Mutations in the Drosophila gene encoding the serine-threonine protein kinase Greatwall have previously been shown to disrupt mitotic progression. To investigate Greatwall's mitotic function, we examined its behavior in Xenopus egg extracts. Greatwall is activated during mitosis by phosphorylation; in vitro evidence indicates that maturation promoting factor (MPF) is an upstream kinase. Conversely, depletion of Greatwall from mitotic extracts rapidly lowers MPF activity due to the accumulation of inhibitory phosphorylations on Cdc2 kinase. Greatwall depletion similarly prevents cycling extracts from entering M phase. The effects of Greatwall depletion can be rescued by the addition of either wild-type (wt) Greatwall or a noninhibitable form of Cdc2 kinase. These results demonstrate that Greatwall participates in an autoregulatory loop that generates and maintains sufficiently high MPF activity levels to support mitosis.

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We have previously shown that Greatwall kinase (Gwl) is required for M phase entry and maintenance in Xenopus egg extracts. Here, we demonstrate that Gwl plays a crucial role in a novel biochemical pathway that inactivates, specifically during M phase, "antimitotic" phosphatases directed against phosphorylations catalyzed by cyclin-dependent kinases (CDKs). A major component of this phosphatase activity is heterotrimeric PP2A containing the B55delta regulatory subunit. Gwl is activated during M phase by Cdk1/cyclin B (MPF), but once activated, Gwl promotes PP2A/B55delta inhibition with no further requirement for MPF. In the absence of Gwl, PP2A/B55delta remains active even when MPF levels are high. The removal of PP2A/B55delta corrects the inability of Gwl-depleted extracts to enter M phase. These findings support the hypothesis that M phase requires not only high levels of MPF function, but also the suppression, through a Gwl-dependent mechanism, of phosphatase(s) that would otherwise remove MPF-driven phosphorylations.

Summary Human CST (CTC1-STN1-TEN1) is an RPA-like complex that is needed for efficient replication through the telomere duplex and genome-wide replication restart after fork stalling. Here we show that STN1/CST has a second function in telomere replication during G-overhang maturation. Analysis of overhang structure after STN1 depletion revealed normal kinetics for telomerase-mediated extension in S-phase but a delay in subsequent overhang shortening. This delay resulted from a defect in C-strand fill-in. Short telomeres exhibited the fill-in defect but normal telomere duplex replication, indicating that STN1/CST functions independently in these processes. Our work also indicates that the requirement for STN1/CST in telomere duplex replication correlates with increasing telomere length and replication stress. Our results provide the first direct evidence that STN1/CST participates in C-strand fill-in. They also demonstrate that STN1/CST participates in two mechanistically separate steps during telomere replication and identify CST as a novel replication factor that solves diverse replication-associated problems.