Binding specificity of the G1/S transcriptional regulators in budding yeast

G1/S transcriptional regulation in the budding yeast Saccharomyces cerevisiae depends on three main transcriptional components, Swi4, Swi6 and Mbp1. These proteins constitute two transcription factor complexes that regulate over 300 G1/S transcripts, namely SBF (Swi4-Swi6) and MBF (Mbp1-Swi6). SBF and MBF are involved in regulating largely non-overlapping sets of G1/S genes via clearly distinct mechanisms

Yeast IME2 Functions Early in Meiosis Upstream of Cell Cycle-Regulated SBF and MBF Targets

BACKGROUND: In Saccharomyces cerevisiae, the G1 cyclin/cyclin-dependent kinase (CDK) complexes Cln1,-2,-3/Cdk1 promote S phase entry during the mitotic cell cycle but do not function during meiosis. It has been proposed that the meiosis-specific protein kinase Ime2, which is required for normal timing of pre-meiotic DNA replication, is equivalent to Cln1,-2/Cdk1. These two CDK complexes directly catalyze phosphorylation of the B-type cyclin/CDK inhibitor Sic1 during the cell cycle to enable its destruction. As a result, Clb5,-6/Cdk1 become activated and facilitate initiation of DNA replication. While Ime2 is required for Sic1 destruction during meiosis, evidence now suggests that Ime2 does not directly catalyze Sic1 phosphorylation to target it for destabilization as Cln1,-2/Cdk1 do during the cell cycle. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated that Sic1 is eventually degraded in meiotic cells lacking the IME2 gene (ime2Δ), supporting an indirect role of Ime2 in Sic1 destruction. We further examined global RNA expression comparing wild type and ime2Δ cells. Analysis of these expression data has provided evidence that Ime2 is required early in meiosis for normal transcription of many genes that are also periodically expressed during late G1 of the cell cycle. CONCLUSIONS/SIGNIFICANCE: Our results place Ime2 at a position in the early meiotic pathway that lies upstream of the position occupied by Cln1,-2/Cdk1 in the analogous cell cycle pathway. Thus, Ime2 may functionally resemble Cln3/Cdk1 in promoting S phase entry, or it could play a role even further upstream in the corresponding meiotic cascade

The Fission Yeast Homeodomain Protein Yox1p Binds to MBF and Confines MBF-Dependent Cell-Cycle Transcription to G1-S via Negative Feedback

The regulation of the G1- to S-phase transition is critical for cell-cycle progression. This transition is driven by a transient transcriptional wave regulated by transcription factor complexes termed MBF/SBF in yeast and E2F-DP in mammals. Here we apply genomic, genetic, and biochemical approaches to show that the Yox1p homeodomain protein of fission yeast plays a critical role in confining MBF-dependent transcription to the G1/S transition of the cell cycle. The yox1 gene is an MBF target, and Yox1p accumulates and preferentially binds to MBF-regulated promoters, via the MBF components Res2p and Nrm1p, when they are transcriptionally repressed during the cell cycle. Deletion of yox1 results in constitutively high transcription of MBF target genes and loss of their cell cycle–regulated expression, similar to deletion of nrm1. Genome-wide location analyses of Yox1p and the MBF component Cdc10p reveal dozens of genes whose promoters are bound by both factors, including their own genes and histone genes. In addition, Cdc10p shows promiscuous binding to other sites, most notably close to replication origins. This study establishes Yox1p as a new regulatory MBF component in fission yeast, which is transcriptionally induced by MBF and in turn inhibits MBF-dependent transcription. Yox1p may function together with Nrm1p to confine MBF-dependent transcription to the G1/S transition of the cell cycle via negative feedback. Compared to the orthologous budding yeast Yox1p, which indirectly functions in a negative feedback loop for cell-cycle transcription, similarities but also notable differences in the wiring of the regulatory circuits are evident

Mutation of the Zebrafish Nucleoporin elys Sensitizes Tissue Progenitors to Replication Stress

The recessive lethal mutation flotte lotte (flo) disrupts development of the zebrafish digestive system and other tissues. We show that flo encodes the ortholog of Mel-28/Elys, a highly conserved gene that has been shown to be required for nuclear integrity in worms and nuclear pore complex (NPC) assembly in amphibian and mammalian cells. Maternal elys expression sustains zebrafish flo mutants to larval stages when cells in proliferative tissues that lack nuclear pores undergo cell cycle arrest and apoptosis. p53 mutation rescues apoptosis in the flo retina and optic tectum, but not in the intestine, where the checkpoint kinase Chk2 is activated. Chk2 inhibition and replication stress induced by DNA synthesis inhibitors were lethal to flo larvae. By contrast, flo mutants were not sensitized to agents that cause DNA double strand breaks, thus showing that loss of Elys disrupts responses to selected replication inhibitors. Elys binds Mcm2-7 complexes derived from Xenopus egg extracts. Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine. These in vivo data indicate a role for Elys in Mcm2-chromatin interactions. Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress

Redundant Mechanisms Prevent Mitotic Entry Following Replication Arrest in the Absence of Cdc25 Hyper-Phosphorylation in Fission Yeast

Following replication arrest the Cdc25 phosphatase is phosphorylated and inhibited by Cds1. It has previously been reported that expressing Cdc25 where 9 putative amino-terminal Cds1 phosphorylation sites have been substituted to alanine results in bypass of the DNA replication checkpoint. However, these results were acquired by expression of the phosphorylation mutant using a multicopy expression vector in a genetic background where the DNA replication checkpoint is intact. In order to clarify these results we constructed a Cdc25(9A)-GFP native promoter integrant and examined its effect on the replication checkpoint at endogenous expression levels. In this strain the replication checkpoint operates normally, conditional on the presence of the Mik1 kinase. In response to replication arrest the Cdc25(9A)-GFP protein is degraded, suggesting the presence of a backup mechanism to eliminate the phosphatase when it cannot be inhibited through phosphorylation

Phosphorylation of the MBF Repressor Yox1p by the DNA Replication Checkpoint Keeps the G1/S Cell-Cycle Transcriptional Program Active

Background: In fission yeast Schizosaccharomyces pombe G1/S cell-cycle regulated transcription depends upon MBF. A negative feedback loop involving Nrm1p and Yox1p bound to MBF leads to transcriptional repression as cells exit G1 phase. However, activation of the DNA replication checkpoint response during S phase results in persistent expression of MBF-dependent genes.Methodology/Principal Findings: This report shows that Yox1p binding to MBF is Nrm1-dependent and that Yox1p and Nrm1p require each other to bind and repress MBF targets. In response to DNA replication stress both Yox1p and Nrm1p dissociate from MBF at promoters leading to de-repression of MBF targets. Inactivation of Yox1p is an essential part of the checkpoint response. Cds1p (human Chk2p) checkpoint protein kinase-dependent phosphorylation of Yox1p promotes its dissociation from the MBF transcription factor. We establish that phosphorylation of Yox1p at Ser114, Thr115 is required for maximal checkpoint-dependent activation of the G1/S cell-cycle transcriptional program.Conclusions/Significance: This study shows that checkpoint-dependent phosphorylation of Yox1p at Ser114, Thr115 results in de-repression of the MBF transcriptional program. The remodeling of the cell cycle transcriptional program by the DNA replication checkpoint is likely to comprise an important mechanism for the avoidance of genomic instability

Purification and analysis of checkpoint protein complexes from Saccharomyces cerevisiae.

The DNA damage-dependent checkpoint of Saccharomyces cerevisiae is a paradigm for eukaryotic checkpoint pathways that regulate cell cycle progression in the presence of insults to the genetic material. In order to better understand this pathway, we undertook a biochemical study of the proteins implicated in its functioning. Analysis of the hydrodynamic properties of a protein in a crude mixture can give insights into possible tertiary organization such as participation in high-molecular-mass protein complexes. We here describe the determination of Stokes radius and sedimentation coefficients for the Rad24 protein, which enabled us to predict that this protein was a component of a protein complex in crude yeast extracts. This led us to develop a protocol to purify this complex to homogeneity in order to determine the component proteins. The methods described here should be applicable to the hydrodynamic analysis and subsequent purification of any soluble protein from organisms amenable to genetic manipulation, such as yeast, as long as the function of that protein is not perturbed by the addition of an epitope tag

Budding yeast Rad9 is an ATP-dependent Rad53 activating machine.

We find budding yeast Rad9 in two distinct, large, and soluble complexes in cell extracts. The larger (> or =850 kDa) complex, found in nondamaged cells, contains hypophosphorylated Rad9, whereas the smaller (560 kDa) complex, which forms after DNA damage, contains hyperphosphorylated Rad9 and Rad53. This smaller Rad9 complex is capable of catalyzing phosphorylation and release of active Rad53 kinase, a process requiring the kinase activity of Rad53. However, Mec1 and Tel1 are no longer required once the 560 kDa complex has been formed. We propose a model whereby Mec1/Tel1-dependent hyperphosphorylation of Rad9 results in formation of the smaller Rad9 complex and recruitment of Rad53. This complex then catalyzes activation of Rad53 by acting as a scaffold that brings Rad53 molecules into close proximity, facilitating Rad53 in trans autophosphorylation and subsequent release of activated Rad53

A novel Rad24 checkpoint protein complex closely related to replication factor C.

Rad24 functions in the DNA damage checkpoint pathway of Saccharomyces cerevisiae. Here, analysis of Rad24 in whole cell extracts demonstrated that its mass was considerably greater than its predicted molecular weight, suggesting that Rad24 is a component of a protein complex. The Rad24 complex was purified to homogeneity. In addition to Rad24, the complex included polypeptides of 40 kDa and 35 kDa. The 40 kDa species was found by mass spectrometry to contain Rfc2 and Rfc3, subunits of replication factor C (RFC), a five subunit protein that is required for the loading of polymerases onto DNA during replication and repair [3]. We hypothesised that other RFC subunits, all of which share sequence homologles with Rad24, might also be components of the Rad24 complex. Reciprocal co-immunoprecipitation studies were performed using extracts prepared from strains containing epitope-tagged RFC proteins. These experiments showed that the small RFC proteins, Rfc2, Rfc3, Rfc4 and Rfc5, interacted with Rad24, whereas the Rfc1 subunit did not. We suggest that this RFC-like Rad24 complex may function as a structure-specific sensor in the DNA damage checkpoint pathway