Mitochondrial DNA synthesis in mouse L cells temperature sensitive in nuclear DNA replication

Sheinin, Rose; Darragh, Pamela; Dubsky, Margaret

doi:10.1139/o77-077

Cited by 13 publications

(4 citation statements)

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“…At later times, semiconservative DNA synthesis is replaced by repair replication (41,42). Temperature inactivation of DNA synthesis is followed within 6 h by inhibition of de novo synthesis of chromatin histones although preformed histones are fully conserved (44). Protoplasmic growth is not blocked and continues over several days, leading eventually to at least a doubling of cell and nuclear volume.…”

Section: Discussionmentioning

confidence: 99%

Structure of interphase nuclei in relation to the cell cycle. Chromatin organization in mouse L cells temperature-sensitive for DNA replication

Setterfield¹,

Sheinin²,

Dardick³

et al. 1978

The Journal of Cell Biology

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Mutant lines of mouse L cells, TS A1S9, and TS C1, show temperature-sensitive (TS) DNA synthesis and cell division when shifted from 34 ~ to 38.5~ With TS A1S9 the decline in DNA synthesis begins after 6-8 h at 38.5~ and is most marked at about 24 h. Most cells in S, G~, or M at temperature upshift complete one mitosis and accumulate in the subsequent interphase at G~ or early S as a result of expression of a primary defect, failure of elongation of newly made small DNA fragments. Heat inactivation of TS C1 cells is more rapid; they fail to complete the interphase in progress at temperature upshift and accumulate at late S or G2. Inhibition of both cell types is reversible on return to 34~ Cell and nuclear growth continues during inhibition of replication.Expression of both TS mutations leads to a marked change in gross organization of chromatin as revealed by electron microscopy. Nuclei of wild-type cells at 34 ~ and 38.5~ and mutant cells at 34~ show a range of aggregation of condensed chromatin from small dispersed bodies to large discrete clumps, with the majority in an intermediate state. In TS cells at 38.5~ condensed chromatin bodies in the central nuclear region become disaggregated into small clumps dispersed through the nucleus. Morphometric estimation of volume of condensed chromatin indicates that this process is not due to complete decondensation of ehromatin fibrils, but rather involves dispersal of large condensed chromatin bodies into finer aggregates and loosening of fibrils within the aggregates. The dispersed condition is reversed in nuclei which resume DNA synthesis when TS cells are downshifted from 38.5 ~ to 34~ The morphological observations are consistent with the hypothesis that condensed chromatin normally undergoes an ordered cycle of transient, localized disaggregation and reaggregation associated with replication. In temperature-inactivated mutants, normal progressive disaggregation presumably occurs, but subsequent lack of chromatin replication prevents reaggregation. 246J, CELL BIOLOGY t~ The Rockefeller University Press.

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Section: Discussionmentioning

confidence: 99%

Structure of interphase nuclei in relation to the cell cycle. Chromatin organization in mouse L cells temperature-sensitive for DNA replication

Setterfield¹,

Sheinin²,

Dardick³

et al. 1978

The Journal of Cell Biology

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“…Thus, when these cells are incubated at the nonpermissive temperature, nuclear semiconservative DNA replica tion is suppressed and repair replication is activated after an interval equiva lent to that required fo r full cell cycling (414). Mitochondrial DNA replication proceeds apparently normally, but at a reduced rate which is coupled to that observed for nuclear DNA synthesis (412). De novo chromatin synthesis is interrupted once the ts defect is fu lly established, although the preformed chromatin is fully conserved (4 15; Sheinin and Lewis, unpublished observations).…”

Section: S Phasementioning

confidence: 99%

Some Aspects of Eukaryotic DNA Replication

Sheinin¹,

Humbert²,

Pearlman³

1978

Annu. Rev. Biochem.

128

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“…The ts AlS9 L cell (1) is mutant in a gene required for nuclear DNA replication (2,3) and for normal progression through the S phase of the cell cycle (4). Temperature-inactivated ts A1M9 cells synthesize "Okazald fragments" and convert them to progeny single-strand DNA of >5 x 106 daltons (2).…”

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confidence: 99%

ts A1S9 locus in mouse L cells may encode a novobiocin binding protein that is required for DNA topoisomerase II activity.

Colwill¹,

Sheinin²

1983

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

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Nuclear novobiocin binding proteins (NBPs) from a set of mouse L cells have been extensively purified by affinity chromatography on novobiocin-Sepharose columns. The NBPs, specifically eluted with 100 ,ug of novobiocin per ml, exhibited equivalent DNA topoisomerase activities (measured as ATP-dependent relaxation or catenation of #X174 replicative-form I DNA substrate) when extracted from equal numbers of wild-type (WT-4) mouse L cells growing logarithmically at 34WC or at 38.50C, from ts A1S9 cells similarly cultivated at the low, permissive temperature or from revertant ts+ AR cells in exponential growth at either temperature. The NBPs isolated from similar numbers of ts A1S9 cells grown to midlogarithmic phase and then incubated for 24 hr at 38.5C (the nonpermissive temperature) showed no topoisomerase II activity. Preliminary NaDodSO4/polyaerylamide gel electrophoretic analysis of enzymatically active material revealed that the NBPs of WT4 and ts+ AR cells grown at 34WC comprised three major polypeptides of 76,000, 74,000, and 30,000 daltons and a number of larger molecular mass components present in trace amounts. The NBP of ts A1S9 cells grown at the permissive temperature was similar, except that the 30,000-kilodalton polypeptide was not detected. Such enzymatically active NBPs from WT-4 and ts+ AR cells were unaffected by 100 1tg of novobiocin per ml, whereas the analogous preparation from ts A1S9 cells was totally inhibited. On the basis of these and other considerations, it is postulated that the ts A1S9 locus of mouse L cells encodes a temperature-sensitive polypeptide that is required for normal DNA topoisomerase I activity.The ts AlS9 L cell (1) is mutant in a gene required for nuclear DNA replication (2, 3) and for normal progression through the S phase of the cell cycle (4). Temperature-inactivated ts A1M9 cells synthesize "Okazald fragments" and convert them to progeny single-strand DNA of >5 x 106 daltons (2). In addition they support full replication of monomeric and multimeric (up to 34 X 107 daltons) polyoma DNA (5, 6). Therefore, it was concluded that the enzyme proteins of polydeoxyribonucleotide guished by their properties and by the mechanism by which they modify the topology of DNA. The DNA topoisomerase I more closely resembles the nicking-closing, w protein activity already well characterized biochemically and genetically in bacteria (12, 13). Whereas the topoisomerase I enzyme has long been recognized to function in eukaryotes (14), evidence for the topoisomerase II eukaryotic analogue for the DNA gyrase of prokaryotes was reported later (15-17) and, indeed, after the studies described herein were initiated. No evidence for abnormal topoisomerase I activity had been seen in earlier studies of temperature-inactivated ts AlS9 cells (cf. refs. 4 and 6; unpublished data). Therefore, attention was focused on DNA topoisomerase II. The first hint that this enzyme might be affected came with the demonstration that ts AlS9 cells are hypersensitive to novobiocin (9) The publication co...

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Mitochondrial DNA synthesis in mouse L cells temperature sensitive in nuclear DNA replication

Cited by 13 publications

References 0 publications

Structure of interphase nuclei in relation to the cell cycle. Chromatin organization in mouse L cells temperature-sensitive for DNA replication

Structure of interphase nuclei in relation to the cell cycle. Chromatin organization in mouse L cells temperature-sensitive for DNA replication

Some Aspects of Eukaryotic DNA Replication

ts A1S9 locus in mouse L cells may encode a novobiocin binding protein that is required for DNA topoisomerase II activity.

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