Friedrich Wanka

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.

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.

Association of nuclear DNA with a rapidly sedimenting structure

Abstract Nuclei were isolated from monolayer cultures of bovine liver cells by use of a Triton containing Tris buffer, and dissolved in 1 M NaCl. Analysis of the lysate by sedimentation through sucrose gradients revealed that a variable proportion of the DNA, sometimes as much as 90%, remained associated with a rapidly sedimenting structure. The rapidly sedimenting material was degraded to smaller fragments by pronase digestion, indicating that proteins are essential to maintain the structural properties conferring the high rate of sedimentation. DNA could be released almost quantitatively by DNAase digestion without causing a significant change of the sedimentation rate of the supporting structure. A limited digestion showed that newly synthesized DNA was more resistant against release from the support. It is suggested that DNA molecules are attached to the rapidly sedimenting structure by one binding site per replicon, and that during replication additional attachment sites are generated by DNA regions at the replication forks.

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.

Selective inhibition by cyclohextimide of nuclear DNA synthesis in synchronous cultures of chlorella

Abstract In synchronous cultures of chlorella pyrenoidosa the synthesis of nuclear DNA was inhibited by more than 90% in the presence of 15 μM cycloheximide. Incorporation of externally applied 14 C-uracil into nuclear DNA was inhibited to about the same extent, while the inhibitor had only little effect on satellite DNA synthesis. The results suggest that continuous protein synthesis is required for nuclear DNA replication.

Ultrastructure of the nuclear matrix from Physarum polycephalum during the mitotic cycle.

We have investigated the ultrastructural changes of the nuclear matrix in nuclei of Physarum polycephalum during the mitotic cycle. Thin sections of high-salt and DNase-extracted nuclei reveal a residual nuclear structure consisting of the lamina and nuclear pore complexes, an internal fibrillogranular matrix, and the residual nucleolus. The lamina remains present until anaphase. The internal matrix is attached to the lamina in interphase but becomes separated from it in prophase. As prophase progresses, the residual nucleolus moves to the periphery of the structure and disintegrates. The internal matrix condenses and takes a more central position in the structure. Its distribution during mitosis follows the known movements of the chromosomes of nuclei in fixed plasmodia. DNase labile threads are continuously attached to the matrix. The results suggest that the internal matrix serves as a carrier of the chromosomal DNA comparable to the function of the chromosomal scaffold of higher eucaryotes.

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, reinitiaton at the same site is excluded. Restoration of the initiable 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.

Enhanced release of nascent single strands from DNA synthesized in the presence of arabinosylcytosine

Arabinosylcytosine at a 1 . 10(-4) molar concentration inhibited thymidine incorporation into DNA by more than 95%. In sucrose gradients the labelled dThd was predominantly found in short DNA chains. Labelled arabinosylcytosince (aC) was incorporated into DNA, as was labelled dThd, indicating that it causes a preferential inhibition of the chain polymerization rather than termination of nascent chains. This was confirmed by the observation of the conversion of short chains to normal size DNA molecules in chase experiments. In the absence of NaCl and at neutral pH a release of more than 50% of the nascent label as single strands was repeatedly observed upon sucrose gradient centrifugation. This release could be significantly reduced by 1 M NaCl, indicating that in the presence of aC the in vivo structure was preserved. This was further confirmed by the fact that aC did not cause detachment of DNA from a rapidly sedimenting nuclear structure. Based on these results, a replication model slightly different from the one suggested by Okazaki, is proposed, in which initiations of new nascent chains can occur at certain distances ahead of the replication fork.

Dissociation of nuclear DNA replication from concomitant protein synthesis in synchronous cultures of chlorella

Abstract The interrelationship between nuclear DNA replication and cytoplasmic protein synthesis in Chlorella was studied by use of hydroxyurea and cycloheximide as specific inhibitors. 15 μM cycloheximide completely blocked the synthesis of protein and depressed DNA and RNA formation by about 90 %. Protein synthesis resumed immediately after removal of the inhibitor, while DNA synthesis was fully restored after a lag time of about 2 h. 10 mM hydroxyurea completely inhibited DNA synthesis. A decrease in protein and RNA synthesis gradually ensued after about 3 h of exposure. DNA synthesis was restored immediately after removal of the inhibitor, but the final DNA levels were markedly lower than in control cultures. During the recovery from the hydroxyurea treatment about 50 % increase of the DNA content was observed in the presence of cycloheximide. The results suggest that simultaneous synthesis of specific proteins required for DNA replication occurs during the S phase. These proteins continue to be synthesized when nuclear DNA replication is stopped by specific inhibitors, and can then be used to support subsequent DNA replication in the absence of protein synthesis.

Decreased DNA synthesis in mammalian cells: After exposure to deoxyadenosine

Abstract The effect of a pre-treatment with 2 mM deoxyadenosine on DNA synthesis in bovine liver cells in vitro was investigated. The incorporation of 3 H-deoxythymidine was strongly depressed after 1 h treatment and recovered only gradually during the following 4 to 6 h. Addition of 50 μM deoxyguanosine during this time effectively counteracted the depression. An autoradiographic study revealed that the depression was due to a decrease in thymidine incorporation per cell rather than a decrease of the proportion of thymidine-incorporating cells. It is concluded from these results that the rate of DNA synthesis is decreased during the initial hours after removal of the deoxy-adenosine because of a continued deficiency of dGTP. This conclusion is supported by the appearance of a high proportion of 3 H-label in replicating intermediates after 1 h of incorporation which is otherwise only observed after pulse labelling for 5 to 10 min.