Functional aspects of the nuclear matrix.

A model is proposed of the way in which the unwinding of the chromosomal DNA loops is controlled during DNA replication. It is based on the observation of a permanent binding of replication origins to the nuclear matrix and of a transient attachment of replicating DNA regions to sites in the immediate neighbourhood. DNA unwinding is controlled while the replicating loops are reeled through the replication binding sites. Also a mechanism is proposed to explain how the once-per-cycle replication of individual replicons can be controlled. DNA synthesis is initiated at single-stranded loops exposed by tandemly repeated DNA sequences at the replication origins. The single-stranded loops turn into fully double-stranded DNA during replication, becoming inaccessible for a second initiation during the same cell cycle. The configuration competent for initiation is restored by specific protein-DNA rearrangements coupled to mitotic condensation of the matrix into chromosomal scaffolds and its reversal.

Control of eukaryotic DNA replication at the chromosomal level.

A hypothesis for the control of eukaryotic DNA replication at the chromosomal level is proposed. The specific regulatory problem arises from the subdivision of the genome into thousands of individually replicating units, each of which must be duplicated a single time during S-phase. The hypothesis is based on the finding of direct repeats at replication origins. Such repeats can adopt, beyond the full-length double helical structure, another configuration exposing two single-stranded loops that provide suitable templates for the initiation of DNA replication. Any further initiation at the same origin is excluded as the single strandedness is eliminated by the replication process. Restoration of the initiable loop structure is proposed to occur by DNA-protein rearrangements involved in chromosome condensation and duplication of the chromosomal protein backbone during mitosis. A possible role of the maturation promoting factor (MPF) is suggested.

Direct repeats at nuclear matrix-associated DNA regions and their putative control function in the replicating eukaryotic genome.

Short DNA regions, known to contain replication origins, were isolated from 2 M NaCl resistant nuclear structures of Physarum polycephalum after predigestion with DNase. Regions of 100 bp average length were cloned and sequenced. About 25% of the clones contained direct repeats of 12 to 16 bp and variable base sequences, that have been shown to possess the potential of playing a crucial role in the control of DNA replication. In one of the two alternative three-dimensional configurations such repeats expose single-stranded loops that can function as sites for the initiation of new DNA strands. As these regions are converted into full-length duplexes by their own replication, reinitiation at the same site is excluded. Restoration of the initiationable configuration is considered to be coupled to structural rearrangements involved in the transient condensation of chromosomes in mitosis. This mechanisms ensures that any part of the entire eukaryotic genome is reproduced just a single time during one cell cycle.

Induction of DNA replication in mammalian G1 nuclei by a heat-inactivible component from permeable S phase cells; an assay system.

The in vitro initiation of DNA replication was studied in permeable mammalian cells by a newly developed procedure. Pairs of monolayer cultures, one synchronized in G1 and the other in S phase, were incubated in a sandwich with assay solution, containing Triton X-100 for permeabilization and [3H]TTP as a tracer. After 1.5 h DNA synthesis was shown to be induced in 36 to 81% of the G1 nuclei. The inducing capacity of the S phase cultures was diminished by at least 50% after a 10 min exposure to 60 degrees C prior to incubation. The suitability application of this in vitro system for testing components that might effect the initiation of DNA replication is shown in an assay with G1 cultures where the addition of up to 1 mM Ap4A led to an increase of DNA synthesizing cells from 4 to 15%.

Nuclear matrix and chromosome scaffold preparations of in vitro cultured bovine liver cells have two proteins in common.

Nuclear matrices and chromosome scaffolds of in vitro cultured bovine liver cells were prepared under conditions that preserve the specific binding of the DNA. Protein compositions were analysed by electrophoresis and peptide mapping. Two slightly acidic polypeptides of apparent molecular masses 47 and 53 kDa were present in nuclear matrix as well as chromosome scaffold preparations. The corresponding matrix and scaffold proteins had identical peptide maps. Their putative function in the spatial organization of the DNA during the cell cycle is considered.

Evidence for the persistence of a decondensed chromosome scaffold in the interphase nucleus.

Nuclei of in vitro cultured bovine liver cells, deprived of the membranes by Triton X-100, were treated with 2 M-NaCl and DNase. Changes in ultrastructure and protein composition were studied at successive steps during treatment. Electron micrographs of nuclei treated with 2 M-NaCl showed a peripheral lamina and an internal system of randomly coiled filaments embedded in a mass of DNA fibres. After partial removal of the DNA the filaments could be seen to serve as backbones for the DNA attachment. Artificial redistribution occurring during fixation with glutaraldehyde suggests that the salt-resistant filaments are not stably cross-bridged into a three-dimensional network. The existence of reversible cross-bridges in vivo cannot be excluded, however. From the available data it is inferred that the filaments represent a decondensed from of the chromosome scaffolds and play a basic role in the organization of the genome throughout the nuclear cycle.

Evidence for contamination of origin DNA isolated after an in vivo treatment of mammalian cells with 4,5',8-trimethylpsoralen.

We have studied the effect of in vivo treatment with trioxsalen on DNA replication in mammalian cells. In vitro cultured bovine liver cells were exposed to two or four cycles of treatment with 45 microM trioxsalen followed by irradiation with long-wave ultraviolet light. Thymidine incorporation was reduced by about 95% during the first hour after a double treatment. A large proportion of the label was released in alkaline sucrose gradients as a low molecular weight fraction (average length about 500 nucleotides) which was supposed to consist of replication origins containing DNA fragments. From the relative quantities of this DNA obtained at different times of the S phase we concluded that it contains a considerable but not precisely determinable proportion of non-origin DNA. We also find that the fraction is contaminated by a large excess of non-replicating bulk DNA.

Chromatin degradation in isolated nuclei of normal and transformed baby hamster kidney cells.

In a transformed cell line, derived from baby hamster kidney cells by treatment with ethylnitrosourea, degradation of DNA in isolated nuclei by endogenous nuclease was studied. Compared to the nontransformed cell line, the nuclear DNA of the transformed cells was found to be degraded to a much greater extent. This was reflected by a markedly lower proportion of DNA attached to the nuclear protein matrix in the transformed compared to the nontransformed cells. These observations can be accounted for by assuming that the chromatin of the transformed cell line has a conformation different from that of the nontransformed cells.

Protein composition of the chromosomal scaffold and interphase nuclear matrix.

Residual protein structures were prepared from isolated chromosomes and interphase nuclei of in vitro cultured bovine liver cells and the protein compositions were analysed. Chromosomes with minimal cytoplasmic contamination were obtained by a simple procedure using a pH 8 isolation medium containing Triton X-100 and polyamines, and residual protein-DNA complexes were prepared by extraction with 2 M NaCl. Residual protein structures were also obtained by digesting isolated chromosomes with staphylococcal nuclease. Protein compositions of both structures as obtained by SDS-polyacrylamide gel electrophoresis were essentially the same. Residual protein structures were prepared from isolated nuclei by the same procedures. The major nuclear matrix proteins, i.e., the lamins A, B, and C, were not found in the chromosomes and chromosome scaffolds. On the other hand, the residual chromosome structures contained two major polypeptides of 37 and 83 kilodalton relative molecular weights that were absent from the nuclear matrix preparations. A few polypeptides with the same or very similar electrophoretic mobilities were found in the residual structures of both the nuclei and the chromosomes.

Newly-initiated DNA isolated from Physarum in early S phase consists of nascent-nascent duplexes.

We have studied the nature of newly initiated DNA released during DNA isolation at the beginning of S phase of Physarum polycephalum. The released DNA was separated from the bulk DNA by sedimentation through sucrose gradients. Gentle shearing strongly enhanced the release of newly initiated DNA. The additionally released material had a larger average molecular weight. Buoyant density analysis after labelling with bromodeoxyuridine (BrdU) revealed that the released DNA consisted of nascent-nascent duplexes for more than 90%. This indicates that the release of newly initiated DNA occurs by branch migration. We conclude that shearing enhances branch migration by destabilization of the double helix.

In vitro DNA replication in association with the nuclear matrix of permeable mammalian cells.

DNA replication has been studied in in vitro cultured bovine liver cells permeabilized in 0.02% Triton X-100. The Km for TTP was 20 microM. The initial incorporation rate at 10 microM TTP concentration was about 12% of the in vivo synthesis and declined very strongly within 1 h. A similar decline of the incorporation rate was found at 0.12 microM TTP concentration. DNAase I digestion of DNA-matrix complexes obtained from isolated nuclei in 2 M NaCl revealed that newly replicated DNA was preferentially bound to the nuclear matrix. A similar digestion with S1 nuclease caused a selective release of short duplexes of Okazaki fragments with the complementary parental strand. The results show that in vivo replication continues in permeabilized cells in an almost unchanged way, except for a gradual decline of its rate which is mainly due to inactivation of one or more essential components.

Attachment of origins of replication to the nuclear matrix and the chromosomal scaffold.

We have investigated the attachment of DNA to the nuclear matrix and chromosomal scaffold in synchronized bovine liver cells. Label incorporated at the onset of the S phase remained preferentially associated with the matrix during the subsequent G1 phase and with a residual protein structure from dehistonized chromosomes during mitosis. On the other hand label incorporated during mid or late S phase was about equally distributed over the DNA molecule after a chase into the G1 phase. These results suggest that DNA is attached to the nuclear matrix and chromosome scaffolds by the origins of replication.

Organization of DNA replication in Physarum polycephalum. Attachment of origins of replicons and replication forks to the nuclear matrix.

We have investigated the attachment of the DNA to the nuclear matrix during the division cycle of the plasmodial slime mold Physarum polycephalum. The DNA of plasmodia was pulse labelled at different times during the S phase and the label distribution was studied by graded DNase digestion of the matrix-DNA complexes prepared from nuclei isolated by extraction with 2 M NaCl. Pulse labelled DNA was preferentially recovered from the matrix bound residual DNA at any time of the S phase. Label incorporated at the onset of the S phase remained preferentially associated with the matrix during the G2 phase and the subsequent S phase. The occurrence of the pulse label in the matrix associated DNA regions was transiently elevated at the onset of the subsequent S phase. Label incorporated at the end of the S phase was located at DNA regions which, in the G2 phase, were preferentially released from the matrix by DNase treatment. From the results and previously reported data on the distribution of attachment sites it can be concluded that origins of replicons or DNA sites very close to them are attached to the matrix during the entire nuclear cycle. The data further indicate that initiations of DNA replication occur at the same origins in successive S phases. Replicating DNA is bound to the matrix, in addition, by the replication fork or a region close to it. This binding is loosened after completion of the replication.

Composition and DNA-binding properties of the nuclear matrix proteins from mammalian cell nuclei.

A rapidly sedimenting DNA-protein complex was isolated from nuclear lysates in 2 M NaCl and characterized with regard to its polypeptide composition and the DNA-binding properties of the purified proteins. The complex consists of the nuclear matrix with attached DNA. Electrophoresis in SDS-polyacrylamide gels revealed two major and five minor polypeptide bands, mainly in the 60 to 75 kDa molecular weight region. The DNA-matrix complex dissociated into free DNA and proteins in the presence of 2 M NaCl and 5 M urea. The proteins could be purified by chromatography on hydroxyapatite and showed a strong tendency to reassociate at 0.15 M NaCl concentration in the absence of urea. DNA was bound to the reassociated proteins at 0.15 M NaCl concentration. Part of the DNA-protein complex was stable at 1 M NaCl concentration. The binding appeared to be random with regard to the DNA sequence.

Inhibition of DNA synthesis in mammalian cells by daunomycin. Preferential inhibition of replicon initiation at low concentrations.

The effect of the intercalating agent daunomycin on DNA synthesis was studied in cultured bovine liver cells. At low daunomycin concentrations (1 and 2 muM) the rate of [3H]thymidine incorporation decreased progressively with the duration of exposure to the inhibitor. This was accompanied by a shift of nascent DNA intermediates of replicon size to higher sedimentation values on sucrose gradients, indicating that daunomycin preferentially affects the initiation of replicating units, both in asynchronous and synchronized cells. At high daunomycin concentrations (12 muM) the rate of chain growth was also markedly reduced. This was indicated by a rapid and nearly complete cessation of the [3H]thymidine incorporation and an accumulation of nascent DNA intermediates of low molecular weight. These observations are discussed in relation to a pre-fork mode of DNA synthesis.

Isolation of a residual protein structure from nuclei of the myxomycete Physarum polycephalum.

A nuclear framework structure has been obtained from isolated interphase nuclei of Physarum polycephalum by extraction with 2.5 M NaCl and subsequent digestion with DNase. Whole-mount electron micrographs showed a nuclear lamina containing residual pore structures associated with the fibrous internal matrix. The matrix was continuous with fibrillar remnants of the nucleolus. The structure was shown to consist of 2 major polypeptides of 23,000 and 36,500 Daltons as well as 30 to 40 minor polypeptides of various molecular weight classes. The 2 major polypeptides were also prominent in preparations of the residual nucleolar material, suggesting that matrix proteins are common to both structures. The predominance of low-molecular-weight polypeptides in Physarum nuclear matrix suggests that there may be significant differences in composition of nuclear structural proteins between lower and higher eukaryotes.

Analysis of the attachment of replicating DNA to a nuclear matrix in mammalian interphase nuclei.

The attachment of replicating DNA to a rapidly sedimenting nuclear structure was investigated by digestion with various nucleases. When DNA was gradually removed by DNase I, pulse label incorporated during either 1 min or during 1 hour in the presence of arabinosylcytosine, remained preferentially attached to the nuclear structure. Single strand specific digestion by nuclease S1 or staphylococcal nuclease at low concentrations caused a release of about 30% of the pulse label, without significantly affecting the attachment of randomly labelled DNA. The released material had a low sedimentation coefficient and contained most of the Okasaki fragments. The remaining pulse label was less accessible to further digestion by double strand specific nuclease activity than the bulk DNA. The results suggest that an attachment of the replication fork to the nuclear structure occurs at sites behind but close to the branch point.