A genetic screen for replication initiation defective (rid) mutants in Schizosaccharomyces pombe

In fission yeast the intra-S phase and DNA damage checkpoints are activated in response to inhibition of DNA replication or DNA damage, respectively. The intra-S phase checkpoint responds to stalled replication forks leading to the activation of the Cds1 kinase that both delays cell cycle progression and stabilizes DNA replication forks. The DNA damage checkpoint, that operates during the G2 phase of the cell cycle delays mitotic progression through activation of the checkpoint kinase, Chk1. Delay of the cell cycle is believed to be essential to allow time for either replication restart (in S phase) or DNA damage repair (in G2). Previously, our laboratory showed that fission yeast cells deleted for the N-terminal half of DNA polymerase ε (Cdc20) are delayed in S phase, but surprisingly require Chk1 rather than Cds1 to maintain cell viability. Several additional DNA replication mutants were then tested for their dependency on Chk1 or Cds1 when grown under semi-permissive temperatures. We discovered that mutants defective in DNA replication initiation are sensitive only to loss of Chk1, whilst mutations that inhibit DNA replication elongation are sensitive to loss of both Cds1 and Chk1. To confirm that the Chk1-sensitive, Cds1-insensitive phenotype (rid phenotype) is specific to mutants defective in DNA replication initiation, we completed a genetic screen for cell cycle mutants that require Chk1, but not Cds1 to maintain cell viability when grown at semi-permissive temperatures. Our screen identified two mutants, rid1-1 and rid2-1, that are defective in Orc1 and Mcm4, respectively. Both mutants show defects in DNA replication initiation consistent with our hypothesis that the rid phenotype is replication initiation specific. In the case of Mcm4, the mutation has been mapped to a highly conserved region of the protein that appears to be required for DNA replication initiation, but not elongation. Therefore, we conclude that the cellular response to inhibition of DNA replication initiation is distinct from blocking DNA replication elongation, and this difference can be exploited to identify mutants specifically defective in DNA replication initiation

Description of a new species of Oligosita Walker (Chalcidoidea: Trichogrammatidae), egg parasitoid of Balclutha brevis Lindberg (Homoptera: Cicadellidae) living on Pennisetum setaceum, from Italy

A new species of Oligosita Walker (Chalcidoidea: Trichogrammatidae), O. balcluthae Viggiani et Laudonia n. sp., is described as a parasitoid of the eggs of Balclutha brevis Lindberg (Homoptera: Cicadellidae) associated with crimson fountain grass, Pennisetum setaceum (Poaceae) in Italy. Morphological features and biology of the new species are discussed and illustrated. The 28S-D2 and ITS2 regions were successfully amplified and sequenced

The CENP-B homolog, Abp1, interacts with the initiation protein Cdc23 (MCM10) and is required for efficient DNA replication in fission yeast

Abp1, and the closely related Cbh1 and Cbh2 are homologous to the human centromere-binding protein CENP-B that has been implicated in the assembly of centromeric heterochromatin. Fission yeast cells lacking Abp1 show an increase in mini-chromosome instability suggesting that Abp1 is important for chromosome segregation and/or DNA synthesis. Here we show that Abp1 interacts with the DNA replication protein Cdc23 (MCM10) in a two-hybrid assay, and that the Δabp1 mutant displays a synthetic phenotype with a cdc23 temperature-sensitive mutant. Moreover, genetic interactions were also observed between abp1(+ )and four additional DNA replication initiation genes cdc18(+), cdc21(+), orc1(+), and orc2(+). Interestingly, we find that S phase is delayed in cells deleted for abp1(+ )when released from a G1 block. However, no delay is observed when cells are released from an early S phase arrest induced by hydroxyurea suggesting that Abp1 functions prior to, or coincident with, the initiation of DNA replication

Elective cancer surgery in COVID-19-free surgical pathways during the SARS-CoV-2 pandemic: An international, multicenter, comparative cohort study

PURPOSE As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19–free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19–free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19–free surgical pathways. Patients who underwent surgery within COVID-19–free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19–free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score–matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19–free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION Within available resources, dedicated COVID-19–free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks

Elective Cancer Surgery in COVID-19-Free Surgical Pathways During the SARS-CoV-2 Pandemic: An International, Multicenter, Comparative Cohort Study.

PURPOSE: As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19-free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS: This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19-free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS: Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19-free surgical pathways. Patients who underwent surgery within COVID-19-free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19-free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score-matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19-free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION: Within available resources, dedicated COVID-19-free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks

Checkpoints in the cell cycle of fission yeast

When cell cycle progression in fission yeast is disrupted, checkpoint controls ensure that the normal sequence of cell cycle events is maintained. Activation of a checkpoint relies on monitoring signals that might involve assembly of macromolecular structures essential for specific cell cycle processes. The past year has seen further elucidation of two new checkpoints operating during the cell cycle of Schizosaccharomyces pombe. One involves the product of the ruml gene and prevents cells from entering mitosis from the pre-Start G 1 interval. The second checkpoint operates during the later stages of the cell cycle and is essential for coupling the events of mitosis and cell division

Programmed cell death in fission yeast

Abstract Recently a metacaspase, encoded by YCA1, has been implicated in a primitive form of apoptosis or programmed cell death in yeast. Previously it had been shown that over-expression of mammalian pro-apoptotic proteins can induce cell death in yeast, but the mechanism of how cell death occurred was not clearly established. More recently, it has been shown that DNA or oxidative damage, or other cell cycle blocks, can result in cell death that mimics apoptosis in higher cells. Also, in fission yeast deletion of genes required for triacylglycerol synthesis leads to cell death and expression of apoptotic markers. A metacaspase sharing greater than 40% identity to budding yeast Yca1 has been identified in fission yeast, however, its role in programmed cell death is not yet known. Analysis of the genetic pathways that influence cell death in yeast may provide insights into the mechanisms of apoptosis in all eukaryotic organisms

Identification and cloning of two putative subunits of DNA polymerase epsilon in fission yeast

DNA polymerase epsilon (Pol ε) is a multi-subunit enzyme required for the initiation of chromosomal DNA replication. Here, we report the cloning of two fission yeast genes, called dpb3 + and dpb4 + that encode proteins homologous to the two smallest subunits of Pol ε. Although Dpb4 is not required for cell viability, Δ dpb4 mutants are synthetically lethal with mutations in four genes required for DNA replication initiation, cdc20 + (encoding DNA Pol ε), cut5 + (homologous to DPB11/TopBP1), sna41 + (homologous to CDC45) and cdc21 + (encoding Mcm4, a component of the pre-replicative complex). In contrast to Dpb4, Dpb3 is essential for cell cycle progression. A glutathione S -transferase pull-down assay indicates that Dpb3 physically interacts with both Dpb2 and Dpb4, suggesting that Dpb3 associates with other members of the Pol ε complex. Depletion of Dpb3 leads to an accumulation of cells in S phase consistent with Dpb3 having a role in DNA replication. In addition, many of the cells have a bi-nucleate or multinucleate phenotype, indicating that cell separation is also inhibited. Finally, we have examined in vivo localization of green fluorescent protein (GFP)-tagged Dpb3 and Dpb4 and found that both proteins are localized to the nucleus consistent with their proposed role in DNA replication. However, in the absence of Dpb3, GFP-Dpb4 appears to be more dispersed throughout the cell, suggesting that Dpb3 may be important in establishing or maintaining normal localization of Dpb4

A novel RING-finger-like protein Ini1 is essential for cell cycle progression in fission yeast

We have cloned a fission yeast (Schizosaccharomyces pombe) homologue of Ini, a novel RING-finger-like protein recently identified in rat that interacts with the connexin43 (cx43) promoter and might be important for the response of the cx43 gene to estrogen. S. pombe cells deleted for ini1(+) fail to form colonies and arrest with an elongated cell phenotype, indicating a cell cycle block. Cell cycle arrest is dependent on expression of Wee1, but not Rad3, suggesting that it occurs independently of the DNA damage checkpoint control. Analysis of mRNA intermediates in cells depleted for Ini1 demonstrates that Ini1 is required for pre-mRNA splicing. We observe an accumulation of pre-mRNA for six of seven genes analysed, suggesting that Ini1 is required for general splicing activity. Interestingly, loss of Ini1 results in cell death that is partially suppressed by elimination of the Wee1 kinase. Therefore, Wee1 might promote cell death in the absence of Ini1

Control of cell polarity in fission yeast by association of Orb6p kinase with the highly conserved protein methyltransferase Skb1p

In the fission yeast Schizosaccharomyces pombe, proper establishment and maintenance of cell polarity require Orb6p, a highly conserved serine/threonine kinase involved in regulating both cell morphogenesis and cell cycle control. Orb6p localizes to the cell tips during interphase and to the cell septum during mitosis. To investigate the mechanisms involved in Orb6p function, we conducted a two-hybrid screen to identify proteins that interact with Orb6p. Using this approach, we identified Skb1p, a highly conserved protein methyltransferase that has been implicated previously in cell cycle control, in the coordination of cell cycle progression with morphological changes, and in hyperosmotic stress response. We found that Skb1p associates with Orb6p in S. pombe cells and that the two proteins interact directly in vitro. Loss of Skb1p exacerbates the phenotype of orb6 mutants, suggesting that Skb1p and Orb6p functionally interact in S. pombe cells. Our results suggest that Skb1p affects the intracellular localization of Orb6p and that loss of Skb1p leads to a redistribution of the Orb6p kinase away from the cell tips. Furthermore, we found that Orb6p kinase activity is strongly increased following exposure to salt shock, suggesting that Orb6p has a role in cell response to hyperosmotic stress. Previous studies have shown that Skb1p interacts with the fission yeast p21-activated kinase homologue Pak1p/Shk1p to regulate cell polarity and cell cycle progression. Our findings identify Orb6p as an additional target for Skb1p and suggest a novel function for Skb1p in the control of cell polarity by regulating the subcellular localization of Orb6p