The spindle checkpoint delays chromosome segregation in response to misaligned sister chromatids during mitosis, thus ensuring the fidelity of chromosome inheritance. Through binding to Cdc20, the Mad2 spindle checkpoint protein inhibits the target of this checkpoint, the ubiquitin protein ligase APC/C(Cdc20). We now show that without cofactor binding or covalent modification Mad2 adopts two distinct folded conformations at equilibrium (termed N1-Mad2 and N2-Mad2). The structure of N2-Mad2 has been determined by NMR spectroscopy. N2-Mad2 is much more potent in APC/C inhibition. Overexpression of a Mad2 mutant that specifically sequesters N2-Mad2 partially blocks checkpoint signaling in living cells. The two Mad2 conformers interconvert slowly in vitro, but interconversion is accelerated by a fragment of Mad1, an upstream regulator of Mad2. Our results suggest that the unusual two-state behavior of Mad2 is critical for spindle checkpoint signaling.

Meiotic crossover (CO) formation between homologous chromosomes (homologues) entails DNA double strand break (DSB) formation, homology search using DSB ends, and synaptonemal complex (SC) formation coupled with DSB repair. Meiotic progression must be prevented until DSB repair and homologue alignment are completed to avoid forming aneuploid gametes. Here we show that mouse HORMAD1 ensures that sufficient numbers of processed DSBs are available for successful homology search. HORMAD1 is needed for normal SC formation and for the efficient recruitment of ATR checkpoint kinase activity to unsynapsed chromatin. The latter phenomenon was proposed to be important in meiotic prophase checkpoints in both sexes.

In yeast, resolution of chiasmata in meiosis I requires proteolytic cleavage along chromosome arms of cohesin's Rec8 subunit by separase. Since activation of separase by the anaphase-promoting complex (APC/C) is supposedly not required for meiosis I in Xenopus oocytes, it has been suggested that animal cells might resolve chiasmata by a separase-independent mechanism related to the so-called "prophase pathway" that removes cohesin from chromosome arms during mitosis. By expressing Cre recombinase from a zona pellucida promoter, we have deleted a floxed allele of separase specifically in mouse oocytes. This prevents removal of Rec8 from chromosome arms and resolution of chiasmata. It also hinders extrusion of the first polar body (PBE) and causes female sterility. mRNA encoding wild-type but not catalytically inactive separase restores chiasma resolution. Both types of mRNA restore PBE. Proteolytic activity of separase is therefore essential for Rec8's removal from chromosome arms and for chiasma resolution but not for PBE.

Errors in meiosis I cause missegregation of chromosomes and can result in the generation of aneuploid embryos with severe birth defects. In human oocytes, failures in spindle checkpoint control may be responsible for the generation of trisomies (e.g., Down Syndrome) due to chromosome missegregation in meiosis I. Up to now, the mechanisms ensuring correct separation of chromosomes in meiosis I remained unknown. Our study shows for the first time that a functional Mad2-dependent spindle checkpoint exists during the first meiotic division in mammalian oocytes.

No abstract

In the budding yeast Saccharomyces cerevisiae, MCM1 encodes an essential DNA-binding protein that regulates transcription of many genes in cooperation with different associated factors. With the help of a conditional expression system, we show that Mcm1 depletion has a distinct effect on cell cycle progression by preventing cells from undergoing mitosis. Genes that normally exhibit a G2-to-M-phase-specific expression pattern, such as CLB1, CLB2, CDC5, SWI5, and ACE2, remain uninduced in the absence of functional Mcm1. In vivo footprinting experiments show that Mcm1, in conjunction with an Mcm1-recruited factor, binds to the promoter regions of SWI5 and CLB2 at sites shown to be involved in cell cycle regulation. However, promoter occupation at these sites is cell cycle independent, and therefore the regulatory system seems to operate on constitutively bound Mcm1 complexes. A gene fusion that provides Mcm1 with a strong transcriptional activation domain causes transcription of SWI5, CLB1, CLB2, and CDC5 at inappropriate times of the cell cycle. Thus, Mcm1 and a cooperating, cell cycle-regulated activation partner are directly involved in the coordinated expression of multiple G2-regulated genes. The arrest phenotype of Mcm1-depleted cells is consistent with low levels of Clb1 and Clb2 kinase. However, constitutive CLB2 expression does not suppress the mitotic defect, and therefore other essential activities required for the G2-to-M transition must also depend on Mcm1 function.

Faithful segregation of homologous chromosomes during the first meiotic division is essential for further embryo development. The question at issue is whether the same mechanisms ensuring correct separation of sister chromatids in mitosis are at work during the first meiotic division. In mitosis, sister chromatids are linked by a cohesin complex holding them together until their disjunction at anaphase. Their disjunction is mediated by Separase, which cleaves the cohesin. The activation of Separase requires prior degradation of its associated inhibitor, called securin. Securin is a target of the APC/C (Anaphase Promoting Complex/Cyclosome), a cell cycle-regulated ubiquitin ligase that ubiquitinates securin at the metaphase-to-anaphase transition and thereby targets it for degradation by the 26S proteasome. After securin degradation, Separase cleaves the cohesins and triggers chromatid separation, a prerequisite for anaphase. In yeast and worms, the segregation of homologous chromosomes in meiosis I depends on the APC/C and Separase activity. Yet, it is unclear if Separase is required for the first meiotic division in vertebrates because APC/C activity is thought to be dispensable in frog oocytes. We therefore investigated if Separase activity is required for correct chromosome segregation in meiosis I in mouse oocytes.

SUMMARYIn female meiosis, chromosome missegregations lead to the generation of aneuploid oocytes and can cause the development of trisomies or infertility. Because mammalian female meiosis I is error prone, the full functionality of control mechanisms, such as the spindle assembly checkpoint (SAC), has been put into question. The SAC monitors the correct orientation, microtubule occupancy and tension on proteinaceous structures named kinetochores. Although it has been shown previously that the SAC exists in meiosis I, where attachments are monopolar, the role of microtubule occupancy for silencing the SAC and the importance of certain essential SAC components, such as the kinase Mps1, are unknown in mammalian oocytes. Using a conditional loss-of-function approach, we address the role of Mps1 in meiotic progression and checkpoint control in meiosis I. Our data demonstrate that kinetochore localization of Mps1 is required for the proper timing of prometaphase and is essential for SAC control, chromosome alignment and aurora C localization in meiosis I. The absence of Mps1 from kinetochores severely impairs chromosome segregation in oocyte meiosis I and, therefore, fertility in mice. In addition, we settle a long-standing question in showing that kinetochore-microtubule attachments are present in prometaphase I at a time when most of the SAC protein Mad2 disappears from kinetochores.