scholarly journals Initiation of DNA replication in nuclei from quiescent cells requires permeabilization of the nuclear membrane.

1994 ◽  
Vol 127 (1) ◽  
pp. 5-14 ◽  
Author(s):  
G H Leno ◽  
R Munshi
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Nuclear Membrane ◽  
Cell Cycle Control ◽  
G1 Phase ◽  
Replication Capacity ◽  
Quiescent Cells ◽  
Nuclear Membranes ◽  
Egg Extract ◽  
Xenopus Egg

We have investigated the replication capacity of intact nuclei from quiescent cells using Xenopus egg extract. Nuclei, with intact nuclear membranes, were isolated from both exponentially growing and contact-inhibited BALB/c 3T3 fibroblasts by treatment of the cells with streptolysin-O. Flow cytometry showed that > 90% of all contact-inhibited cells and approximately 50% of the exponential cells were in G0/G1-phase at the time of nuclear isolation. Intact nuclei were assayed for replication in the extract by incorporation of [alpha-32P]dATP or biotin-dUTP into nascent DNA. Most nuclei from exponential cells replicated in the egg extract, consistent with previous results showing that intact G1 nuclei from HeLa cells replicate in this system. In contrast, few nuclei from quiescent cells replicated in parallel incubations. However, when the nuclear membranes of these intact quiescent nuclei were permeabilized with lysophosphatidylcholine prior to addition to the extract, nearly all the nuclei replicated under complete cell cycle control in a subsequent incubation. The ability of LPC-treated quiescent nuclei to undergo DNA replication was reversed by resealing permeable nuclear membranes with Xenopus egg membranes prior to extract incubation demonstrating that the effect of LPC treatment is at the level of the nuclear membrane. These results indicate that nuclei from G1-phase cells lose their capacity to initiate DNA replication following density-dependent growth arrest and suggest that changes in nuclear membrane permeability may be required for the initiation of replication upon re-entry of the quiescent cell into the cell cycle.

scholarly journals Site-specific initiation of DNA replication in Xenopus egg extract requires nuclear structure.

1995 ◽  
Vol 15 (6) ◽  
pp. 2942-2954 ◽  
Author(s):  
D M Gilbert ◽  
H Miyazawa ◽  
M L DePamphilis
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Dihydrofolate Reductase ◽  
Nuclear Membrane ◽  
S Phase ◽  
G1 Phase ◽  
Site Specific ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Xenopus Egg

Previous studies have shown that Xenopus egg extract can initiate DNA replication in purified DNA molecules once the DNA is organized into a pseudonucleus. DNA replication under these conditions is independent of DNA sequence and begins at many sites distributed randomly throughout the molecules. In contrast, DNA replication in the chromosomes of cultured animal cells initiates at specific, heritable sites. Here we show that Xenopus egg extract can initiate DNA replication at specific sites in mammalian chromosomes, but only when the DNA is presented in the form of an intact nucleus. Initiation of DNA synthesis in nuclei isolated from G1-phase Chinese hamster ovary cells was distinguished from continuation of DNA synthesis at preformed replication forks in S-phase nuclei by a delay that preceded DNA synthesis, a dependence on soluble Xenopus egg factors, sensitivity to a protein kinase inhibitor, and complete labeling of nascent DNA chains. Initiation sites for DNA replication were mapped downstream of the amplified dihydrofolate reductase gene region by hybridizing newly replicated DNA to unique probes and by hybridizing Okazaki fragments to the two individual strands of unique probes. When G1-phase nuclei were prepared by methods that preserved the integrity of the nuclear membrane, Xenopus egg extract initiated replication specifically at or near the origin of bidirectional replication utilized by hamster cells (dihydrofolate reductase ori-beta). However, when nuclei were prepared by methods that altered nuclear morphology and damaged the nuclear membrane, preference for initiation at ori-beta was significantly reduced or eliminated. Furthermore, site-specific initiation was not observed with bare DNA substrates, and Xenopus eggs or egg extracts replicated prokaryotic DNA or hamster DNA that did not contain a replication origin as efficiently as hamster DNA containing ori-beta. We conclude that initiation sites for DNA replication in mammalian cells are established prior to S phase by some component of nuclear structure and that these sites can be activated by soluble factors in Xenopus eggs.

scholarly journals Reversible effects of nuclear membrane permeabilization on DNA replication: evidence for a positive licensing factor

1993 ◽  
Vol 122 (5) ◽  
pp. 985-992 ◽  
Author(s):  
D Coverley ◽  
CS Downes ◽  
P Romanowski ◽  
RA Laskey
Keyword(s):  
Dna Replication ◽  
Nuclear Membrane ◽  
Direct Evidence ◽  
Membrane Permeabilization ◽  
Membrane Repair ◽  
Xenopus Egg Extract ◽  
Nuclear Membranes ◽  
Egg Extract ◽  
Licensing Factor ◽  
Xenopus Egg

We have investigated the mechanism which prevents reinitiation of DNA replication within a single cell cycle by exploiting the observation that intact G2 HeLa nuclei do not replicate in Xenopus egg extract, unless their nuclear membranes are first permeabilized (Leno et al., 1992). We have asked if nuclear membrane permeabilization allows escape of a negative inhibitor from the replicated nucleus or entry of a positive activator as proposed in the licensing factor hypothesis of Blow and Laskey (1988). We have distinguished these possibilities by repairing permeabilized nuclear membranes after allowing soluble factors to escape. Membrane repair of G2 nuclei reverses the effects of permeabilization arguing that escape of diffusible inhibitors is not sufficient to allow replication, but that entry of diffusible activators is required. Membrane repair has no significant effect on G1 nuclei. Pre-incubation of permeable G2 nuclei in the soluble fraction of egg extract before membrane repair allows semiconservative DNA replication of these nuclei when incubated in complete extract. Addition of the same fraction after membrane repair has no effect. Our results provide direct evidence for a positively acting "licensing" activity which is excluded form the interphase nucleus by the nuclear membrane. Nuclear membrane permeabilization and repair can be used as an assay for licensing activity which could lead to its purification and subsequent analysis of its action within the nucleus.

DNA replication and cell cycle control in Xenopus egg extracts

1989 ◽  
Vol 1989 (Supplement 12) ◽  
pp. 197-212 ◽  
Author(s):  
C. J. HUTCHISON ◽  
D. BRILL ◽  
R. COX ◽  
J. GILBERT ◽  
I. KILL ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cell Cycle Control ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Cell cycle control of DNA replication

1995 ◽  
pp. 73-89 ◽  
Author(s):  
Rati Fotedar ◽  
Arun Fotedar
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cell Cycle Control

scholarly journals Cell cycle arrest of cdc mutants and specificity of the RAD9 checkpoint.

Genetics ◽  
1993 ◽  
Vol 134 (1) ◽  
pp. 63-80 ◽  
Author(s):  
T A Weinert ◽  
L H Hartwell
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Dna Replication ◽  
Cell Cycle Control ◽  
Dna Lesions ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
A Cell ◽  
G2 Phase ◽  
Rad Mutants

Abstract In eucaryotes a cell cycle control called a checkpoint ensures that mitosis occurs only after chromosomes are completely replicated and any damage is repaired. The function of this checkpoint in budding yeast requires the RAD9 gene. Here we examine the role of the RAD9 gene in the arrest of the 12 cell division cycle (cdc) mutants, temperature-sensitive lethal mutants that arrest in specific phases of the cell cycle at a restrictive temperature. We found that in four cdc mutants the cdc rad9 cells failed to arrest after a shift to the restrictive temperature, rather they continued cell division and died rapidly, whereas the cdc RAD cells arrested and remained viable. The cell cycle and genetic phenotypes of the 12 cdc RAD mutants indicate the function of the RAD9 checkpoint is phase-specific and signal-specific. First, the four cdc RAD mutants that required RAD9 each arrested in the late S/G2 phase after a shift to the restrictive temperature when DNA replication was complete or nearly complete, and second, each leaves DNA lesions when the CDC gene product is limiting for cell division. Three of the four CDC genes are known to encode DNA replication enzymes. We found that the RAD17 gene is also essential for the function of the RAD9 checkpoint because it is required for phase-specific arrest of the same four cdc mutants. We also show that both X- or UV-irradiated cells require the RAD9 and RAD17 genes for delay in the G2 phase. Together, these results indicate that the RAD9 checkpoint is apparently activated only by DNA lesions and arrests cell division only in the late S/G2 phase.

Yeast L double-stranded ribonucleic acid is synthesized during the G1 phase but not the S phase of the cell cycle

1981 ◽  
Vol 1 (8) ◽  
pp. 673-679
Author(s):  
V A Zakian ◽  
D W Wagner ◽  
W L Fangman
Keyword(s):  
Cell Cycle ◽  
Deoxyribonucleic Acid ◽  
Cell Cycle Control ◽  
S Phase ◽  
G1 Phase ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Phase Synthesis ◽  
Ribonucleic Acids ◽  
Alpha Factor

The cytoplasm of Saccharomyces cerevisiae contains two major classes of protein-encapsulated double-stranded ribonucleic acids (dsRNA's), L and M. Replication of L and M dsRNA's was examined in cells arrested in the G1 phase by either alpha-factor, a yeast mating pheromone, or the restrictive temperature for a cell cycle mutant (cdc7). [3H]uracil was added during the arrest periods to cells prelabeled with [14C]uracil, and replication was monitored by determining the ratio of 3H/14C for purified dsRNA's. Like mitochondrial deoxyribonucleic acid, both L and M dsRNA's were synthesized in the G1 arrested cells. The replication of L dsRNA was also examined during the S phase, using cells synchronized in two different ways. Cells containing the cdc7 mutation, treated sequentially with alpha-factor and then the restrictive temperature, enter a synchronous S phase when transferred to permissive temperature. When cells entered the S phase, synthesis of L dsRNA ceased, and little or no synthesis was detected throughout the S phase. Synthesis of L dsRNA was also observed in G1 phase cells isolated from asynchronous cultures by velocity centrifugation. Again, synthesis ceased when cells entered the S phase. These results indicate that L dsRNA replication is under cell cycle control. The control differs from that of mitochondrial deoxyribonucleic acid, which replicates in all phases of the cell cycle, and from that of 2-micron DNA, a multiple-copy plasmid whose replication is confined to the S phase.

Role of transcriptional and posttranscriptional regulation in expression of histone genes in Saccharomyces cerevisiae

1987 ◽  
Vol 7 (2) ◽  
pp. 614-621
Author(s):  
D E Lycan ◽  
M A Osley ◽  
L M Hereford
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Replication ◽  
Posttranscriptional Regulation ◽  
Cell Cycle Control ◽  
Dimensional Structure ◽  
Histone Genes ◽  
Transcriptional Regulatory Mechanisms ◽  
Rna Levels

We analyzed the role of posttranscriptional mechanisms in the regulation of histone gene expression in Saccharomyces cerevisiae. The rapid drop in histone RNA levels associated with the inhibition of ongoing DNA replication was postulated to be due to posttranscriptional degradation of histone transcripts. However, in analyzing the sequences required for this response, we showed that the coupling of histone RNA levels to DNA replication was due mostly, if not entirely, to transcriptional regulatory mechanisms. Furthermore, deletions which removed the negative, cell cycle control sequences from the histone promoter also uncoupled histone transcription from DNA replication. We propose that the arrest of DNA synthesis prematurely activates the regulatory pathway used in the normal cell cycle to repress transcription. Although posttranscriptional regulation did not appear to play a significant role in coupling histone RNA levels to DNA replication, it did affect the levels of histone RNA in the cell cycle. Posttranscriptional regulation could apparently restore much of the periodicity of histone RNA accumulation in cells which constitutively transcribed the histone genes. Unlike transcriptional regulation, periodic posttranscriptional regulation appears to operate on a clock which is independent of events in the mitotic DNA cycle. Posttranscriptional recognition of histone RNA must require either sequences in the 3' end of the RNA or an intact three-dimensional structure since H2A- and H2B-lacZ fusion transcripts, containing only 5' histone sequences, were insensitive to posttranscriptional controls.

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