Preliminary characterization of the temperature-sensitive defect in DNA replication in a mutant mouse L cell

Sheinin, Rose

doi:10.1016/0092-8674(76)90254-3

Cited by 64 publications

(42 citation statements)

References 19 publications

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“…sumption of the coexistence of two cycles: the nuclear phase has been suggested not only in the vitamin B12 growth in size and DNA rep1ication are folate deficiency (30,40), drug treatment (1,2,43), in mutants (42), and in acquired abnormality in syntheses of nated. .…”

Section: Discussionmentioning

confidence: 99%

DNA flow cytometry of control Euglena and cell cycle blockade of vitamin B12‐starved cells

Lefort‐Tran¹,

Bré²,

Pouphile³

et al. 1987

Cytometry

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Vitamin B E starvation inIn severe anaemia that is due to B12 avitaminosis, the block of mitosis and the maturation of the erythrocyte line is followed by a diminution of red blood cells, while large cells with abnormal nuclei ~fmegaloblasts") appear in the blood and the bone marrow. These giant cells have a high RNA/DNA ratio (7,26). Defective DNA synthesis in the megaloblastic state has been reviewed (30,40). In vitamin B12-deprived Euglena cells, divisions slow down and then stop, while RNA and protein content, number of chloroplasts, and mitochondria continue to increase (14). For the study of biological and biochemical disorders induced by vitamin B12 deficiency, Euglena cells, whose alterations resemble those observed in the megaloblasts, are a very suitable model for observing where "DNA replication cycle" is decoupled from the "growth cycle" (36), as they are easy to cultivate and even to synchronize. In the recovering cells, after addition of vitamin B12, the DNA replication is completed, followed by cell divisions (9). Two relatively synchronous divisions occur immediately without a new G1 phase (15,32,41).In the model of the metabolic cell cycle block, biochemical analyses have given a mean DNA content of 1.8 or twice that of G1 cells (5,161, with no information on cell heterogeneity. In contrast, microcytofluorometry or flow cytometric analyses can provide data on DNA content of individual cells. These two types of analysis complement each other and can lead to a better understanding of the cell cycle arrest, in light of independent results concerning the dispersed structure of chromatin in starved giant cells (4,6,12), the modification of histones (101, and the nucleosomal organization in control and vitamin B12-starved chromatin (241, all of which could affect DNA stainability.The fluorochrome Hoechst 33258 for DNA labeling was chosen in present studies in order to avoid, as far as possible, nonstoichiometric DNA stainability owing to differences in the conformation of the chromatin (23).Initial studies that used microfluorometry contributed to a better knowledge of the material because individual cells could be observed, and so, for example, mitotic cells could be distinguished from cells in GB. Later, flow cytofluorometric measurements allowed analysis of large samples, and thus statistically more reliable results could be obtained. The data indicated that starved Euglena cells were distributed in the S and G2 phases. No cells were found either in the G1 phase or in the M phase. The unbalanced growth of vitamin B12-deficient cells was characterized by a block in the DNA replication and another block at the transition from G2 to M.The significance of cell arrest is discussed in relation to DNA replication. MATERIALS AM) METHODSControl Cells Euglena gracilis strain Z (Cambridge culture collection No. 1224-5D) were cultivated, either on a mineral

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Section: Discussionmentioning

confidence: 99%

DNA flow cytometry of control Euglena and cell cycle blockade of vitamin B12‐starved cells

Lefort‐Tran¹,

Bré²,

Pouphile³

et al. 1987

Cytometry

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“…Previous reports have described the separation of this replication system into multiple fractions (9), the existence of a pre-elongation step that most likely includes pre-elongation-complex formation (26), and the role of pol a and primase in determining host specificity for viral replication (8). Various replication mutants, including those with conditional lesions in pol a, have been isolated from a variety of rodent cell lines (27)(28)(29)(30). Thus, both biochemical and genetic approaches using the in vitro Py replication system should contribute further to the understanding of DNA replication in animal cells.…”

Section: Methodsmentioning

confidence: 99%

Species-specific in vitro synthesis of DNA containing the polyoma virus origin of replication.

Murakami¹,

Eki²,

Yamada³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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In vitro replication of DNA containing the polyoma (Py) virus orign of replication has been carried out with cell-free extracts prepared from mouse FM3A cells. The in vitro system required the Py virus-encoded large tumor (T) antigen, DNA containing the Py virus origin of replication, ATP, and an ATP-regenerating system. The replication reaction was inhibited by aphidicolin, suggesting the involvement of DNA polymerase a in this system. Simian virus 40 (SV40) T antigen could not substitute for the Py T antigen. Cell extracts prepared from HeLa cells, a source that replicates SV40 DNA in the presence of SV40 T antigen, replicated Py DNA poorly. Cell-free extracts of monkey and human cells that replicate SV40 DNA have been described (4-7). The in vitro replication of SV40 DNA requires, in addition to suitably prepared cell extracts, circular DNA containing the viral replication origin (ori) and purified SV40 large tumor (T) antigen. Since SV40 T antigen and DNA containing the SV40 replication origin are the only viral components required, this system should be useful for identifying and characterizing the eukaryotic proteins involved in DNA replication. In previous reports (7-9), it was shown that DNA polymerase a (pol a) and DNA primase are essential for the replication of SV40 DNA in vitro and that the source of these enzymes is important in determining whether SV40 DNA replication will occur. In contrast to extracts of human cells, mouse cell extracts supplemented with SV40 T antigen did not support replication of DNA containing the SV40 origin unless supplemented with the pol a-primase complex from HeLa cells.In this report we describe the establishment of a system that replicates Py DNA in vitro; it requires mouse cell extract, the Py replication origin, and Py T antigen. In addition, we have confirmed and further defined the important role played by pol a-primase in determining the species specificity of papovavirus DNA replication. MATERIALS AND METHODSPurification of Py T Antigen. Py T antigen was purified from CV-1 cells infected with the helper-dependent recombinant adenovirus vector Ad-SVR587 (10) as follows. Cells were infected with wild-type adenovirus [2-5 plaque-forming units (pfu) per cell] and Ad-SVR587 recombinant virus (a gift from S. Mansour, T. Grodzicker, and R. Tjian) (5-10 pfu per cell). After incubation for 36 hr at 370C, cells were scraped from thirty 150-mm plates into -3 ml of cold Dulbecco's phosphate-buffered saline (PBS: Ca2+-and Mg2+-free), washed twice with cold PBS, suspended in 10 volumes of pH 9.0 buffer [20 mM Tris HCl, pH 9.0/0.2 M NaCl/1 mM EDTA/1 mM dithiothreitol/10% (vol/vol) glycerol/1% (vol/vol) Nonidet P-40 (NP-40)/1 mM phenylmethylsulfonyl fluoride (PMSF)], and lysed on ice for 10: min. The lysate was centrifuged for 10 min at 2000 rpm in a Sorvall H-6000A rotor, and the supernatant was then centrifuged for 10 min at 20,000 rpm in a Sorvall SS34 rotor. The supernatant was mixed with 0.5 volume of pH 6.8 buffer (0.1 M Tris'HCl, pH 6.8/1 mM EDTA/1 mM dithiothre...

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“…No temperature-sensitive mammalian topoisomerase II mutants are yet available; but in the mouse mutant line tsA159, in which a temperature-sensitive mutation at another locus inhibits topoisomerase II activity (9), cells are arrested at the nonpermissive temperature after a few hours of progression into S phase (10). Mammalian topoisomerase II function has also been studied with the aid of enzyme inhibitors, such as etoposide or its congener teniposide, for lack of temperature-sensitive mutants.…”

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confidence: 99%

Inhibitors of DNA topoisomerase II prevent chromatid separation in mammalian cells but do not prevent exit from mitosis.

Downes

Mullinger

Johnson

1991

Proc. Natl. Acad. Sci. U.S.A.

159

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DNA topoisomerase II (EC 5.99.1.3) is necessary for chromosome condensation and disjunction in yeast but not for other functions. In mammalian cells, it has been reported to be necessary for progression toward mitosis but not for transit through mitosis. We have found, on the contrary, that specific inhibition of topoisomerase II (but not of topoisomerase I) interferes with mammalian mitotic progression. Metaphase is prolonged, and anaphase separation of chromatids is completely inhibited, in cells given high concentrations of topoisomerase II inhibitors; nevertheless these cells attempt cleavage, sometimes generating nucleate and anucleate daughters. Lower concentrations of inhibitors interfere with anaphase and produce abnormalities of segregation. DNA topoisomerase II activity is therefore necessary for mammalian chromatid separation, but it is not tightly coupled to the control of other mitotic events.In eukaryotic cells DNA topoisomerase II (EC 5.99.1.3) is a major component of the mitotic chromosome core (1), with sites of action adjacent to the attachment points of supercoiled DNA loops (2). This structural role is conserved in all known cases.In budding and in fission yeast, temperature-sensitive top2 mutants show no defect in normal interphase cell functions, including cell cycle progression, at the restrictive temperature; but they are arrested in mitosis (3, 4). In top2 mutants of the fission yeast Schizosaccharomyces pombe, chromosome condensation is partly achieved at the restrictive temperature, but it produces long entangled prophase-like structures, which become fully condensed when the topoisomerase II activity is restored. If topoisomerase II is inactivated after chromosome condensation, the cells remain blocked at metaphase, unable to achieve chromatid separation (4). After release from such arrest, yeast cells show chromosome nondisjunction (5); meiotic nondisjunction can be similarly produced (6). This requirement for topoisomerase II in mitotic condensation and in separation of sister chromatids may be due to the need to separate sister strands of DNA, which inevitably become concatenated during replication but are not decatenated until later (7). Other cellular DNA topoisomerases appear to be adequate for other necessary manipulations of DNA topology but cannot perform this decatenation activity; in top2 mutants, concatenated plasmids accumulate at the restrictive temperature (3,8).Though the topoisomerase II function is necessary for chromatid segregation in fission yeast, other aspects of yeast mitosis continue without it. Temperature-sensitive top2 cells at the restrictive temperature divide into two portions, joined by chromatid fibers that prevent total cytokinesis; in top2 cdcll double mutants, in which septum formation is also blocked, cells reform single active nuclei after an abortive mitosis and enter a new round of replication (8).In mammalian cells the role of topoisomerase II in cycle progression has been reported to be rather different from its role in yeast. No tempe...

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Preliminary characterization of the temperature-sensitive defect in DNA replication in a mutant mouse L cell

Cited by 64 publications

References 19 publications

DNA flow cytometry of control Euglena and cell cycle blockade of vitamin B12‐starved cells

DNA flow cytometry of control Euglena and cell cycle blockade of vitamin B12‐starved cells

Species-specific in vitro synthesis of DNA containing the polyoma virus origin of replication.

Inhibitors of DNA topoisomerase II prevent chromatid separation in mammalian cells but do not prevent exit from mitosis.

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