Abstract
The nucleus/cytoplasm (N/C) ratio controls S phase dynamics in many biological systems, most notably the abrupt remodeling of the cell cycle that occurs at the midblastula transition in early Xenopus laevis embryos. After an initial series of rapid cleavage cycles consisting only of S and M phases, a critical N/C ratio is reached, which causes a sharp increase in the length of S phase as the cell cycle is reconfigured to resemble somatic cell cycles. How the N/C ratio determines the length of S phase has been a longstanding problem in developmental biology. Using Xenopus egg extracts, we show that DNA replication at high N/C ratio is restricted by one or more limiting substances. We report here that the protein phosphatase PP2A, in conjunction with its B55α regulatory subunit, becomes limiting for replication origin firing at high N/C ratio, and this in turn leads to reduced origin activation and an increase in the time required to complete S phase. Increasing the levels of PP2A catalytic subunit or B55α experimentally restores rapid DNA synthesis at high N/C ratio. Inversely, reduction of PP2A or B55α levels sharply extends S phase even in low N/C extracts. These results identify PP2A-B55α as a link between DNA replication and N/C ratio in egg extracts and suggest a mechanism that may influence the onset of the midblastula transition in vivo.
Highlights
The duration of S phase in Xenopus is controlled by the nucleus/cytoplasm ratio through unknown mechanisms
S Phase Elongation in Xenopus Egg Extracts Containing High N/C Ratio—To investigate the mechanism for regulation of cell cycle duration by the N/C ratio, we sought to recapitulate in egg extracts the S phase lengthening that occurs at high N/C ratio in vivo
Control of DNA Replication by the N/C Ratio through Titration of phosphatase 2A (PP2A)-B55␣ Complexes—In this study, we have examined how the length of S phase is controlled by the N/C ratio in Xenopus egg extracts
Summary
The duration of S phase in Xenopus is controlled by the nucleus/cytoplasm ratio through unknown mechanisms. One longstanding model for how the N/C ratio affects S phase in Xenopus suggests that a positively acting DNA replication factor is titrated from the cytoplasm by the exponentially increasing number of nuclei during the rapid cleavage divisions. Work using egg extracts has shown that increased N/C ratio results in lengthening of replicon size, which reflects an expansion of the distance between active origins of replication This indicates that the N/C ratio influences S phase duration by limiting the number of active origins. At high N/C ratio, PP2A activity becomes limiting due to titration of its key regulatory subunit, the B55␣ protein, and this drop in PP2A activity is the critical event that allows for the extension of S phase These results provide a mechanistic basis for how the N/C ratio is sensed to regulate S phase duration during early development
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