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1.
Addition of purified human topoisomerase I (topo I) to simian virus 40 T antigen-driven in vitro DNA replication reactions performed with topo I-deficient extracts results in a greater than 10-fold stimulation of completed molecules as well as a more than 3-fold enhancement of overall DNA replication. To further characterize this stimulation, we first demonstrate that bovine topo I but not Escherichia coli topo I can also enhance DNA replication. By using several human topo I mutants, we show that a catalytically active form of topo I is required. To delineate whether topo I influences the initiation or the elongation step of replication, we performed delayed pulse, pulse-chase, and delayed pulse-chase experiments. The results illustrate that topo I cannot promote the completion of partially replicated molecules but is needed from the beginning of the reaction to initiate replication. Competitive inhibition experiments with the topo I binding T antigen fragment 1-246T and a catalytically inactive topo I mutant suggest that part of topo I's stimulation of replication is mediated through a direct interaction with T antigen. Collectively, our data indicate that topo I enhances the synthesis of fully replicated DNA molecules by forming essential interactions with T antigen and stimulating initiation. 相似文献
2.
Exogenously added simian virus 40 (SV40) DNA can be replicated semiconservatively in vitro by a mixture of a soluble extract of HeLa cell nuclei and the cytoplasm from SV40-infected CosI cells. When cloned DNA was used as a template, the clone containing the SV40 origin of DNA replication was active, but a clone lacking the SV40 origin was inactive. The major products of the in vitro reaction were form I and form II SV40 DNAs and a small amount of form III. DNA synthesis in extracts began at or near the in vivo origin of SV40 DNA synthesis and proceeded bidirectionally. The reaction was inhibited by the addition of anti-large T hamster serum, aphidicolin, or RNase but not by ddNTP. Furthermore, this system was partially reconstituted between HeLa nuclear extract and the semipurified SV40 T antigen instead of the CosI cytoplasm. It is clear from these two systems that the proteins containing SV40 T antigen change the nonspecific repair reaction performed by HeLa nuclear extract alone to the specific semiconservative DNA replication reaction. These results show that these in vitro systems closely resemble SV40 DNA replication in vivo and provide an assay that should be useful for the purification and subsequent characterization of viral and cellular proteins involved in DNA replication. 相似文献
3.
4.
Studies of simian virus 40 (SV40) DNA replication in vitro have identified a small (approximately 30-nucleotide) RNA-DNA hybrid species termed primer-DNA. Initial experiments indicated that T antigen and the polymerase alpha-primase complex are required to form primer-DNA. Proliferating cell nuclear antigen, and presumably proliferating cell nuclear antigen-dependent polymerases, is not needed to form this species. Herein, we present an investigation of the stages at which primer-DNA functions during SV40 DNA replication in vitro. Hybridization studies indicate that primer-DNA is initially formed in the origin region and is subsequently synthesized in regions distal to the origin. At all time points, primer-DNA is synthesized from templates for lagging-strand DNA replication. These studies indicate that primer-DNA functions during both initiation and elongation stages of SV40 DNA synthesis. Results of additional experiments suggesting a precursor-product relationship between formation of primer-DNA and Okazaki fragments are presented. 相似文献
5.
High yield of endoreduplication induced by ICRF-193: a topoisomerase II catalytic inhibitor 总被引:4,自引:0,他引:4
Previous studies have demonstrated that phenolic compounds, including genistein (4',5,7-trihydroxyisoflavone) and resveratrol (3,4',5-trihydroxystilbene), are able to protect against carcinogenesis in animal models. This study was undertaken to examine the ability of genistein and resveratrol to inhibit reactive oxygen species (ROS)-mediated strand breaks in phi X-174 plasmid DNA. H(2)O(2)/Cu(II) and hydroquinone/Cu(II) were used to cause oxidative DNA strand breaks in the plasmid DNA. We demonstrated that the presence of genistein at micromolar concentrations resulted in a marked inhibition of DNA strand breaks induced by either H(2)O(2)/Cu(II) or hydroquinone/Cu(II). Genistein neither affected the Cu(II)/Cu(I) redox cycle nor reacted with H(2)O(2) suggest that genistein may directly scavenge the ROS that participate in the induction of DNA strand breaks. In contrast to the inhibitory effects of genistein, the presence of resveratrol at similar concentrations led to increased DNA strand breaks induced by H(2)O(2)/Cu(II). Further studies showed that in the presence of Cu(II), resveratrol, but not genistein was able to cause DNA strand breaks. Moreover, both Cu(II)/Cu(I) redox cycle and H(2)O(2) were shown to be critically involved in resveratrol/copper-mediated DNA strand breaks. The above results indicate that despite their similar in vivo anticarcinogenic effects, genistein and resveratrol appear to exert different effects on oxidative DNA damage in vitro. 相似文献
6.
Effects of VM26, a specific inhibitor of type II DNA topoisomerase, on SV40 chromatin replication in vitro. 总被引:1,自引:0,他引:1
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We have examined the influence of VM26 (teniposide), a specific inhibitor of mammalian type II DNA topoisomerase, on the replication of SV40 minichromosomes in vitro. The replication system we used consists of replicative intermediate SV40 chromatin as substrate which is converted to mature SV40 chromatin in the presence of ATP, deoxynucleotides and a protein extract from uninfected cells. The addition of 100 microM VM26 to this system reduces DNA synthesis to 70 to 80 percent of the control and leads to an accumulation of 'late replicative intermediates'. The VM26 induced block of replication was not released by the addition of large quantities of type I DNA topoisomerase. We conclude, that type II DNA topoisomerase is essential for the final replication steps leading from late Cairns structures of replicative intermediates to monomeric minichromosomes. It appears that type I DNA topoisomerase can function as a swivelase during most of the replicative elongation phase, but must later be replaced by type II DNA topoisomerase. 相似文献
7.
Effects of ICRF-193, a topoisomerase II inhibitor, on metaphase chromosome preparations were examined. A short-time exposure of this drug to human HL60 cells in a suspension culture before harvest resulted in obtaining more extended metaphase chromosomes. The length of chromosome 6 identified by fluorescence in situ hybridization was twice as long with this drug treatment. Together with effectiveness for adherent HepG2 cells, these results suggest that treatments with ICRF-193 provide a simple and reliable method for extended metaphase chromosome preparations from cultured cells. 相似文献
8.
In vitro initiation of DNA replication in simian virus 40 chromosomes 总被引:15,自引:0,他引:15
R S Decker M Yamaguchi R Possenti M K Bradley M L DePamphilis 《The Journal of biological chemistry》1987,262(22):10863-10872
A soluble system has been developed that can initiate DNA replication de novo in simian virus 40 (SV40) chromatin isolated from virus-infected monkey cells as well as in circular plasmid DNA containing a functional SV40 origin of replication (ori). Initiation of DNA replication in SV40 chromatin required the soluble fraction from a high-salt nuclear extract of SV40-infected cells, a low-salt cytosol fraction, polyethylene glycol, and a buffered salts solution containing all four standard deoxyribonucleoside triphosphates. Purified SV40 large tumor antigen (T-ag) partially substituted for the high-salt nucleosol, and monoclonal antibodies directed against SV40 T-ag inhibited DNA replication. Replication began at ori and proceeded bidirectionally to generate replicating DNA intermediates in which the parental strands remained covalently closed, as observed in vivo. Partial inhibition of DNA synthesis by aphidicolin resulted in accumulation of newly initiated replicating intermediates in this system, a phenomenon not observed under conditions that supported completion of replication only. However, conditions that were optimal for initiation of replication repressed conversion of late-replicating intermediates into circular DNA monomers. Most surprising was the observation that p-n-butylphenyl-dGTP, a potent and specific inhibitor of DNA polymerase-alpha, failed to inhibit replication of SV40 chromatin under conditions that completely inhibited replication of plasmid DNA containing the SV40 ori and either purified or endogenous DNA polymerase-alpha activity. In contrast, all of these DNA synthesis activities were inhibited equally by aphidicolin. Therefore, DNA replication in mammalian cells is carried out either by DNA polymerase-alpha that bears a unique association with chromatin or by a different enzyme such as DNA polymerase-delta. 相似文献
9.
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. 相似文献
10.
Non-specific termination of simian virus 40 DNA replication. 总被引:4,自引:0,他引:4
Axial X-ray diffraction patterns have been studied from relaxed, contracted and rigor vertebrate striated muscles at different sarcomere lengths to determine which features of the patterns depend on the interaction of actin and myosin. The intensity of the myosin layer lines in a live, relaxed muscle is sometimes less in a stretched muscle than in the muscle at rest-length; the intensity depends not only on the sarcomere length but on the time that has elapsed since dissection of the muscle. The movement of cross-bridges giving rise to these intensity changes are not caused solely by the withdrawal of actin from the A-band.When a muscle contracts or passes into rigor many changes occur that are independent of the sarcomere length: the myosin layer lines decrease in intensity to about 30% of their initial value when the muscle contracts, and disappear completely when the muscle passes into rigor. Both in contracting and rigor muscles at all sarcomere lengths the spacings of the meridional reflections at 143 Å and 72 Å are 1% greater than from a live relaxed muscle at rest-length. It is deduced that the initial movement of cross-bridges from their positions in resting muscle does not depend on the interaction of each cross-bridge with actin, but on a conformational change in the backbone of the myosin filament: occurring as a result of activation. The possibility is discussed that the conformational change occurs because the myosin filament, like the actin filament, has an activation control mechanism. Finally, all the X-ray diffraction patterns are interpreted on a model in which the myosin filament can exist in one of two possible states: a relaxed state which gives a diffraction pattern with strong myosin layer lines and an axial spacing of 143.4 Å, and an activated state which gives no layer lines but a meridional spacing of 144.8 Å. 相似文献
11.
Inhibition of DNA topoisomerase II by ICRF-193 induces polyploidization by uncoupling chromosome dynamics from other cell cycle events 总被引:11,自引:1,他引:10
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《The Journal of cell biology》1994,126(6):1341-1351
ICRF-193, a novel noncleavable, complex-stabilizing type topoisomerase (topo) II inhibitor, has been shown to target topo II in mammalian cells (Ishida, R., T. Miki, T. Narita, R. Yui, S. Sato, K. R. Utsumi, K. Tanabe, and T. Andoh. 1991. Cancer Res. 51:4909-4916). With the aim of elucidating the roles of topo II in mammalian cells, we examined the effects of ICRF-193 on the transition through the S phase, when the genome is replicated, and through the M phase, when the replicated genome is condensed and segregated. Replication of the genome did not appear to be affected by the drug because the scheduled synthesis of DNA and activation of cdc2 kinase followed by increase in mitotic index occurred normally, while VP-16, a cleavable, complex-stabilizing type topo II inhibitor, inhibited all these processes. In the M phase, however, late stages of chromosome condensation and segregation were clearly blocked by ICRF-193. Inhibition at the stage of compaction of 300-nm diameter chromatin fibers to 600-nm diameter chromatids was demonstrated using the drug during premature chromosome condensation (PCC) induced in tsBN2 baby hamster kidney cells in early S and G2 phases. In spite of interference with M phase chromosome dynamics, other mitotic events such as activation of cdc2 kinase, spindle apparatus reorganization and disassembly and reassembly of nuclear envelopes occurred, and the cells traversed an unusual M phase termed "absence of chromosome segregation" (ACS)-M phase. Cells then continued through further cell cycle rounds, becoming polyploid and losing viability. This effect of ICRF-193 on the cell cycle was shown to parallel that of inactivation of topo II on the cell cycle of the ts top2 mutant yeast. The results strongly suggest that the essential roles of topo II are confined to the M phase, when the enzyme decatenates intertwined replicated chromosomes. In other phases of the cycle, including the S phase, topo II may thus play a complementary role with topo I in controlling the torsional strain accumulated in various genetic processes. 相似文献
12.
Catenation of DNA by eucaryotic topoisomerase II associated with simian virus 40 minichromosomes 总被引:3,自引:1,他引:2
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After incubation of purified SV40 minichromosomes with superhelical DNA molecules either of SV40 or plasmid origin, a catenation of monomeric DNA via dimers and multimers to large networks was observed. The catenation reaction was stimulated by the DNA condensing agent spermidine with ATP as an energy donor and was dependent on the presence of magnesium ions. The reaction could be blocked by inhibitors of topoisomerase II such as novobiocin and nalidixic acid. Relaxed covalently closed circular DNA was catenated to networks in the presence of ATP as the energy donor. 相似文献
13.
Involvement of DNA polymerase alpha in simian virus 40 DNA replication. 总被引:28,自引:0,他引:28
14.
Differential increase in topoisomerase II in simian virus 40-infected cells. 总被引:2,自引:1,他引:1
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The time course of expression of topoisomerase I, topoisomerase II, and simian virus 40 (SV40) large tumor (T) antigen was determined in whole-cell extracts of uninfected versus SV40-infected TC7 cells. After a minor increase, the level of topoisomerase I remained fairly constant throughout the time course in both uninfected and SV40-infected cells. In contrast, the level of topoisomerase II increased markedly in SV40-infected cells but not in uninfected cells following the appearance of SV40 T antigen. 相似文献
15.
The distributions of DNA cleavage sites induced by topoisomerase II in the presence or absence of specific drugs were mapped in the simian virus 40 genome. The drugs studied were 5-iminodaunorubicin, amsacrine (m-AMSA), teniposide (VM-26) and 2-methyl-9-hydroxyellipticinium; each produced a distinctive pattern of enhanced cleavage. Consistently intense cleavage, both in the presence and in the absence of drugs, occurred in the nuclear matrix-associated region. Since topoisomerase II is a major constituent of the nuclear matrix, and cleavage complexes include a covalent link between topoisomerase II and DNA, the findings suggest that topoisomerase II may function to attach DNA to the nuclear matrix. Cleavage usually occurred on both DNA strands with the expected four base-pair 5' stagger, and strong sites tended to occur within A/T runs such as have been associated with binding to the nuclear scaffold. Intense cleavage was present also in the replication termination region, but was absent from the vicinity of the replication origin. Cleavage intensities were found to change with time in a manner that depended both on the site and on the drug, suggesting that topoisomerase II can move along the DNA from a kinetically preferred site to a thermodynamically preferred site. 相似文献
16.
Initiation of simian virus 40 DNA replication in vitro: identification of RNA-primed nascent DNA chains. 总被引:4,自引:0,他引:4
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J Taljanidisz R S Decker Z S Guo M L DePamphilis N Sarkar 《Nucleic acids research》1987,15(19):7877-7888
Cell-free extracts of simian virus 40 (SV40)-infected CV-1 cells can initiate large tumor antigen dependent bidirectional replication in circular DNA molecules containing a functional SV40 origin of replication (ori). To determine whether or not DNA replication under these conditions involves RNA-primed DNA synthesis, replication was carried out in the presence of 5-mercuri-deoxycytidine triphosphate to label nascent DNA chains. Newly synthesized mercurated DNA was isolated by its affinity for thiol-agarose, and the 5'-ends of the isolated chains were radiolabeled to allow identification of RNA primers. At least 50% of the isolated chains contained 4 to 7 ribonucleotides covalently linked to their 5'-end; 80% of the oligoribonucleotides began with adenosine and 19% began with guanosine. About 60% of the nascent DNA chains annealed to the SV40 ori region, and about 80% of these chains were synthesized in the same direction as early mRNA. These results are consistent with the properties of SV40 DNA replication in vivo and support a model for initiation of SV40 DNA replication in which DNA primase initiates DNA synthesis on that strand of ori that encodes early mRNA. 相似文献
17.
Effects of VM26 (teniposide), a specific inhibitor of type II DNA topoisomerase, on SV40 DNA replication in vivo. 总被引:8,自引:2,他引:6
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Simian Virus 40 (SV40) infected cells were pulse labeled with (3H) thymidine and chased either in the absence or in the presence of the cytotoxic drug VM26 (teniposide). We investigated the structure of labeled SV40 DNA and found that VM26 had no significant effect on replicative chain elongation but strongly inhibited the conversion of late replication intermediates to mature DNA daughter molecules. The late replicative SV40 DNA intermediates which accumulate in VM26 treated cells contained essentially full length labeled DNA strands. These newly synthesized strands were not part of two catenated interlocked SV40 monomers suggesting that the block occurred prior to the final ligation reaction. Since VM26 is known to be a specific inhibitor of DNA topoisomerase II we conclude that this enzyme is dispensable for the chain elongation of replicating SV40 DNA, but that it is essential for the termination of SV40 DNA replication cycles. 相似文献
18.
DNA topoisomerase I was required for bidirectional DNA replication in an in vitro system for Simian virus 40 (SV40) DNA replication with purified proteins in which the replication fork moved at the rate of 260 nucleotides/min on average. DNA topoisomerase I purified from camptothecin-resistant human lymphoblastoid cells, which confers high resistance of cellular DNA replication to camptothecin [Andoh, T., Ishii, K., Suzuki, Y., Ikegami, Y., Kusunoki, Y., Takemoto, Y. & Okada, K. (1987) Proc. Natl Acad. Sci. USA 84, 5565-5569], was characterized using this system. The activity of stimulating bidirectional DNA replication was comparable between two topoisomerase I from parental and resistant cells, i.e. in its dose-response relationship and in its time course for DNA synthesis. Camptothecin severely inhibited the leading as well as the lagging strand synthesis in the reaction containing the wild type topoisomerase I but not the mutant type topoisomerase I. The mutant type topoisomerase I was over 125-fold as resistant to camptothecin as the wild type topoisomerase I. These results are in good agreement with those on the sensitivity of cellular DNA synthesis to camptothecin in the resistant cells. These findings suggest that topoisomerase I is involved in cellular DNA replication as a swivelase and the mutation conferring camptothecin-resistance on the enzyme does not affect its functional efficiency in this system. 相似文献
19.
Simian virus 40 (SV40) T antigen can efficiently initiate SV40 origin-dependent DNA synthesis in crude extracts of HeLa cells. Therefore, initiation of SV40 DNA synthesis can be analyzed in detail. We present evidence that antibodies which neutralize proliferating cell nuclear antigen (PCNA) inhibit but do not abolish pulse-labeling of nascent DNA. The lengths of DNA products formed after a 5-s pulse in the absence and presence of anti-PCNA serum averaged 150 and 34 nucleotides, respectively. The small DNAs formed in the presence of anti-PCNA serum underwent little or no increase in size during further incubation periods. The addition of PCNA to reaction mixtures inhibited with anti-PCNA serum largely reversed the inhibitory effect of the antiserum. The small nascent DNAs formed in the presence or absence of anti-PCNA serum products arose from the replication of lagging strands. These results suggest that a PCNA-dependent elongation reaction participates in the synthesis of lagging strands as well as leading strands. We also present evidence that in crude extracts of HeLa cells, DNA synthesis generally does not initiate within the core origin. Initiation of DNA synthesis outside of a genetically defined origin region has not been previously described in a eukaryotic replication system but appears to be a common feature of initiation events in many prokaryotic organisms. Additional results presented indicate that in the absence of nucleoside triphosphates other than ATP, the preinitiation complex remains within or close to the SV40 origin. 相似文献
20.
Essentially all of the Okazaki fragments on replicating Simian virus 40 (SV40)DNA could be grouped into one of three classes. Class I Okazaki fragments (about 20%) were separated from longer nascent DNA chains by a single phosphodiester bond interruption (nick) and were quantitatively identified by treating purified replicating DNA with Escherichia coli DNA ligase and then measuring the fraction of Okazaki fragments joined to longer nascent DNA chains. Similarly, class II Okazaki fragments (about 30%) were separated by a region of single-stranded DNA template (gap) that could be filled and sealed by T4 DNA polymerase plus E. coli DNA ligase, and class III fragments (about 50%) were separated by RNA primers that could be removed with E. coli DNA olymerase I, allowing the fragments to be joined with E. coli DNA ligase. These results were obtained with replicating SV40 DNA that had been briefly labeled with radioactive precursors in either intact cells or isolated nuclei. When isolated nuclei were further incubated in the presence of cytosol, all of the Okazaki fragments were converted into longer DNA strands as expected for intermediates in DNA synthesis. However, when washed nuclei were incubated in the abscence of cytosol, both class I and class II Okazaki fragments accumulated despite the excision of RNA primers: class III Okazaki fragments and RNA-DNA covalent linkages both disappeared at similar rates. These data demonstrate the existence of RNA primers in whole cells as well as in isolated nuclei, and identify a unique gap-filling step that is not simply an extension of the DNA chain elongation process concomitant with the excision of RNA primers. One or more factos found in cytosol, in addition to DNA polymerase alpha, are specifically involved in the gap-filling and ligation steps. The sizes of mature Okazaki fragments (class I) and Okazaki fragments whose synthesis was completed by T4 DNA polymerase were measured by gel electrophoresis and found to be broadly distributed between 40 and 290 nucleotides with an average length of 135 nucleotides. Since 80% and 90% of the Okazaments does not occur at uniformly spaced intervals along the DNA template. During the excision of RNA primers, nascent DNA chains with a single ribonucleotide covalently attached to the 5' terminus were identified as transient intermediates. These intermediates accumulated during excision of RNA primers in the presence of adenine 9-beta-D-arabinoside 5'-triphosphate, and those Okazaki fragments blocked by RNA primers (class III) were found to have originated the farthest from the 5' ends of long nascent DNA strands. Thus, RNA primers appear to be excised in two steps with the second step, removal of the final ribonucleotide, being stimulated by concomitant DNA synthesis. These and other data were used to construct a comprehensive metabolic pathway for the initiation, elongation, and maturation of Okazaki fragments at mammalian DNA replication forks. 相似文献