Meiotic nuclear divisions in budding yeast require PP2ACdc55-mediated antagonism of Net1 phosphorylation by Cdk

During meiosis, one round of deoxyribonucleic acid replication is followed by two rounds of nuclear division. In Saccharomyces cerevisiae, activation of the Cdc14 early anaphase release (FEAR) network is required for exit from meiosis I but does not lead to the activation of origins of replication. The precise mechanism of how FEAR regulates meiosis is not understood. In this paper, we report that premature activation of FEAR during meiosis caused by loss of protein phosphatase PP2ACdc55 activity blocks bipolar spindle assembly and nuclear divisions. In cdc55 meiotic null (cdc55-mn) cells, the cyclin-dependent kinase (Cdk)–counteracting phosphatase Cdc14 was released prematurely from the nucleolus concomitant with hyperphosphorylation of its nucleolar anchor protein Net1. Crucially, a mutant form of Net1 that lacks six Cdk phosphorylation sites rescued the meiotic defect of cdc55-mn cells. Expression of a dominant mutant allele of CDC14 mimicked the cdc55-mn phenotype. We propose that phosphoregulation of Net1 by PP2ACdc55 is essential for preventing precocious exit from meiosis I

Meiotic nuclear divisions in budding yeast require PP2ACdc55-mediated antagonism of Net1 phosphorylation by Cdk

During meiosis, one round of deoxyribonucleic acid replication is followed by two rounds of nuclear division. In Saccharomyces cerevisiae, activation of the Cdc14 early anaphase release (FEAR) network is required for exit from meiosis I but does not lead to the activation of origins of replication. The precise mechanism of how FEAR regulates meiosis is not understood. In this paper, we report that premature activation of FEAR during meiosis caused by loss of protein phosphatase PP2ACdc55 activity blocks bipolar spindle assembly and nuclear divisions. In cdc55 meiotic null (cdc55-mn) cells, the cyclin-dependent kinase (Cdk)–counteracting phosphatase Cdc14 was released prematurely from the nucleolus concomitant with hyperphosphorylation of its nucleolar anchor protein Net1. Crucially, a mutant form of Net1 that lacks six Cdk phosphorylation sites rescued the meiotic defect of cdc55-mn cells. Expression of a dominant mutant allele of CDC14 mimicked the cdc55-mn phenotype. We propose that phosphoregulation of Net1 by PP2ACdc55 is essential for preventing precocious exit from meiosis I

Regulation of Clb1 during meiosis in saccharomyces cerevisiae

Meiosis is a specialised form of cell division in which diploid cells divide to form four non-identical spores containing half the genetic complement of the parent. During this cell division program, much of the usual machinery regulating cell division is put to alternate use to allow the cells to undergo an extra round of division without an intervening phase of DNA synthesis. In particular, the end of the first division, meiosis I, must be regulated differently than the end of the mitotic division. We used the model organism Saccharomyces cerevisiae to determine some of these differences in regulation. The cell division program is driven by the sequential association of cyclins with the CDK (cyclin dependent kinase), leading to waves of kinase activity. Exit from mitosis requires the downregulation of CDK activity, and is coordinated by two signalling networks, the FEAR (Cdc14 Early Anaphase Release) network and the MEN (Mitotic Exit Network). Both networks initiate the release of the phosphatase Cdc14 from its inhibitor, Net1, to counter CDK activity. Exit from meiosis I similarly relies on Cdc14 activity, but is driven only by the FEAR network. Experimental results showed that the phosphorylation state and subcellular localisation of the meiotic cyclin, C1b1, are altered in meiosis I. We investigated this relationship and aimed to determine the kinase responsible. We used modelling techniques to explore several rationales for the specific regulation of C1b1. We examined the functional significance of C1b1 localisation, using localisation mutants, and made an investigation into Cdc14 release in meiosis I

Fluorescent Single-Stranded DNA Binding Protein as a Probe for Sensitive, Real-Time Assays of Helicase Activity

The formation and maintenance of single-stranded DNA (ssDNA) are essential parts of many processes involving DNA. For example, strand separation of double-stranded DNA (dsDNA) is catalyzed by helicases, and this exposure of the bases on the DNA allows further processing, such as replication, recombination, or repair. Assays of helicase activity and probes for their mechanism are essential for understanding related biological processes. Here we describe the development and use of a fluorescent probe to measure ssDNA formation specifically and in real time, with high sensitivity and time resolution. The reagentless biosensor is based on the ssDNA binding protein (SSB) from Escherichia coli, labeled at a specific site with a coumarin fluorophore. Its use in the study of DNA manipulations involving ssDNA intermediates is demonstrated in assays for DNA unwinding, catalyzed by DNA helicases