DNA replication, the core histones can be assembled on both sides of the replication fork by an active, replication-linked mechanism that does not require a template of preexisting nucleosomes.     

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.     

The effect of the simple repeating d(CG.GC)n, d(CA.GT)n, and d(A.T)n DNA sequences on the nucleosomal organization of SV40 minichromosomes.

The effect of several simple repeating DNA sequences--d(CG.GC)5, d(CA.GT)30, and d(A.T)60--on the nucleosomal organization of the SV40 minichromosome is analyzed. These three different sequences were cloned at the Hpa II site of SV40 (position 346) which occurs at the 3' border of the nucleosome-free SV40 control region. Our results show that neither the d(A.T)60 sequence nor the d(CG.GC)5 sequence appear to have any relevant effect on the nucleosomal organization of the region of the minichromosome surrounding the inserted repeated sequence. Both sequences are hypersensitive to micrococcal nuclease cleavage in the minichromosome, indicating that they are not organized into nucleosomes. On the other hand, the d(CA.GT)30 sequence is found organized as nucleosomes and causes the delocation of nucleosomes in the minichromosomal region close to the inserted repeated sequence.     

Preirradiation of host cells does not alter blockage of simian virus 40 replication forks by pyrimidine dimers

Do damage-inducible responses in mammalian cells alter the interaction of lesions with replication forks? We have previously demonstrated that preirradiation of the host cell mitigates UV inhibition of SV40 DNA replication; this mitigation can be detected within the first 30 min after the test irradiation. Here we test the hypotheses that this mitigation involves either (1) rapid dimer removal, (2) rapid synthesis of daughter strands past lesions (trans-dimer synthesis), or (3) continued progression of the replication fork beyond a dimer. Cells preirradiated with UV were infected with undamaged SV40, and the effects of UV upon viral DNA synthesis were measured within the first hour after a subsequent test irradiation. In preirradiated cells, as well as in non-preirradiated cells, pyrimidine dimers block elongation of daughter strands; daughter strands grow only to a size equal to the interdimer distance along the parental strands. There is, within this first hour after UV, no evidence for trans-dimer synthesis, nor for more rapid dimer removal either in the bulk of the parental DNA or in molecules in the replication pool. Progression of the replication forks was analyzed by electron microscopy of replicating SV40 molecules. Dimers block replication-fork progression in preirradiated cells to the same extent as in non-preirradiated cells. These experiments argue strongly against the hypotheses that preirradiation of host cells results in either the rapid removal of dimers, trans-dimer synthesis, or continued replication-fork progression beyond dimers.     

Nucleosome positioning at the replication fork.

In order to determine the time required for nucleosomes assembled on the daughter strands of replication forks to assume favoured positions with respect to DNA sequence, psoralen cross-linked replication intermediates purified from preparative two-dimensional agarose gels were analysed by exonuclease digestion or primer extension. Analysis of sites of psoralen intercalation revealed that nucleosomes in the yeast Saccharomyces cerevisiae rDNA intergenic spacer are positioned shortly after passage of the replication machinery. Therefore, both the 'old' randomly segregated nucleosomes as well as the 'new' assembled histone octamers rapidly position themselves (within seconds) on the newly replicated DNA strands, suggesting that the positioning of nucleosomes is an initial step in the chromatin maturation process.     

Hairpin structures are the primary amplification products: a novel mechanism for generation of inverted repeats during gene amplification.

Early events of DNA amplification which occur during perturbed replication were studied by using simian virus 40 (SV40)-transformed Chinese hamster cells (CO60) as a model system. The amplification is observed shortly after carcinogen treatment, and the amplified sequences contain molecules organized as inverted repeats (IRs). SV40 amplification in vitro was studied by using extracts from carcinogen-treated CO60 cells. In the amplified DNA the SV40 origin region was rereplicated, while more distal sequences were not replicated even once. Using several experimental procedures such as sucrose gradients, "snap-back" assay, and two-dimensional gel electrophoresis, we show that the overreplicated DNA contains IRs which are synthesized de novo as hairpins or stem-loop structures which were detached from the template molecules. The fully replicated SV40 molecules synthesized by the HeLa extracts do not contain such IRs. We propose "U-turn replication" as a novel mechanism for gene amplification, accounting for the generation of extrachromosomal inverted duplications as a result of perturbed replication and template switching of the DNA polymerases.