PubMed · 5008365
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J R Curtis. 1972-01-01. Tetracycline and renal function.. https://doi.org/10.1136/bmj.1.5791.52-a
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In proliferating cells, DNA synthesis must be performed with extreme precision. We show that groups of replicons, labeled together as replicon clusters, form stable units of chromosome structure. HeLa cells were labeled with 5-bromodeoxyuridine (BrdU) at different times of S phase. At the onset of S phase, clusters of replicons were activated in each of approximately 750 replication sites. The majority of these replication "foci" were shown to be individual replicon clusters that remained together, as stable cohorts, throughout the following 15 cell cycles. In individual cells, the same replication foci were labeled with BrdU and 5-iododeoxyuridine at the beginning of different cell cycles. In DNA fibers, 95% of replicons in replicon clusters that were labeled at the beginning of one S phase were also labeled at the beginning of the next. This shows that a subset of origins are activated both reliably and efficiently in different cycles. The majority of replication forks activated at the onset of S phase terminated 45-60 min later. During this interval, secondary replicon clusters became active. However, while the activation of early replicons is synchronized at the onset of S phase, different secondary clusters were activated at different times. Nevertheless, replication foci pulse labeled during any short interval of S phase were stable for many cell cycles. We propose that the coordinated replication of related groups of replicons, that form stable replicon clusters, contributes to the efficient activation and propagation of S phase in mammalian cells.
Acentric, autonomously replicating extrachromosomal structures called double-minute chromosomes (DMs) frequently mediate oncogene amplification in human tumors. We show that DMs can be removed from the nucleus by a novel micronucleation mechanism that is initiated by budding of the nuclear membrane during S phase. DMs containing c-myc oncogenes in a colon cancer cell line localized to and replicated at the nuclear periphery. Replication inhibitors increased micronucleation; cell synchronization and bromodeoxyuridine-pulse labeling demonstrated de novo formation of buds and micronuclei during S phase. The frequencies of S-phase nuclear budding and micronucleation were increased dramatically in normal human cells by inactivating p53, suggesting that an S-phase function of p53 minimizes the probability of producing the broken chromosome fragments that induce budding and micronucleation. These data have implications for understanding the behavior of acentric DNA in interphase nuclei and for developing chemotherapeutic strategies based on this new mechanism for DM elimination.
BACKGROUND: Embryonic stem (ES) cells can contribute precursors to all adult cell lineages. Consequently, damage to ES cell genomes may cause serious developmental malfunctions. In somatic cells, cell-cycle checkpoints limit DNA damage by preventing DNA replication under conditions that may produce chromosomal aberrations. The tumor suppressor p53 is involved in such checkpoint controls and is also required to avoid a high rate of embryonic malformations. We characterized the cell-cycle and DNA-damage responses of ES cells to elucidate the mechanisms that prevent accumulation or transmission of damaged genomes during development. RESULTS: ES cells derived from wild-type mice did not undergo cell-cycle arrest in response to DNA damage or nucleotide depletion, although they synthesized abundant quantities of p53. The p53 protein in ES cells was cytoplasmic and translocated inefficiently to the nucleus upon nucleotide depletion. Expression of high levels of active p53 from an adenovirus vector could not trigger cell cycle arrest. Instead, ES cells that sustained DNA damage underwent p53-independent apoptosis. The antimetabolite-induced p53-dependent arrest response was restored in ES cells upon differentiation. CONCLUSIONS: Cell-cycle regulatory pathways in early embryos differ significantly from those in differentiated somatic cells. In undifferentiated ES cells, p53 checkpoint pathways are compromised by factors that affect the nuclear localization of p53 and by the loss of downstream factors that are necessary to induce cell-cycle arrest. A p53-independent programmed cell death pathway is effectively employed to prevent cells with damaged genomes from contributing to the developing organism. The p53-mediated checkpoint controls become important when differentiation occurs.