Bub3 interaction with Mad2, Mad3 and Cdc20 is mediated by WD40 repeats and does not require intact kinetochores.

The kinetochore checkpoint pathway, involving the Mad1, Mad2, Mad3, Bub1, Bub3 and Mps1 proteins, prevents anaphase entry and mitotic exit by inhibiting the anaphase promoting complex activator Cdc20 in response to monopolar attachment of sister kinetochores to spindle fibres. We show here that Cdc20, which had previously been shown to interact physically with Mad2 and Mad3, associates also with Bub3 and association is up-regulated upon checkpoint activation. Moreover, co-fractionation experiments suggest that Mad2, Mad3 and Bub3 may be concomitantly present in protein complexes with Cdc20. Formation of the Bub3-Cdc20 complex requires all kinetochore checkpoint proteins but, surprisingly, not intact kinetochores. Conversely, point mutations altering the conserved WD40 motifs of Bub3, which might be involved in the formation of a beta-propeller fold devoted to protein-protein interactions, disrupt its association with Mad2, Mad3 and Cdc20, as well as proper checkpoint response. We suggest that Bub3 could serve as a platform for interactions between kinetochore checkpoint proteins, and its association with Mad2, Mad3 and Cdc20 might be instrumental for checkpoint activation.

Correct spindle elongation at the metaphase/anaphase transition is an APC-dependent event in budding yeast.

At the metaphase to anaphase transition, chromosome segregation is initiated by the splitting of sister chromatids. Subsequently, spindles elongate, separating the sister chromosomes into two sets. Here, we investigate the cell cycle requirements for spindle elongation in budding yeast using mutants affecting sister chromatid cohesion or DNA replication. We show that separation of sister chromatids is not sufficient for proper spindle integrity during elongation. Rather, successful spindle elongation and stability require both sister chromatid separation and anaphase-promoting complex activation. Spindle integrity during elongation is dependent on proteolysis of the securin Pds1 but not on the activity of the separase Esp1. Our data suggest that stabilization of the elongating spindle at the metaphase to anaphase transition involves Pds1-dependent targets other than Esp1.

Role of the kinetochore protein Ndc10 in mitotic checkpoint activation in Saccharomyces cerevisiae.

Mitotic checkpoints delay cell cycle progression in response to alterations in the mitotic apparatus, thus ensuring correct chromosome segregation. While improper spindle orientation activates the Bub2/Bfa1-dependent checkpoint in budding yeast, delaying exit from mitosis, lack of bipolar kinetochore-microtubule attachment activates a signal transduction cascade that prevents both anaphase onset and exit from mitosis by inhibiting the Cdc20/APC (Anaphase Promoting Complex)-mediated proteolysis of securin and inactivation of mitotic cyclin-dependent kinases (CDKs), respectively. Proteolysis of the securin Pdsl is necessary to liberate the separase Esp1, which then triggers sister chromatid separation, whereas inactivation of mitotic CDKs is a prerequisite for exit from mitosis and for starting a new round of DNA replication in the next cell cycle. In budding yeast, this latter checkpoint response involves the proteins Mad1, 2, 3, Bub1 and Bub3, whose vertebrate counterparts localize to unattached kinetochores. Mutations that alter other kinetochore proteins result in mitotic checkpoint activation, while the ndc10-1 mutation not only impairs kinetochore function, but also disrupts the checkpoint response, indicating a role for Ndc10 in this process. Here we present evidence that Ndc10 is not part of the Bub2/Bfa1-dependent pathway, and its role in the checkpoint response might also be different from that of the other Mad and Bub proteins. Indeed, Ndc10, unlike other mitotic checkpoint proteins, is not required for the mitotic block induced by overexpression of the Mpsl protein kinase, which is implicated in mitotic checkpoint control. Furthermore, the delay in mitotic exit caused by non-degradable Pds1, which does not require Mad and Bub proteins, depends on Ndc10 function. We propose that a pathway involving Ndc10 might monitor defects in the mitotic apparatus independently of the Mad and Bub proteins. Since the Espl separase is required for exit from mitosis in both ndc10-1 and nocodazole-treated mad2delta cells, the two signal transduction cascades might ultimately converge on the inactivation of Esp1.

Budding yeast Bub2 is localized at spindle pole bodies and activates the mitotic checkpoint via a different pathway from Mad2.

The mitotic checkpoint blocks cell cycle progression before anaphase in case of mistakes in the alignment of chromosomes on the mitotic spindle. In budding yeast, the Mad1, 2, 3, and Bub1, 2, 3 proteins mediate this arrest. Vertebrate homologues of Mad1, 2, 3, and Bub1, 3 bind to unattached kinetochores and prevent progression through mitosis by inhibiting Cdc20/APC-mediated proteolysis of anaphase inhibitors, like Pds1 and B-type cyclins. We investigated the role of Bub2 in budding yeast mitotic checkpoint. The following observations indicate that Bub2 and Mad1, 2 probably activate the checkpoint via different pathways: (a) unlike the other Mad and Bub proteins, Bub2 localizes at the spindle pole body (SPB) throughout the cell cycle; (b) the effect of concomitant lack of Mad1 or Mad2 and Bub2 is additive, since nocodazole-treated mad1 bub2 and mad2 bub2 double mutants rereplicate DNA more rapidly and efficiently than either single mutant; (c) cell cycle progression of bub2 cells in the presence of nocodazole requires the Cdc26 APC subunit, which, conversely, is not required for mad2 cells in the same conditions. Altogether, our data suggest that activation of the mitotic checkpoint blocks progression through mitosis by independent and partially redundant mechanisms.

The beta4 integrin interactor p27(BBP/eIF6) is an essential nuclear matrix protein involved in 60S ribosomal subunit assembly.

p27(BBP/eIF6) is an evolutionarily conserved protein that was originally identified as p27(BBP), an interactor of the cytoplasmic domain of integrin beta4 and, independently, as the putative translation initiation factor eIF6. To establish the in vivo function of p27(BBP/eIF6), its topographical distribution was investigated in mammalian cells and the effects of disrupting the corresponding gene was studied in the budding yeast, Saccharomyces cerevisiae. In epithelial cells containing beta4 integrin, p27(BBP/eIF6) is present in the cytoplasm and enriched at hemidesmosomes with a pattern similar to that of beta4 integrin. Surprisingly, in the absence and in the presence of the beta4 integrin subunit, p27(BBP/eIF6) is in the nucleolus and associated with the nuclear matrix. Deletion of the IIH S. cerevisiae gene, encoding the yeast p27(BBP/eIF6) homologue, is lethal, and depletion of the corresponding gene product is associated with a dramatic decrease of the level of free ribosomal 60S subunit. Furthermore, human p27(BBP/eIF6) can rescue the lethal effect of the iihDelta yeast mutation. The data obtained in vivo suggest an evolutionarily conserved function of p27(BBP/eIF6) in ribosome biogenesis or assembly rather than in translation. A further function related to the beta4 integrin subunit may have evolved specifically in higher eukaryotic cells.

Is the yeast anaphase promoting complex needed to prevent re-replication during G2 and M phases?

The Anaphase Promoting Complex (APC) is required for anaphase progression and B-type cyclin proteolysis. The recent finding that inactivation of the APC allows 'over-replication' of DNA has led to the proposal that the APC might also be required for preventing reduplication of chromosomes during G2 and M phases. In this report we re-investigate the phenotype of apc mutant cells and find that they do not re-replicate their DNA during the period taken for wild-type cells to traverse G2 and M phases. apc mutants do, however, gradually increase their DNA content after long periods of cell cycle arrest. Such DNA synthesis occurs almost exclusively in the cytoplasm and neither occurs in cells lacking mitochondrial DNA nor depends on Cdc6, a protein which is essential for the initiation of chromosomal but not mitochondrial DNA replication. ARS1, a chromosomal replication origin, is not re-fired in cells deprived of APC function, confirming that the 'over-replicated' DNA in apc mutant cells is of mitochondrial origin. Furthermore, we find that APC function is required to promote but not to prevent re-replication in ndc10 mutant cells. We therefore propose that the APC is not involved in preventing re-duplication of chromosomes during G2 and M phases.

Cell cycle regulation of S phase entry in Saccharomyces cerevisiae.

Eukaryotic DNA replication is restricted to a narrow window of the cell cycle called S phase, and occurs once and only once during each cell cycle. The combination of genetic and biochemical approaches in the budding yeast Saccharomyces cerevisiae has proven extremely helpful for studying the cell cycle regulation of S phase entry. This review will try to summarise the most recent discoveries which led to a new model to explain how entry into S phase is regulated in eukaryotic cells.

Activation of S-phase-promoting CDKs in late G1 defines a "point of no return" after which Cdc6 synthesis cannot promote DNA replication in yeast.

In eukaryotic cells, DNA replication is confined to a discrete period of the cell cycle and does not usually recur until after anaphase. In the budding yeast Saccharomyces cerevisiae, assembly of pre-replication complexes (pre-RCs) at future origins as cells exit mitosis (or later during G1 is necessary for subsequent initiation of DNA replication triggered by activation in late G1 of Cdc28/Cdk1 kinases associated with B-type cyclins Clb1-Clb6. The absence of pre-RCs during G2 and M phases could explain why origins of DNA replication fire only once during the cell cycle, even though S-phase-promoting Cdks remain active from the beginning of S phase through the end of M phase. Formation of pre-RCs and their maintenance during G1 depend on the synthesis and activity of an unstable protein encoded by CDC6. We find that Cdc6 synthesis can only promote DNA replication in a restricted window of the cell cycle: between destruction of Clbs after anaphase and activation of Clb5/ and Clb6/Cdk1 in late G1. The latter corresponds to a "point of no return," after which Cdc6 synthesis can no longer promote DNA replication. Cdc6 protein can be made throughout the cell cycle and, in certain circumstances, can accumulate within the nuclei of G2 and M phase cells without inducing re-replication. Thus, control over Cdc6 degradation and/or nuclear localization is not crucial for preventing origin re-firing. Our data are consistent with the notion that cells can no longer incorporate de novo synthesized Cdc6 into pre-RCs once C1b/Cdk1 kinases have been activated. We show that Cdc6p associates with Clb/Cdk1 kinases from late G1 until late anaphase, which might be important for inhibiting pre-RC assembly during S, G2, and M phases. Inhibition of pre-RC assembly by the same kinases that trigger initiation explains how origins are prevented from re-firing until Clb kinases are destroyed after anaphase.

An essential role for the Cdc6 protein in forming the pre-replicative complexes of budding yeast.

Origins of DNA replication in Saccharomyces cerevisiae are bound by two protein complexes during the cell cycle. Post-replicative complexes closely resemble those generated in vitro by purified origin recognition complex (ORC), which is essential for DNA replication in vivo. Pre-replicative complexes (pre-RCs) are characterized by an extended region of nuclease protection overlapping the ORC footprint. We show here that the Cdc6 protein (Cdc6p), which is necessary for origin firing in vivo, is essential for the establishment and maintenance of pre-RCs, suggesting that it is a component of these complexes. Without Cdc6p, G1 origins closely resemble post-replicative origins, providing evidence that ORC is also a component of pre-RCs. These results suggest that pre-RCs play an essential role in initiating DNA replication and support a two-step mechanism for the assembly of functional initiation complexes.

Cdc6 is an unstable protein whose de novo synthesis in G1 is important for the onset of S phase and for preventing a 'reductional' anaphase in the budding yeast Saccharomyces cerevisiae.

S phase entry depends on cyclin-dependent kinases whose activation during late G1 due partly to the synthesis of unstable cyclin subunits. We identify here a second type of unstable protein, Cdc6, whose synthesis during G1 is important for initiation of DNA replication. The CDC6 gene is normally transcribed at the end of mitosis, but in cells with a prolonged G1 phase there is a second burst of transcription in late G1. The former is due to Swi5, while the latter is due to MBF or SBF transcription factors. Small G1 cells that cannot synthesize Cdc6 in late G1 progress through S phase very slowly. Cells that transcribe CDC6 neither at the end of mitosis nor in late G1 fail to replicate DNA but, despite this, undergo mitosis and produce daughter cells with fractional DNA contents. This 'reductional' anaphase occurs with almost wild-type kinetics and depends on the activity of G2 cyclins. Thus, cells that fail to duplicate chromosomes due to a cdc6 defect cannot prevent the onset of mitosis, unlike other mutants with replication defects. We show, by fluorescence in situ hybridization, that chromosomes which remain unduplicated due to a lack of Cdc6 synthesis are segregated intact to spindle poles during the 'reductional' anaphase.

Genes involved in sister chromatid separation are needed for B-type cyclin proteolysis in budding yeast.

B-type cyclin destruction is necessary for exit from mitosis and the initiation of a new cell cycle. Through the isolation of mutants, we have identified three essential yeast genes, CDC16, CDC23, and CSE1, which are required for proteolysis of the B-type cyclin CLB2 but not of other unstable proteins. cdc23-1 mutants are defective in both entering and exiting anaphase. Their failure to exit anaphase can be explained by defective cyclin proteolysis. CDC23 is required at the metaphase/anaphase transition to separate sister chromatids, and we speculate that it might promote proteolysis of proteins that hold sister chromatids together. Proteolysis of CLB2 is initiated in early anaphase, but a fraction of CLB2 remains stable until anaphase is complete.

Positive cis-acting regulatory sequences mediate proper control of POL1 transcription in Saccharomyces cerevisiae.

The 5'ACGCGT3' MluI motif, which is found in the upstream region of several yeast DNA-synthesis genes which are periodically expressed during the mitotic cell-cycle, is present twice in the 5' non-coding region of the DNA-polymerase alpha gene (POL1). Deletion of the most distal repeat does not affect POL1 transcription, while the adjacent 40 base-pair (bp) downstream sequence is necessary both for the proper level and the fluctuation of POL1 mRNA. This region contains the 5'ACGCGTCGCGT3' sequence, which is sufficient to control periodic transcription of a CYC1-lacZ reporter gene with the same kinetics observed for POL1. The adjacent 29 bp AT-rich region does not show any activity by itself, but it acts synergistically in conjunction with at least one MluI hexamer to stimulate CYC1-lacZ expression. By further deletion analysis, DNA sequences necessary to initiate POL1 transcription at the proper sites have also been identified.

Control of DNA synthesis genes in budding yeast: involvement of the transcriptional modulator MOT1 in the expression of the DNA polymerase alpha gene.

Periodic transcription during the cell cycle of the budding yeast DNA polymerase alpha gene (POL1) requires the cis-acting element 5' ACGCGT 3', which has been found in the 5' non-coding region of all the DNA synthesis genes analyzed so far. Search for trans-acting mutations affecting POL1 expression led to the isolation of the temperature-sensitive reg1033 mutant, that showed increased levels of both DNA polymerase alpha and delta gene transcripts. Cloning of the REG1033 gene demonstrated that it is essential for cell viability and required for proper expression of the POL1 gene. DNA sequence comparison established that the REG1033 gene is identical to MOT1, a gene encoding a presumptive DNA helicase which modulates transcription of several yeast genes.

The yeast DNA polymerase-primase complex: genes and proteins.

The yeast DNA polymerase-primase complex is composed of four polypeptides designated p180, p74, p58 and p48. All the genes coding for these polypeptides have now been cloned. By protein sequence comparison we found that yeast DNA polymerase I (alpha) shares three major regions of homology with several DNA polymerases. A fourth region, called region P, is conserved in yeast and human DNA polymerase alpha. The site of a temperature-sensitive mutation in the POL1 gene which causes decreased stability of the polymerase-primase complex has been sequenced and falls in this region. We hypothesize that region P is important for protein-protein interactions. Highly selective biochemical methods might be similarly important to distinguish functional domains in the polymerase-primase complex. An autocatalytic affinity labeling procedure has been applied to map the active center of yeast DNA primase. From this approach we conclude that both primase subunits (p48 and p58) participate in the formation of the catalytic site of the enzyme.