Control of DNA Replication by the Nucleus to Cytoplasm Ratio
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CitationMurphy, Christopher. 2012. Control of DNA Replication by the Nucleus to Cytoplasm Ratio. Doctoral dissertation, Harvard University.
AbstractXenopus embryos begin development by undergoing a series of extremely rapid cell divisions that occur without growth, gap phases, or cell cycle checkpoints. This cell cycle program, which allows the fertilized egg to rapidly subdivide its contents into many separate cells, is made possible by the extraordinary ability of these embryos to replicate DNA quickly. After a dozen such divisions, the time required to complete S phase and complete the cell cycle increases sharply amidst other embryonic changes during the midblastula transition (MBT). Successful completion of the MBT is essential for viability, but the mechanism responsible for actuating these changes remains unknown. Previous work has shown that the onset of the MBT is dependent upon the embryo reaching a critical nucleus to cytoplasm (N/C) ratio, but it is unclear how this controls cell cycle lengthening. Here, we use Xenopus egg extracts to investigate the mechanism responsible for S phase lengthening at the MBT. As in embryos, high N/C extracts exhibit lengthened S phases, and this is due to both reduced utilization of origins of replication and reduced replication fork progression. Although recent work has suggested that developmental activation of the ATR/Chk1 pathway may provide the stimulus for cell cycle remodeling at the MBT, we find that this pathway is not activated more efficiently at high N/C ratio. Rather, the Chk1 phosphorylation observed at high N/C is simply the aggregated, basal checkpoint activity associated with normal replication in a large number of nuclei. Instead, we provide evidence that the reduced replication rates at high N/C ratio are the result of the depletion of maternal factors by the increased number of nuclei, and these factors are involved in both the initiation of replication and replication fork progression. We provide evidence that protein phosphatase 2A (PP2A) activity is the limiting factor for origin firing in high N/C extracts. Likewise, partial depletion of PP2A is sufficient to prevent the high levels of origin firing observed in low N/C extracts. These results suggest a mechanism by which PP2A levels control the rate of origin firing in Xenopus egg extracts and in Xenopus embryos at the MBT.
Citable link to this pagehttp://nrs.harvard.edu/urn-3:HUL.InstRepos:10288629
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