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As a result of exposure to stress conditions, mutations, or defects during synthesis, cellular proteins are prone to misfold. To cope with such partially denatured proteins, cells mount a regulated transcriptional response involving the Hsf1 transcription factor, which drives the synthesis of molecular chaperones and other stress-relieving proteins. Here, we show that the fission yeast Schizosaccharomyces pombe orthologues of human BAG-1, Bag101, and Bag102, are Hsp70 co-chaperones that associate with 26S proteasomes. Only a subgroup of Hsp70-type chaperones, including Ssa1, Ssa2, and Sks2, binds Bag101 and Bag102 and key residues in the Hsp70 ATPase domains, required for interaction with Bag101 and Bag102, were identified. In humans, BAG-1 overexpression is typically observed in cancers. Overexpression of bag101 and bag102 in fission yeast leads to a strong growth defect caused by triggering Hsp70 to release and activate the Hsf1 transcription factor. Accordingly, the bag101-linked growth defect is alleviated in strains containing a reduced amount of Hsf1 but aggravated in hsp70 deletion strains. In conclusion, we propose that the fission yeast UBL/BAG proteins release Hsf1 from Hsp70, leading to constitutive Hsf1 activation and growth defects.

Nucleotide synthesis is a universal response to DNA damage, but how this response facilitates DNA repair and cell survival is unclear. Here we establish a role for DNA damage-induced nucleotide synthesis in homologous recombination (HR) repair in fission yeast. Using a genetic screen, we found the Ddb1–Cul4Cdt2 ubiquitin ligase complex and ribonucleotide reductase (RNR) to be required for HR repair of a DNA double-strand break (DSB). The Ddb1–Cul4Cdt2 ubiquitin ligase complex is required for degradation of Spd1, an inhibitor of RNR in fission yeast. Accordingly, deleting spd1+ suppressed the DNA damage sensitivity and the reduced HR efficiency associated with loss of ddb1+ or cdt2+. Furthermore, we demonstrate a role for nucleotide synthesis in postsynaptic gap filling of resected ssDNA ends during HR repair. Finally, we define a role for Rad3 (ATR) in nucleotide synthesis and HR through increasing Cdt2 nuclear levels in response to DNA damage. Our findings support a model in which break-induced Rad3 and Ddb1–Cul4Cdt2 ubiquitin ligase-dependent Spd1 degradation and RNR activation promotes postsynaptic ssDNA gap filling during HR repair.

Cullin‐4 forms a scaffold for multiple ubiquitin ligases. In Schizosaccharomyces pombe , the Cullin‐4 homologue (Pcu4) physically associates with Ddb1 and the COP9 signalosome (CSN). One target of this complex is Spd1. Spd1 regulates ribonucleotide reductase (RNR) activity. Spd1 degradation during S phase, or following DNA damage of G2 cells, results in the nuclear export of the small RNR subunit. We demonstrate that Cdt2, an unstable WD40 protein, is a regulatory subunit of Pcu4–Ddb1–CSN ubiquitin ligase. cdt2 deletion stabilises Spd1 and prevents relocalisation of the small RNR subunit from the nucleus to the cytoplasm. cdt2 + is periodically transcribed by the Cdc10/DSC1 transcription factor during S phase and transiently transcribed following DNA damage of G2 cells, corresponding to Spd1 degradation profiles. Cdt2 co‐precipitates with Spd1, and Cdt2 overexpression results in constitutive Spd1 degradation. We propose that Cdt2 incorporation into the Pcu4–Ddb1–CSN complex prompts Spd1 targeting and subsequent degradation and that Cdt2 is a WD40 repeat adaptor protein for Cullin‐4‐based ubiquitin ligase.

The human UV-damaged DNA-binding protein Ddb1 associates with cullin 4 ubiquitin ligases implicated in nucleotide excision repair (NER). These complexes also contain the signalosome (CSN), but NER-relevant ubiquitination targets have not yet been identified. We report that fission yeast Ddb1, Cullin 4 (Pcu4), and CSN subunits Csn1 and Csn2 are required for degradation of the ribonucleotide reductase (RNR) inhibitor protein Spd1. Ddb1-deficient cells have >20-fold increased spontaneous mutation rate. This is partly dependent on the error-prone translesion DNA polymerases. Spd1 deletion substantially reduced the mutation rate, suggesting that insufficient RNR activity accounts for ∼50% of observed mutations. Epistasis analysis indicated that Ddb1 contributed to mutation avoidance and tolerance to DNA damage in a pathway distinct from NER. Finally, we show that Ddb1/Csn1/Cullin 4-mediated Spd1 degradation becomes essential when cells differentiate into meiosis. These results suggest that Ddb1, along with Cullin 4 and the signalosome, constitute a major pathway controlling genome stability, repair, and differentiation via RNR regulation.

The protein called p97 in mammals and Cdc48 in budding and fission yeast is a homo-hexameric, ring-shaped, ubiquitin-dependent ATPase complex involved in a range of cellular functions, including protein degradation, vesicle fusion, DNA repair, and cell division. The cdc48+ gene is essential for viability in fission yeast, and point mutations in the human orthologue have been linked to disease. To analyze the function of p97/Cdc48 further, we performed a screen for cold-sensitive suppressors of the temperature-sensitive cdc48-353 fission yeast strain. In total, 29 independent pseudo revertants that had lost the temperature-sensitive growth defect of the cdc48-353 strain were isolated. Of these, 28 had instead acquired a cold-sensitive phenotype. Since the suppressors were all spontaneous mutants, and not the result of mutagenesis induced by chemicals or UV irradiation, we reasoned that the genome sequences of the 29 independent cdc48-353 suppressors were most likely identical with the exception of the acquired suppressor mutations. This prompted us to test if a whole genome sequencing approach would allow us to map the mutations. Indeed genome sequencing unambiguously revealed that the cold-sensitive suppressors were all second site intragenic cdc48 mutants. Projecting these onto the Cdc48 structure revealed that while the original temperature-sensitive G338D mutation is positioned near the central pore in the hexameric ring, the suppressor mutations locate to subunit-subunit and inter-domain boundaries. This suggests that Cdc48-353 is structurally compromized at the restrictive temperature, but re-established in the suppressor mutants. The last suppressor was an extragenic frame shift mutation in the ufd1 gene, which encodes a known Cdc48 co-factor. In conclusion, we show, using a novel whole genome sequencing approach, that Cdc48-353 is structurally compromized at the restrictive temperature, but stabilized in the suppressors.

In yeasts, small intrinsically disordered proteins (IDPs) modulate ribonucleotide reductase (RNR) activity to ensure an optimal supply of dNTPs for DNA synthesis. The Schizosaccharomyces pombe Spd1 protein can directly inhibit the large RNR subunit (R1), import the small subunit (R2) into the nucleus and induce an architectural change in the R1-R2 holocomplex. Here, we report the characterization of Spd2, a protein with sequence similarity to Spd1. We show that Spd2 is a CRL4(Cdt2)-controlled IDP that functions together with Spd1 in the DNA damage response and in modulation of RNR architecture. However, Spd2 does not regulate dNTP pools and R2 nuclear import. Furthermore, deletion of spd2 only weakly suppresses the Rad3(ATR) checkpoint dependency of CRL4(Cdt2) mutants. However, when we raised intracellular dNTP pools by inactivation of RNR feedback inhibition, deletion of spd2 could suppress the checkpoint dependency of CRL4(Cdt2) mutant cells to the same extent as deletion of spd1. Collectively, these observations suggest that Spd1 on its own regulates dNTP pools, whereas in combination with Spd2 it modulates RNR architecture and sensitizes cells to DNA damage.

Cullin4, Ddb1 and Cdt2 are core subunits of the ubiquitin ligase complex CRL4(Cdt2), which controls genome stability by targeting Spd1 for degradation during DNA replication and repair in fission yeast. Spd1 has an inhibitory effect on ribonucleotide reductase (RNR), the activity of which is required for deoxynucleotide (dNTP) synthesis. The failure to degrade Spd1 in mutants where CRL4(Cdt2) is defective leads to DNA integrity checkpoint activation and dependency. This correlates with a lower dNTP pool. Pools are restored in a spd1-deleted background and this also suppresses checkpoint activation and dependency. We hypothesized that fission yeast with RNR hyperactivity would display a mutator phenotype on their own, but also possibly repress aspects of the phenotype associated with the inability to target Spd1 for degradation. Here, we report that a mutation in the R1 subunit of ribonucleotide reductase cdc22 (cdc22-D57N), which alleviated allosteric feedback inhibition, caused a highly elevated dNTP pool that was further increased by deleting spd1. The Δspd1 cdc22-D57N double mutant had elevated mutation rates and was sensitive to damaging agents that cause DNA strand breaks, demonstrating that Spd1 can protect the genome when dNTP pools are high. In ddb1-deleted cells, cdc22-D57N also potently elevated RNR activity, but failed to allow cell growth independently of the intact checkpoint. Our results provide evidence that excess Spd1 interferes with other functions in addition to its inhibitory effect on ribonucleotide reduction to generate replication stress and genome instability.

In both Schizosaccharomyces pombe and Saccharomyces cerevisiae, Mms22 and Mms1 form a complex with important functions in the response to DNA damage, loss of which leads to perturbations during replication. Furthermore, in S. cerevisiae, Mms1 has been suggested to function in concert with a Cullin-like protein, Rtt101/Cul8, a potential paralog of Cullin 4. We performed epistasis analysis between Δmms1 and mutants of pathways with known functions in genome integrity, and measured the recruitment of homologous recombination proteins to blocked replication forks and recombination frequencies. We show that, in S. pombe, the functions of Mms1 and the conserved components of the Cullin 4 ubiquitin ligase, Pcu4 and Ddb1, do not significantly overlap. Furthermore, unlike in S. cerevisiae, the function of the H3K56 acetylase Rtt109 is not essential for Mms1 function. We provide evidence that Mms1 function is particularly important when a single strand break is converted into a double strand break during replication. Genetic data connect Mms1 to a Mus81 and Rad22(Rad52) dependent, but Rhp51 independent, branch of homologous recombination. This is supported by results demonstrating that Mms1 is recruited to a site-specific replication fork barrier and that, in a Δmms1 strain, Rad22(Rad52) and RPA recruitment to blocked forks are reduced, whereas Rhp51 recruitment is unaffected. In addition, Mms1 appears to specifically promote chromosomal rearrangements in a recombination assay. These observations suggest that Mms1 acts to channel repair of perturbed replication into a particular sub-pathway of homologous recombination.

The correct levels of deoxyribonucleotide triphosphates and their relative abundance are important to maintain genomic integrity. Ribonucleotide reductase (RNR) regulation is complex and multifaceted. RNR is regulated allosterically by two nucleotide-binding sites, by transcriptional control, and by small inhibitory proteins that associate with the R1 catalytic subunit. In addition, the subcellular localization of the R2 subunit is regulated through the cell cycle and in response to DNA damage. We show that the fission yeast small RNR inhibitor Spd1 is intrinsically disordered and regulates R2 nuclear import, as predicted by its relationship to Saccharomyces cerevisiae Dif1. We demonstrate that Spd1 can interact with both R1 and R2, and show that the major restraint of RNR in vivo by Spd1 is unrelated to R2 subcellular localization. Finally, we identify a new behavior for RNR complexes that potentially provides yet another mechanism to regulate dNTP synthesis via modulation of RNR complex architecture.

Background Fission yeast cells undergo sexual differentiation in response to nitrogen starvation. In this process haploid M and P cells first mate to form diploid zygotes, which then enter meiosis and sporulate. Prior to mating, M and P cells communicate with diffusible mating pheromones that activate a signal transduction pathway in the opposite cell type. The pheromone signalling orchestrates mating and is also required for entry into meiosis. Results Here we use DNA microarrays to identify genes that are induced by M-factor in P cells and by P-factor in M-cells. The use of a cyr1 genetic background allowed us to study pheromone signalling independently of nitrogen starvation. We identified a total of 163 genes that were consistently induced more than two-fold by pheromone stimulation. Gene disruption experiments demonstrated the involvement of newly discovered pheromone-induced genes in the differentiation process. We have mapped Gene Ontology (GO) categories specifically associated with pheromone induction. A direct comparison of the M- and P-factor induced expression pattern allowed us to identify cell-type specific transcripts, including three new M-specific genes and one new P-specific gene. Conclusion We found that the pheromone response was very similar in M and P cells. Surprisingly, pheromone control extended to genes fulfilling their function well beyond the point of entry into meiosis, including numerous genes required for meiotic recombination. Our results suggest that the Ste11 transcription factor is responsible for the majority of pheromone-induced transcription. Finally, most cell-type specific genes now appear to be identified in fission yeast.

Synthesis of dNTPs is required for both DNA replication and DNA repair and is catalyzed by ribonucleotide reductases (RNR), which convert ribonucleotides to their deoxy forms [1, 2]. Maintaining the correct levels of dNTPs for DNA synthesis is important for minimising the mutation rate [3-7], and this is achieved by tight regulation of ribonucleotide reductase [2, 8, 9]. In fission yeast, ribonucleotide reductase is regulated in part by a small protein inhibitor, Spd1, which is degraded in S phase and after DNA damage to allow up-regulation of dNTP supply [10-12]. Spd1 degradation is mediated by the activity of the CRL4Cdt2 ubiquitin ligase complex [5, 13, 14]. This has been reported to be dependent on modulation of Cdt2 levels which are cell cycle regulated, peaking in S phase, and which also increase after DNA damage in a checkpoint-dependent manner [7, 13]. We show here that Cdt2 levels fluctuations are not sufficient to regulate Spd1 proteolysis and that the key step in this event is the interaction of Spd1 with the polymerase processivity factor PCNA, complexed onto DNA. This mechanism thus provides a direct link between DNA synthesis and ribonucleotide reductase regulation.