Association of newly replicated DNA with the nuclear matrix of Physarum polycephalum.

We have studied the role of the nuclear matrix in DNA replication in a naturally synchronized eucaryote, Physarum polycephalum. When P. polycephalum. When P. polycephalum macroplasmodia were pulse labeled with 3H-thymidine, the DNA remaining tightly associated with the matrix was highly enriched in newly synthesized DNA. This enrichment was found both in nuclei that had just initiated DNA replication as well as in nuclei isolated later during S phase. Pulse chase experiments showed that the association of newly replicated DNA with the matrix is transient, since most of the newly replicated DNA could be chased from the matrix by incubating pulse labeled macroplasmodia in media containing unlabeled thymidine. Studies measuring the size distribution of the matrix DNA supported the hypothesis that replication forks are attached to the nuclear matrix. Reconstitution controls indicated that these results were unlikely to be due to preferential, nonspecific binding of nascent DNA to the matrix during the extraction procedures. These results with P. polycephalum in combination with previous studies in non-synchronized rodent cells, suggest that the association of newly replicated DNA with the nuclear matrix may be a general feature of eucaryotic DNA replication.     

The anchoring of newly synthesized adenovirus DNA to the nuclear matrix

After adenovirus infected HeLa cells were pulse labeled and pulse-chase labeled with 3H-thymidine, the nuclear matrix and DNA remaining tightly bound to the matrix were obtained by sequential cell fractionation. Measuring the radioactivity of labeled DNA indicated that newly synthesized viral DNA specifically attach to the nuclear matrix and the amount of binding DNA is in direct proportion to the viral DNA replication activity: then the DNA gradually detach from the matrix and are involved in viral assembly. Electron microscopic autoradiography of the extracted cells showed the virion and viral DNA associated with the nuclear matrix, and thus further confirmed the anchoring of newly synthesized viral DNA to the nuclear matrix.     

Nuclear matrix support of DNA replication

In higher eukaryotic cells, DNA is tandemly arranged into 10(4) replicons that are replicated once per cell cycle during the S phase. To achieve this, DNA is organized into loops attached to the nuclear matrix. Each loop represents one individual replicon with the origin of replication localized within the loop and the ends of the replicon attached to the nuclear matrix at the bases of the loop. During late G1 phase, the replication origins are associated with the nuclear matrix and dissociated after initiation of replication in S phase. Clusters of several replicons are operated together by replication factories, assembled at the nuclear matrix. During replication, DNA of each replicon is spooled through these factories, and after completion of DNA synthesis of any cluster of replicons, the respective replication factories are dismantled and assembled at the next cluster to be replicated. Upon completion of replication of any replicon cluster, the resulting entangled loops of the newly synthesized DNA are resolved by topoisomerases present in the nuclear matrix at the sites of attachment of the loops. Thus, the nuclear matrix plays a dual role in the process of DNA replication: on one hand, it represents structural support for the replication machinery and on the other, provides key protein factors for initiation, elongation, and termination of the replication of eukaryotic DNA.     

Molecular Weight of Deoxyribonucleic Acid Synthesized During Initiation of Chromosome Replication in Escherichia coli

Alkaline sucrose gradients were used to study the molecular weight of deoxyribonucleic acid (DNA) synthesized during the initiation of chromosome replication in Escherichia coli 15 TAU-bar. The experiments were conducted to determine whether newly synthesized, replication origin DNA is attached to higher-molecular-weight parental DNA. Little of the DNA synthesized after readdition of required amino acids to cells previously deprived of the amino acids was present in DNA with a molecular weight comparable to that of the parental DNA. The newly synthesized, low-molecular-weight DNA rapidly appeared in higher-molecular-weight material, but there was an upper limit to the size of this intermediate-molecular-weight DNA. This limit was not observed when exponentially growing cells converted newly synthesized DNA to higher-molecular-weight material. The size of the intermediate-molecular-weight DNA was related to the age of the replication forks, and the size increased as the replication forks moved further from the replication origin. The results indicate that the newly synthesized replication origin DNA is not attached to parental DNA, but it is rapidly attached to the growing strands that extend from the replication fork to the replication origin, or to the other replication fork if replication is bidirectional. Experiments are reported which demonstrate that the DNA investigated was from the vicinity of the replication origin and was not plasmid DNA or DNA from random positions on the chromosome.     

Replication forks are associated with the nuclear matrix.

It has been proposed that DNA in eukaryotic cells is synthesized via replication complexes that are fixed to a proteinaceous nuclear matrix. This model has not been universally accepted because the matrix and its associated DNA are usually prepared under hypertonic conditions that could facilitate non-specific aggregation of macromolecules. We therefore investigated whether different ionic conditions can significantly affect the association of nascent DNA with the nuclear matrix in cultured mammalian cells. Matrices were prepared either by a high salt method or by hypotonic or isotonic LIS extraction. Chromosomal DNA was subsequently removed by digestion with either DNAse I or EcoRI. With all methods of preparation, we found that newly synthesized DNA preferentially partitioned with the nuclear matrix. Furthermore, when the matrix-attached DNA fraction was analyzed by two-dimensional gel electrophoresis, we found that it was markedly enriched for replication forks. We therefore conclude that attachment of DNA to the matrix in the vicinity of replication forks is not induced by conditions of high ionic strength, and that replication may, indeed, occur on or near the skeletal framework provided by the nuclear matrix. From a practical standpoint, our findings suggest a strategy for greatly increasing the sensitivity of two important new gel electrophoretic methods for the direct mapping of replication fork movement through defined chromosomal domains in mammalian cells.     

Is the nuclear matrix the site of DNA replication in eukaryotic cells?

Four types of experiment were carried out to test the recently proposed model of matrix-bound replication in eukaryotic cells. In experiments with pulse-labelling we found preferential association of newly replicated DNA with the matrix only when the procedure for isolation includes first high-salt treatment of isolated nuclei and then digestion with nucleases, or when prior to digestion the nuclei have been stored for a prolonged time. In both cases, however, evidence was found that this preferential association is due to a secondary, artifactual binding of the newly replicated chromatin region to the matrix elements. Pulse-chase experiments and experiments with continuous labelling were carried out to answer the question whether during replication the DNA is reeled through the replication complexes, i.e., whether newly replicated DNA is temporarily or permanently associated with the matrix. The results showed that at that time the matrix DNA does not move from its site of attachment. Since, according to the model of matrix-bound replication, the forks are assumed to be firmly anchored to high-salt resistant proteinaceous matrix structures, the chromatin fragments isolated with endonuclease not recognizing newly replicated DNA and purified by sucrose gradient centrifugation should be free of replication intermediates. The electronmicroscopic analysis of such fragments revealed the existence of intact replication micro-bubbles. Moreover, the fragments with replication configurations appeared as smooth chromatin fibres not attached to elements characteristic for the matrix. All these experiments suggest that the nuclear skeleton is not a native site of DNA replication in eukaryotic cells.     

Structural topography of simian virus 40 DNA replication.

Applying an in situ cell fractionation procedure, we analyzed structural systems of the cell nucleus for the presence of mature and replicating simian virus 40 (SV40) DNA. Replicating SV40 DNA intermediates were tightly and quantitatively associated with the nuclear matrix, indicating that elongation processes of SV40 DNA replication proceed at this structure. Isolated nuclei as well as nuclear matrices were able to continue SV40 DNA elongation under replication conditions in situ, arguing for a coordinated and functional association of SV40 DNA and large T molecules at nuclear structures. SV40 DNA replication also was terminated at the nuclear matrix. While the bulk of newly synthesized, mature SV40 DNA molecules then remained at this structure, some left the nuclear matrix and accumulated at the chromatin.     

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.     

Replication of polyoma DNA in nuclear extracts and nucleoprotein complexes.

Viral nucleoprotein complexes containing radioactive form l DNA or replicative intermediates were extracted from nuclei isolated from polyoma-infected 3T6 fibroblasts, pulse labelled with [³H]thymidine. Such extracts incorporated labelled dGTP into viral DNA, similar to intact isolated nuclei, but at a decreased rate and for shorter periods. The two kinds of nucleoprotein complexes containing form l DNA or replicative intermediates were separated and purified. Each complex retained some capacity to incorporate labelled dGTP and this reaction was stimulated by ATP. The new DNA consisted mainly of short strands hydrogen-bonded to the template. With replicative intermediate complexes incorporation occurred at random into different parts of the viral DNA, while form l complexes incorporated dGTP preferentially into a region around the origin of replication. A crude preparation of T-antigen stimulated the incorporation. The amount of synthesis was low and it was not possible to decide with certainty whether some of the incorporation observed with form 1 complexes represented initiation of new rounds of replication or whether it represented elongation of early replicative intermediates.     

Kinetoplast DNA replication: mechanistic differences between Trypanosoma brucei and Crithidia fasciculata

Kinetoplast DNA, the mitochondrial DNA of trypanosomatid parasites, is a network containing several thousand minicircles and a few dozen maxicircles. We compared kinetoplast DNA replication in Trypanosoma brucei and Crithidia fasciculata using fluorescence in situ hybridization and electron microscopy of isolated networks. One difference is in the location of maxicircles in situ. In C. fasciculata, maxicircles are concentrated in discrete foci embedded in the kinetoplast disk; during replication the foci increase in number but remain scattered throughout the disk. In contrast, T. brucei maxicircles generally fill the entire disk. Unlike those in C. fasciculata, T. brucei maxicircles become highly concentrated in the central region of the kinetoplast after replication; then during segregation they redistribute throughout the daughter kinetoplasts. T. brucei and C. fasciculata also differ in the pattern of attachment of newly synthesized minicircles to the network. In C. fasciculata it was known that minicircles are attached at two antipodal sites but subsequently are found uniformly distributed around the network periphery, possibly due to a relative movement of the kinetoplast disk and two protein complexes responsible for minicircle synthesis and attachment. In T. brucei, minicircles appear to be attached at two antipodal sites but then remain concentrated in these two regions. Therefore, the relative movement of the kinetoplast and the two protein complexes may not occur in T. brucei.     

Structural requirements for simian virus 40 replication and virion maturation.

The nuclear matrix plays an important role in simian virus 40 (SV40) DNA replication in vivo, since functional replication complexes containing large T and replicating SV40 minichromosomes are anchored to this structure (R. Schirmbeck and W. Deppert, J. Virol. 65:2578-2588, 1991). In the present study, we have analyzed the course of events leading from nuclear matrix-associated replicating SV40 minichromosomes to fully replicated minichromosomes and, further, to their encapsidation into mature SV40 virions. Pulse-chase experiments revealed that newly replicated SV40 minichromosomes accumulated at the nuclear matrix and were directly encapsidated into DNase-resistant SV40 virions at this nuclear structure. Alternatively, a small fraction of newly replicated minichromosomes left the nuclear matrix to associate with the cellular chromatin. During the course of infection, progeny virions continuously were released from the nuclear matrix to the cellular chromatin and into the cytoplasm-nucleoplasm. The bulk of SV40 progeny virions, however, remained at the nuclear matrix until virus-induced cell lysis.     

Caffeine-induced reorganization of DNA replicating system occurs on or near nuclear matrix in HeLa cells

Since caffeine reorganizes the DNA replicating system, with several consequences, we studied the effect of caffeine on the DNA replication which normally occurs on or near the nuclear matrix in a variety of eukaryotic cells. When HeLa cells, treated with or without the DNA-damaging agent, neocarzinostatin, were postincubated in the presence or absence of caffeine and then pulse-labeled with [3H]thymidine, the DNA remaining tightly associated with the matrix was enriched in the newly synthesized DNA at the same level as that seen in untreated cells. The nuclear matrix-bound DNA polymerase alpha activity was also the same in these cells. Therefore, in the presence of caffeine, DNA replication, with or without DNA damage, also occurs on or near the nuclear matrix, as is the case in normal DNA replication.     

Initiation of DNA replication at a nuclear matrix-attached chromatin fraction

It is still unclear what nuclear components support initiation of DNA replication. To address this issue, we developed a cell-free replication system in which the nuclear matrix along with the residual matrix-attached chromatin was used as a substrate for DNA replication. We found out that initiation occurred at late G1 residual chromatin but not at early G1 chromatin and depended on cytosolic and nuclear factors present in S phase cells but not in G1 cells. Initiation of DNA replication occurred at discrete replication foci in a pattern typical for early S phase. To prove that the observed initiation takes place at legitimate DNA replication origins, the in vitro synthesized nascent DNA strands were isolated and analyzed. It was shown that they were enriched in sequences from the core origin region of the early firing, dihydrofolate reductase origin of replication ori-beta and not in distal to the origin sequences. A conclusion is drawn that initiation of DNA replication occurs at discrete sub-chromosomal structures attached to the nuclear matrix.     

Permanent attachment of replication origins to the nuclear matrix in BHK-cells.

The position of replication origins and replication forks relative to the nuclear matrix was analysed by autoradiography. Analysis of 2M NaCl-extracted extracted BHK-nuclei, prepared on coverslips, showed that after brief pulses grains were exclusively found over the central core of the residual nuclei, which corresponds to positions in the nuclear matrix. In asynchronous cells these grains were found to migrate into the DNA-halo surrounding the matrix during a subsequent chase. When the pulse had been administered to synchronous cells at the onset of S-phase, it was observed, however, that in the majority of the structures no such migration had occurred. From this, and from the fact that label incorporated later in S-phase could be chased into the halo, we conclude that, contrary to DNA in replication forks, DNA containing replication origins is permanently attached to the nuclear matrix.     

Characterization of Simian virus 40 DNA component II during viral DNA replication

Replicating molecules of Simian virus 40 DNA labeled during a short pulse with [³H]thymidine have been fractionated by ultracentrifugation methods and the open circular form (DNA component II) has been characterized. The pulse-labeled DNA component II is a relatively small constituent (1 to 3%) of the pool of replicating molecules. Examination of the circular (18 S) and linear (16 S) strands of DNA component II by alkaline sedimentation and by degradation using exonuclease III of Escherichia coli reveals that the newly synthesized DNA is principally in the linear strand. Cleavage of pulse-labeled DNA component II by an fi⁺, R-factor restriction endonuclease from E. coli demonstrates that the interruption in the pulse-labeled strand is specifically located at the termination point for replication.During a chase period of 20 minutes the amount of DNA component II increases to about 6 to 8% of the total labeled viral DNA. The kinetics of formation of superhelical, DNA component I and disappearance of replicative intermediates are linear during the chase period. After several hours of continuous labeling of replicating viral DNA, the DNA component II pool consists mainly of molecules labeled in both strands with the interruption non-specifically located in either strand. These molecules probably arise by the random introduction of single-strand breaks in newly synthesized DNA component I. During short periods of continuous labeling with [³H]thymidine, the ratio of DNA components I to II increases as a function of the pulse duration. These results support a model for 8V 40 DNA replication in which the open circular form is a precursor of the superhelical form.     

The NS-1 polypeptide of minute virus of mice is covalently attached to the 5'' termini of duplex replicative-form DNA and progeny single strands.

When A9 cells are infected with minute virus of mice, a small proportion of the virally coded NS-1 polypeptide becomes covalently attached to newly synthesized viral DNA. Antisera directed against NS-1 will specifically precipitate two forms of monomer duplex replicative-form DNA, multimeric duplex intermediates and progeny single strands, and restriction analysis of the duplex forms in these precipitates reveals that NS-1 is exclusively associated with extended-form conformers of the genomic termini. Pulse-labeled viral DNA, harvested at various times in a highly synchronized infection, can be almost quantitatively precipitated with any one of a series of antisera directed against different protein domains distributed throughout the NS-1 molecule but not with antibodies directed against other viral proteins. In each case the interaction with NS-1 can be shown to involve both termini of duplex DNA and single-strand forms, suggesting that in each case a full-length (83-kilodalton) copy of NS-1 is present. Precipitation of the replicating viral DNA with an antibody directed against a synthetic 16-amino-acid peptide containing the sequence at the extreme carboxy terminus of NS-1 can be quantitatively and specifically inhibited with the immunizing peptide in its unconjugated form, showing that the antibodies responsible for precipitating viral DNA are directed against the NS-1 sequence itself and not against a trace contaminant. Exonuclease digestion studies show that the association effectively blocks the 5' ends of the DNA molecules. Very little (less than 0.1%) of the newly synthesized [35S]methionine-labeled NS-1 made in highly synchronized cells during a 15-min pulse early in infection (6.25 to 6.5 h into the S phase) becomes associated with viral DNA immediately. However, pulse-chase experiments show that later in infection (10 to 13 h into the S phase), when viral DNA replication is reaching its peak, a few percent of the molecules in these preexisting pools of NS-1 do become covalently attached to the newly replicated DNA. Isolated viral DNA-protein complexes labeled with [35S]methionine in this way can be obtained by fractionation of the immunoprecipitated complexes on Sepharose CL4B in sodium dodecyl sulfate. Digestion of the purified complexes with nuclease releases an 83-kilodalton molecule which exactly comigrates with authentic NS-1 in sodium dodecyl sulfate-polyacrylamide gels.