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1.
X-irradiation of mammalian cells with moderate doses (100-1000 rads) inhibits the initiation of DNA replicons. This inhibition is observed as depressed amounts of radioactivity at low molecular weights when the DNA from the cells is analysed by velocity sedimentation in alkaline sucrose gradients at 30 min after irradiation. There is no detectable effect on chain elongation and joining of those molecules that do initiate replication; this is indicated by the presence of the same amounts of radioactivity in nascent DNA molecules of high molecular weights from control and irradiated cells. The labeling of DNA molecules that initiated replication before irradiation continues unhindered for more than 60 min after irradiation, which is observed as peaks of radioactivity at high S values in alkaline sucrose gradients from irradiated cells. These data indicate that DNA replication in mammalian cells proceeds by continuous joining of nascent molecules that initiate almost simultaneously at origins at various distances from one another. Some of the interorigin distances are much greater than others, implying that large replicons make up a significant component of mammalian DNA. 相似文献
2.
G D Pearson 《Journal of virology》1975,16(1):17-26
Replicating chromosomes, called intermediate DNA, have been extracted from the adenovirus replication complex. Compared to mature molecules, intermediate DNA had a greater buoyant density in CsCl gradients and ethidium bromide-cesium chloride gradients. Digestion of intermediate DNA with S1 endonuclease, but not with RNase, abolished the difference in densities. These properties suggest that replicating molecules contain extensive regions of parental single strands. Although intermediate DNA sedimented faster than marker viral DNA in neutral sucrose gradients, single strands longer than unit length could not be detected after alkaline denaturation. Integral size classes of nascent chains in intermediate DNA suggest a relationship between units of replication and the nucleoprotein structure of the virus chromosome. Adenovirus DNA was replicated at a rate of 0.7 x 10-6 daltons/min. Although newly synthesized molecules had the same sedimentation coefficient and buoyant density as mature chromosomes, they still contained single-strand interruptions. Complete joining of daughter strands required an additional 15 to 20 min. 相似文献
3.
4.
Evidence for contamination of origin DNA isolated after an in vivo treatment of mammalian cells with 4,5',8-trimethylpsoralen 总被引:1,自引:0,他引:1
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. 相似文献
5.
《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1986,867(4):187-194
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 μM 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. 相似文献
6.
Single-cell and DNA fiber autoradiography, cytophotometry and velocity sedimentation in alkaline sucrose gradients were used to analyse DNA replication and nascent replicon maturation in 5-fluorodeoxyuridine (FUdR)-synchronized cells of Pisum sativum. The replicon size was not significantly changed by the protracted FUdR treatment. When the synchronized cells were released from the inhibitor, labeled with [3H]TdR for 30 min, and chased in medium containing 1 × 10−6 M or lower concentrations of cold thymidine, DNA replication stopped after approx. 25% of the genome had replicated, and the nascent strands failed to grow above 9–12 × 106 D single-stranded (ss) DNA. When the cells were chased in medium with 1 × 10−5 M cold thymidine, the DNA content of the labeled cells steadily increased with time and the size of the nascent molecules grew continuously until replicon size was achieved; then they were accumulated at replicon size until the cells arrived in late S or G2. When the FUdR-synchronized cells were chased in medium containing 1 × 10−4 M cold thymidine, the size of the nascent strands increased continuously with time, indicating that some neighbouring nascent replicons were joined as soon as they completed their replication. These observations led us to postulate that in FUdR-synchronized cells the rates of chain elongation, cell progression through the S phase and nascent replicon maturation are controlled by thymidine availability. 相似文献
7.
8.
We have used a thymidine auxotroph of the simple eukaryote, Dictyostelium discoideum and alkaline sucrose gradients of isolated nuclei to study alterations in DNA synthesis following irradiation of replicating haploid cells with 254 nm UV light. Three responses were characterized using pulse-chase protocols: (1) Lags in DNA synthesis as measured by the amount of label incorporated were 4, 9, and 20 h after 10, 50, and 200 J/m2. (2) The DNA synthesized during a 15-min pulse immediately after irradiation was of lower single strand molecular weight: 7, 3.5, and 3 x 10(6) dalton after 0, 50, and 200 J/m2. (3) The time required for maturation of the nascent DNA to full-sized single strands of about 2 x 10(8) dalton was 45-50 min for unirradiated cells, 3 h after 10 J/m2, and 20 h after 200 J/m2. The DNA of the irradiated cells did not mature uniformly during these delays; instead, a period of no increase in size was followed by a rapid, nearly control rate of maturation. We conclude: (a) at least some UV lesions block elongation of replicons; (b) the elongation of the replicons and their subsequent joining to yield mature high molecular weight DNA occurs after most of the lesions are repaired; (c) the timing of the different aspects of recovery suggest that initiation of replication is also inhibited. 相似文献
9.
The size distribution of newly made DNA and the dynamics of size maturation of progeny DNA molecules were studied in the synchronous S and G2 phases of Physarum polycephalum. Pulse labeling of DNA and analysis of the products on alkaline sucrose gradients showed that synthesis of primary replication units (which will also be referred to as “Okazaki” fragments) occurred throughout the S period. Pulse and pulse-chase experiments revealed a distinct pattern of size maturation. An apparently linear increase in molecular weight of progeny DNA molecules during the first hour of the S phase occurred at a rate of approximately 4–5 × 105 daltons per min at 26°C, corresponding to the joining of 6–8 Okazaki fragments. The resulting 35–45S (1.1–2.2 × 107 daltons) DNA molecules may correspond to the Physarum “replicon.” The further size increases of the newly made DNA appear to occur in steps, possibly reflecting a clustering of isochronous replicons along the chromatide. These observations are discussed with regard to mechanisms of DNA replication and size maturation. 相似文献
10.
Replicon sizes in mammalian cells as estimated by an x-ray plus bromodeoxyuridine photolysis method.
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A new method is described for estimating replicon sizes in mammalian cells. Cultures were pulse labeled with [3H]thymidine ([3H]TdR) and bromodeoxyuridine (BrdUrd) for up to 1 h. The lengths of the resulting labeled regions of DNA, Lobs, were estimated by a technique wherein the change in molecular weight of nascent DNA strands, induced by 313 nm light, is measured by velocity sedimentation in alkaline sucrose gradients. If cells are exposed to 1,000 rads of X-rays immediately before pulse labeling, initiation of replicon operation is blocked, although chain elongation proceeds almost normally. Under these conditions Lobs continues to increase only until operating replicons have completed their replication. This value for Lobs then remains constant as long as the block to initiation remains and represents an estimate for the average size of replicons operating in the cells before X-irradiation. For human diploid fibroblasts and human HeLa cells this estimated average size is approximately 17 micron, whereas for Chinese hamster ovary cells, the average replicon size is about 42 micron. 相似文献
11.
The histone deacetylase inhibitor trichostatin A alters the pattern of DNA replication origin activity in human cells 总被引:6,自引:1,他引:5
Eukaryotic chromatin structure limits the initiation of DNA replication spatially to chromosomal origin zones and temporally to the ordered firing of origins during S phase. Here, we show that the level of histone H4 acetylation correlates with the frequency of replication initiation as measured by the abundance of short nascent DNA strands within the human c-myc and lamin B2 origins, but less well with the frequency of initiation across the β-globin locus. Treatment of HeLa cells with trichostatin A (TSA) reversibly increased the acetylation level of histone H4 globally and at these initiation sites. At all three origins, TSA treatment transiently promoted a more dispersive pattern of initiations, decreasing the abundance of nascent DNA at previously preferred initiation sites while increasing the nascent strand abundance at lower frequency genomic initiation sites. When cells arrested in late G1 were released into TSA, they completed S phase more rapidly than untreated cells, possibly due to the earlier initiation from late-firing origins, as exemplified by the β-globin origin. Thus, TSA may modulate replication origin activity through its effects on chromatin structure, by changing the selection of initiation sites, and by advancing the time at which DNA synthesis can begin at some initiation sites. 相似文献
12.
Arnold Fridland 《Biochemical and biophysical research communications》1977,74(1):72-78
Cytosine arabinoside inhibited DNA synthesis in human lymphoblasts by inhibiting the initiation of DNA replication units. This effect was observed by a decrease in the incorporation of (3H) thymidine into low molecular weight DNA analyzed by velocity sedimentation in alkaline sucrose gradients. In contrast, there was no detectable effect on chain elongation and joining of those molecules that initiated replication before addition of the drug. These data indicate that cytosine arabinoside acts preferentially at the level of initiation of DNA replication rather than chain elongation. 相似文献
13.
Separation of the Herpesvirus Deoxyribonucleic Acid Duplex into Unique Fragments and Intact Strand on Sedimentation in Alkaline Gradients 总被引:15,自引:12,他引:3
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Deoxyribonucleic acid (DNA) extracted from herpes simplex virions forms multiple partially overlapping bands upon denaturation and centrifugation in alkaline sucrose density gradients. The most rapidly sedimenting DNA corresponds to an intact strand 48 x 10(6) daltons in molecular weight. In this study, we analyzed the DNA fragments generated in alkaline sucrose gradients with respect to size and uniqueness of base sequences. The distribution of sedimentation constants of the various fragments obtained in numerous gradients showed that the fragments smaller than the whole strand fall into six distinct classes ranging in molecular weight from 10 x 10(6) to 39 x 10(6) daltons. Four types of DNA strands can be reconstructed from the whole strand and six fragments on the basis of their molecular weights. DNA from each of the bands self-hybridizes to a lower extent than unfractionated viral DNA, indicating that each of the bands preferentially contains sequences from one unique strand. The data permit reconstruction of four possible types of DNA duplexes differing in the positions of the strand interruptions. Analysis of viral DNA extracted from nuclei of cells labeled with (3)H-thymidine for intervals from 3 to 120 min showed that nascent DNA is invariably attached to small fragments and that the fragments become elongated only upon prolonged incubation of cells. The experiments suggest that viral DNA replication begins at numerous initiation sites along each strand and that the elongation beyond the size of the replication unit involves repair or ligation, or both. Since newly made DNA yields more fragments than viral DNA extracted from mature virions, it is suggested that the fragmentation of mature DNA on denaturation with alkali arises from incomplete processing of specific initiation sites. Comparison of viral DNA extracted from nuclei with that extracted from mature cytoplasmic virions in cells labeled for 120 min indicates that packaged DNA is not randomly selected from among the nuclear DNA population but rather represents DNA molecules which in alkaline gradients yield a minimal number of fragments. 相似文献
14.
The distribution pattern of 5-bromodeoxyuridine-labelled DNA from salivary glands of Rhynchosciara angelae upon caesium chloride gradient centrifugation was studied with DNA of different molecular weights. This pattern suggests a very low rate of DNA chain growth in polytene chromosomes. The rate of DNA chain growth was found to be 0.025 μm/min at 24 °C. The result was obtained through the development of a mathematical expression which took into account the distribution of the 5-bromodeoxyuridine-labelled DNA in CsCl gradients.DNA pulse-labelled for a short time sediments more slowly in alkaline sucrose gradients than DNA which has been labelled during a prolonged incubation. However, in neutral sucrose gradients the pattern of banding is the same for both DNAs. This indicates a discontinuity in the newly synthesized DNA strand, but not in the template strand. The transition of slow sedimenting to fast sedimenting DNA observed in alkaline sucrose gradients, occurs very slowly, as would be expected for a slow rate of DNA chain growth.The data obtained provided a means of comparing the number of replication points with the rate of DNA chain elongation and the length of S phase in Rhynchosciara. 相似文献
15.
Mapping initiation sites of DNA replication in vivo using polymerase chain reaction amplification of nascent strand segments. 总被引:14,自引:2,他引:12
We describe a sensitive method for mapping replication initiation sites near regions of sequenced genomic DNA in vivo. It is based on selective amplification of sets of segments in purified nascent DNA strands and subsequent determination of the lengths of these strands required to include each member of the set. We demonstrate the ability of this method to accurately map a well-defined origin, that of replicating SV40 DNA. Pulse-labeled DNA from infected CV-1 cells was size-fractionated on an alkaline sucrose gradient and newly-synthesized strands purified by immunoprecipitation using anti-BrdU antibodies. Three pairs of synthetic oligonucleotide primers were used to amplify three SV40 segments, using the polymerase chain reaction (PCR), at known distances from the origin. Lengths of the nascent DNA strands that allow amplification were determined by hybridization to probes homologous to the amplified segments and used to calculate position of the origin. Experiments with a mix of SV40 and human HeLa cell DNA demonstrate the applicability of the method to mapping origins present at the level of single-copy genomic sequences in mammalian cells. 相似文献
16.
The in vivo replication origin of the yeast 2 microns plasmid 总被引:102,自引:0,他引:102
We have used two-dimensional neutral/alkaline agarose gel electrophoresis to separate the nascent strands of replicating yeast 2 micron plasmid DNA molecules according to extent of replication, away from nonreplicating molecules and parental strands. Analysis of the lengths of nascent strands by sequential hybridization with short probes shows that replication proceeds bidirectionally from a single origin at map position 3700 +/- 100, coincident with the genetically mapped ARS element. The two recombinational isomers of 2 microns plasmid (forms A and B) replicate with equal efficiency. These results suggest that ARS elements may prove to be replication origins for chromosomal DNA. 相似文献
17.
In the higher plant Pisum sativum maturation of nascent DNA is blocked by cycloheximide, but only after 4-8 replicons are joined. 总被引:1,自引:1,他引:0
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Velocity sedimentation in alkaline sucrose gradients, single cell autoradiography and cytophotometry were used to determine if protein synthesis is required for the maturation of nascent replicons to chromosomal-sized molecules in cultured pea-root cells. The results obtained showed that cycloheximide at 5 and 10 microgram/ml, added either before or during labeling with tritiated thymidine, blocked maturation of nascent DNA at an intermediate size of 72-140 X 10(6) daltons single-stranded DNA. To reach this size, nascent replicons - which are 18 X 10(6) daltons single-stranded DNA each - were replicated and groups of 4-8 replicons were joined even though protein synthesis was reduced to 15% of the control. Further maturation of the nascent molecules to chromosomal size, however, was prevented and this resulted in the accumulation of nascent molecules in the 72-140 X 10(6) daltons range. The experiments also showed that the joining of nascent replicons is not an absolute function of late S or G2 phase of the cell cycle, since cells treated with cycloheximide and blocked in mid-S phase had nascent DNA of a size corresponding to 4-8 joined replicons. Finally, the results support the hypothesis that at least one step in the process of nascent DNA maturation may require replication, during late-S phase, of DNA segments that are interspersed within replicon-clusters that replicate early in the S phase. 相似文献
18.
Single-stranded replication intermediates of ribosomal DNA replicons of pea. 总被引:4,自引:2,他引:2
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Replication of ribosomal DNA replicons in cells of Pisum sativum (cv. Alaska) occurs bidirectionally by displacement loops. Replication is initiated on opposite parental strands and nascent chains are elongated moving 5'----3' along each parental template. Replicative intermediates were analyzed by 2-dimensional agarose gel electrophoresis under neutral--neutral and neutral--alkaline conditions. Southern blots of ribosomal DNA fragments separated in the second dimension under neutral conditions show slowly migrating replicative fragments that hybridize with specific probes in a manner consistent with bidirectional replication. The replicative fragments are present in root meristems with cells in S phase; they are absent or few in number in meristems with cells in G2 phase. The following observations indicate that the replicative fragments are single stranded. The apparent length of the replicative fragments is not the same when separated under neutral and alkaline conditions. They contain rDNA without breaks and they do not exhibit the smaller nascent chains expected from replication bubbles and forks. They are not cleaved by restriction enzymes that require duplex DNA as substrate and they are digestible by S1 nuclease. 相似文献
19.
Synchronized Chinese hamster ovary (CHO) cells treated with (+/-)7 beta,8 alpha- dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-dihydrobenzo[a]pyrene (BP diol epoxide I) were used to test the 'block-gap' model of replicative bypass repair in mammalian cells. One feature of the model is that carcinogenic or mutagenic DNA adducts act as blocks to the DNA replication fork on the leading strand. Using synchronized CHO cells, the rate of S phase progression by BrdUrd labeling of newly replicated DNA was measured. The rate of S phase progression was reduced by 22% and 42%, when the cells were treated at the G1/S boundary with 0.33 and 0.66 microM BP diol epoxide I, respectively. Using the pH step alkaline elution assay, it was found that the reduced rate of S phase progression was due to a delay in the appearance of multiple replicon size nascent DNA. This observation was consistent with the frequency of BP-DNA adducts present in the leading strand. A second feature of the 'block-gap' model is that the adduct-induced blockage on the lagging strand will produce gaps. It was determined by the use of high-resolution agarose gel electrophoresis, that the ligation of Okazaki size replication intermediates was blocked in a dose-dependent manner in BP diol epoxide I treated, synchronized CHO cells. These data are consistent with a block to the leading strand of DNA replication at DNA-carcinogen adducts. An inhibition of the ligation of Okazaki size fragments by BP diol epoxide I implies a block to replication of the DNA lagging strand leading to gap formation. The data presented here are, therefore, supportive of the 'block-gap' model of replicative bypass repair in carcinogen damaged mammalian cells. 相似文献
20.
The kinetics of DNA replication were analyzed in the second S phase following UV irradiation of Chinese hamster ovary cells synchronized at the beginning of S phase. The cells were synchronized by treating cells selected in mitosis with hydroxyurea for 9 h. Following UV irradiation, the cells were allowed to progress until the next mitosis; at which time they were resynchronized at the beginning of the second S phase by the same procedure. The kinetics of DNA replication were determined by measuring the proportion of DNA which achieved hybrid buoyant density on CsCl density gradients as a function of the time of incubation in the presence of 5-bromodeoxyuridine.The results of these experiments showed that even though the rate of DNA replication is substantially depressed during the first S phase following UV irradiation with a fluence of 5 J/m2, the rate has recovered to the extent that it is indistinguishable from the unirradiated control by the time the cells have entered their second S phase. It was concluded from these observations that the lesions in DNA which caused the rate of DNA replication to be initially depressed during the first S phase have been either removed or modified such that they no longer are able to cause a reduction in the rate of DNA replication in the second S phase following UV irradiation. 相似文献