Role of Ndt80, Sum1, and Swe1 as targets of the meiotic recombination checkpoint that control exit from pachytene and spore formation in Saccharomyces cerevisiae

The meiotic recombination checkpoint, which is triggered by defects in recombination or chromosome synapsis, arrests sporulating cells of Saccharomyces cerevisiae at pachytene by preventing accumulation of active Clb-Cdc28. We compared the effects of manipulating the three known targets of the meiotic recombination checkpoint, NDT80, SWE1, and SUM1, in dmc1-arrested cells. Ndt80 is an activator of a set of middle sporulation-specific genes (MSGs), which includes CLB genes and genes involved in spore wall formation; Swe1 inhibits Clb-Cdc28 activity; and Sum1 is a repressor of NDT80 and some MSGs. Activation of the checkpoint leads to inhibition of Ndt80 activity and to stabilization of Swe1 and Sum1. Thus, dmc1-arrested cells fail to express MSGs, arrest at pachytene, and do not form spores. Our study shows that dmc1/dmc1 sum1/sum1 cells expressed MSGs prematurely and at high levels, entered the meiotic divisions efficiently, and in some cases formed asci containing mature spores. In contrast, dmc1/dmc1 swe1/swe1 cells expressed MSGs at a very low level, were inefficient and delayed in entry into the meiotic divisions, and never formed mature spores. We found that cells of dmc1/dmc1 sum1/sum1 ndt80/ndt80 and dmc1/dmc1 swe1/swe1 ndt80/ndt80 strains arrested at pachytene and that dmc1/dmc1 or dmc1/dmc1 swe1/swe1 cells overexpressing NDT80 were less efficient in bypassing checkpoint-mediated arrest than dmc1/dmc1 sum1/sum1 cells. Our results are consistent with previous suggestions that increased Clb-Cdc28 activity, caused by mutation of SWE1 or by an NDT80-dependent increase in CLB expression, allows dmc1/dmc1 cells to exit pachytene and that subsequent upregulation of Ndt80 activity by a feedback mechanism promotes entry into the meiotic divisions. Spore morphogenesis, however, requires efficient and timely activation of MSGs, which we speculate was achieved in dmc1/dmc1 sum1/sum1 cells by premature expression of NDT80.     

The pachytene checkpoint

The pachytene checkpoint prevents meiotic nuclear division in cells that fail to complete meiotic recombination and chromosome synapsis. This control mechanism prevents chromosome missegregation that would lead to the production of aneuploid gametes. The pachytene checkpoint requires a subset of proteins that function in the mitotic DNA damage checkpoint. In budding yeast, the pachytene checkpoint also requires meiosis-specific chromosomal proteins and, unexpectedly, proteins concentrated in the nucleolus. Progress has been made in identifying components of the cell-cycle machinery that are impacted by the checkpoint.     

Sum1p, the origin recognition complex, and the spreading of a promoter-specific repressor in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, Sum1p is a promoter-specific repressor. A single amino acid change generates the mutant Sum1-1p, which causes regional silencing at new loci where wild-type Sum1p does not act. Thus, Sum1-1p is a model for understanding how the spreading of repressive chromatin is regulated. When wild-type Sum1p was targeted to a locus where mutant Sum1-1p spreads, wild-type Sum1p did not spread as efficiently as mutant Sum1-1p did, despite being in the same genomic context. Thus, the SUM1-1 mutation altered the ability of the protein to spread. The spreading of Sum1-1p required both an enzymatically active deacetylase, Hst1p, and the N-terminal tail of histone H4, consistent with the spreading of Sum1-1p involving sequential modification of and binding to histone tails, as observed for other silencing proteins. Furthermore, deletion of the N-terminal tail of H4 caused Sum1-1p to return to loci where wild-type Sum1p acts, consistent with the SUM1-1 mutation increasing the affinity of the protein for H4 tails. These results imply that the spreading of repressive chromatin proteins is regulated by their affinities for histone tails. Finally, this study uncovered a functional connection between wild-type Sum1p and the origin recognition complex, and this relationship also contributes to mutant Sum1-1p localization.     

The spindle checkpoint of the yeast Saccharomyces cerevisiae requires kinetochore function and maps to the CBF3 domain

We have measured the activity of the spindle checkpoint in null mutants lacking kinetochore activity in the yeast Saccharomyces cerevisiae. We constructed deletion mutants for nonessential genes by one-step gene replacements. We constructed heterozygous deletions of one copy of essential genes in diploid cells and purified spores containing the deletion allele. In addition, we made gene fusions for three essential genes to target the encoded proteins for proteolysis (degron alleles). We determined that Ndc10p, Ctf13p, and Cep3p are required for checkpoint activity. In contrast, cells lacking Cbf1p, Ctf19p, Mcm21p, Slk19p, Cse4p, Mif2p, Mck1p, and Kar3p are checkpoint proficient. We conclude that the kinetochore plays a critical role in checkpoint signaling in S. cerevisiae. Spindle checkpoint activity maps to a discreet domain within the kinetochore and depends on the CBF3 protein complex.     

The S-phase checkpoint and its regulation in Saccharomyces cerevisiae

Cells are never more vulnerable than during DNA replication, which represents a major moment of potential genetic instability. Genotoxic insults induce many different forms of DNA damage that may interfere with the ability of cells to properly duplicate their genome. Primary damage may in turn undergo structural transformations during DNA replication, thus generating secondary lesions that may be even more dangerous. Cells experiencing replication of damaged DNA or replication blocks activate an S-phase checkpoint response that assures the fidelity and completion of DNA replication before cells enter M-phase. The S-phase checkpoint pathway regulates not only progress through the cell cycle but also DNA repair and DNA replication itself.     

Reversible pachytene arrest of Saccharomyces cerevisiae at elevated temperature

Summary The temperature sensitivity of sporulation in a well-characterized yeast strain lacking any known temperature sensitive genes has been investigated. Cytological observations by electron microscopy demonstrate that cells incubated in sporulation medium at a temperature inhibitory to sporulation became arrested in meiotic prophase. The stage of arrest was identified as pachytene by the presence of duplicated (but unseparated) spindle pole bodies and synaptonemal complex. Transfer of the arrested culture to lower temperature permitted resumption of meiosis and sporulation; transfer to vegetative medium resulted in reversion to mitotic division. Genetic analysis of cells that had reverted to mitosis revealed that commitment to intragenic recombination had occurred by the time of arrest. Prolonged incubation at the elevated temperature resulted in the enhancement of intragenic recombination above normal levels, suggesting that some aspect of recombination continued to occur during the pachytene arrest. Evidence is presented that DNA replication, although depressed overall in the arrested cultures, had occurred to completion in many arrested cells.     

The pachytene checkpoint in S. cerevisiae depends on Swe1-mediated phosphorylation of the cyclin-dependent kinase Cdc28

Mutants defective in meiotic recombination and synaptonemal complex formation undergo checkpoint-mediated arrest in mid-meiotic prophase. In S. cerevisiae, this checkpoint requires Swe1, which phosphorylates and inactivates the cyclin-dependent kinase Cdc28. A swe1 deletion allows mutants that normally arrest in meiotic prophase to sporulate at wild-type levels, though sporulation is delayed. This delay is eliminated by overproducing Clb1, the major cyclin required for meiosis I. The Swe1 protein accumulates and is hyperphosphorylated in checkpoint-arrested cells. Our results suggest that meiotic arrest is mediated both by increasing Swe1 activity and limiting cyclin production, with Swe1 being the primary downstream target of checkpoint control. The requirement for Swe1 distinguishes the pachytene checkpoint from the DNA damage checkpoints operating in vegetative cells.     

The Saccharomyces cerevisiae checkpoint gene BUB1 encodes a novel protein kinase.

Normal cell multiplication requires that the events of mitosis occur in a carefully ordered fashion. Cells employ checkpoints to prevent cycle progression until some prerequisite step has been completed. To explore the mechanisms of checkpoint enforcement, we previously screened for mutants of Saccharomyces cerevisiae which are unable to recover from a transient treatment with a benzimidazole-related microtubule inhibitor because they fail to inhibit subsequent cell cycle steps. Two of the identified genes, BUB2 and BUB3, have been cloned and described (M. A. Hoyt, L. Totis, and B. T. Roberts, Cell 66:507-517, 1991). Here we present the characterization of the BUB1 gene and its product. Genetic evidence was obtained suggesting that Bub1 and Bub3 are mutually dependent for function, and immunoprecipitation experiments demonstrated a physical association between the two. Sequence analysis of BUB1 revealed a domain with similarity to protein kinases. In vitro experiments confirmed that Bub1 possesses kinase activity; Bub1 was able to autophosphorylate and to catalyze phosphorylation of Bub3. In addition, overproduced Bub1 was found to localize to the cell nucleus.     

The SNF1 kinase complex from Saccharomyces cerevisiae phosphorylates the transcriptional repressor protein Mig1p in vitro at four sites within or near regulatory domain 1.

Mig1p is a zinc finger protein required for repression of glucose-regulated genes in budding yeast. On removal of medium glucose, gene repression is relieved via a mechanism that requires the SNF1 protein kinase complex. We show that Mig1p expressed as a glutathione-S-transferase fusion in bacteria is readily phosphorylated by the SNF1 kinase in vitro. Four phosphorylation sites were identified, i.e. Ser-222, Ser-278, Ser-311 and Ser-381. The latter three are exact matches to the recognition motif we previously defined for SNF1 and lie within regions shown to be required for SNF1-dependent derepression and nuclear-to-cytoplasmic translocation.     

Dual role for Saccharomyces cerevisiae Tel1 in the checkpoint response to double-strand breaks

The main responder to DNA double-strand breaks (DSBs) in mammals is ataxia telangiectasia mutated (ATM), whereas DSB-induced checkpoint activation in budding yeast seems to depend primarily on the ATM and Rad-3-related (ATR) orthologue Mec1. Here, we show that Saccharomyces cerevisiae Tel1, the ATM orthologue, has two functions in checkpoint response to DSBs. First, Tel1 participates, together with the MRX complex, in Mec1-dependent DSB-induced checkpoint activation by increasing the efficiency of single-stranded DNA accumulation at the ends of DSBs, and this checkpoint function can be overcome by overproducing the exonuclease Exo1. Second, Tel1 can activate the checkpoint response to DSBs independently of Mec1, although its signalling activity only becomes apparent when several DSBs are generated. Furthermore, we provide evidence that the kinetics of DSB resection can influence Tel1 activation, indicating that processing of the DSB termini might influence the transition from Tel1/ATM- to Mec1/ATR-dependent checkpoint.