Geminin Stabilizes Cdt1 during Meiosis in Xenopus Oocytes

Yadushyla Narasimhachar,Martine Coué

doi:10.1074/jbc.m109.008854

Yadushyla Narasimhachar, Martine Coué

Open Access

https://doi.org/10.1074/jbc.m109.008854

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Abstract

During the mitotic cell cycle, Geminin can act both as a promoter and inhibitor of initiation of DNA replication. As a promoter, Geminin stabilizes Cdt1 and facilitates its accumulation leading to the assembly of the pre-replication complex on DNA. As an inhibitor, Geminin prevents Cdt1 from loading the mini-chromosome maintenance complex onto pre-replication complexes in late S, G(2), and M phases. Here we show that during meiosis Geminin functions as a stabilizer of Cdt1 promoting its accumulation for the early division cycles of the embryo. Depletion of Geminin in Xenopus immature oocytes leads to a decrease of Cdt1 protein levels during maturation and after activation of these oocytes. Injection of exogenous recombinant Geminin into the depleted oocytes rescues Cdt1 levels demonstrating that Geminin stabilizes Cdt1 during meiosis and after fertilization. Furthermore, Geminin-depleted oocytes did not replicate their DNA after meiosis I indicating that Geminin does not act as an inhibitor of initiation of DNA replication between meiosis I and meiosis II.

Highlights

The recruitment of additional Geminin molecules to this complex on the chromatin blocks further pre-RC formation. These results indicate that the stoichiometry of Cdt1 and Geminin in this complex regulates its activity as a promoter or inhibitor of pre-RC assembly and DNA replication [23, 26]
Geminin and Cdt1 Levels Increase during Meiotic Maturation—The level of Geminin and Cdt1 in Xenopus oocytes before and during the maturation process was determined by Western blot analysis
Our results indicate that the levels of Geminin and Cdt1 were extremely low in stage VI oocytes arrested in prophase of meiosis I (Fig. 1, lane 1), but they increased significantly during the maturation process (Fig. 1, lanes 2– 8)

Summary

Introduction

Cdt1 protein levels increased during maturation suggest- absence of cycloheximide treatment, an excess of exogenous ing that injection of sense oligonucleotides did not affect their Geminin did not affect the maturation process as indicated by accumulation or the maturation process (Fig. 3A, lanes 1– 8). This finding reinforces the injection of antisense oligonucleotides suggesting that Geminin idea that Geminin does not affect the rate of Cdt1 synthesis but is not required for MPF activity and the maturation process protects Cdt1 against degradation during meiosis II.

Results

Conclusion