Break-induced ATR and Ddb1-Cul4(Cdt)² ubiquitin ligase-dependent nucleotide synthesis promotes homologous recombination repair in fission yeast.

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-Cul4(Cdt)² ubiquitin ligase complex and ribonucleotide reductase (RNR) to be required for HR repair of a DNA double-strand break (DSB). The Ddb1-Cul4(Cdt)² 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-Cul4(Cdt)² ubiquitin ligase-dependent Spd1 degradation and RNR activation promotes postsynaptic ssDNA gap filling during HR repair.

The DNA damage checkpoint pathway promotes extensive resection and nucleotide synthesis to facilitate homologous recombination repair and genome stability in fission yeast.

DNA double-strand breaks (DSBs) can cause chromosomal rearrangements and extensive loss of heterozygosity (LOH), hallmarks of cancer cells. Yet, how such events are normally suppressed is unclear. Here we identify roles for the DNA damage checkpoint pathway in facilitating homologous recombination (HR) repair and suppressing extensive LOH and chromosomal rearrangements in response to a DSB. Accordingly, deletion of Rad3(ATR), Rad26ATRIP, Crb2(53BP1) or Cdc25 overexpression leads to reduced HR and increased break-induced chromosome loss and rearrangements. We find the DNA damage checkpoint pathway facilitates HR, in part, by promoting break-induced Cdt2-dependent nucleotide synthesis. We also identify additional roles for Rad17, the 9-1-1 complex and Chk1 activation in facilitating break-induced extensive resection and chromosome loss, thereby suppressing extensive LOH. Loss of Rad17 or the 9-1-1 complex results in a striking increase in break-induced isochromosome formation and very low levels of chromosome loss, suggesting the 9-1-1 complex acts as a nuclease processivity factor to facilitate extensive resection. Further, our data suggest redundant roles for Rad3ATR and Exo1 in facilitating extensive resection. We propose that the DNA damage checkpoint pathway coordinates resection and nucleotide synthesis, thereby promoting efficient HR repair and genome stability.

Failed gene conversion leads to extensive end processing and chromosomal rearrangements in fission yeast.

Loss of heterozygosity (LOH), a causal event in cancer and human genetic diseases, frequently encompasses multiple genetic loci and whole chromosome arms. However, the mechanisms by which such extensive LOH arises, and how it is suppressed in normal cells is poorly understood. We have developed a genetic system to investigate the mechanisms of DNA double-strand break (DSB)-induced extensive LOH, and its suppression, using a non-essential minichromosome, Ch(16), in fission yeast. We find extensive LOH to arise from a new break-induced mechanism of isochromosome formation. Our data support a model in which Rqh1 and Exo1-dependent end processing from an unrepaired DSB leads to removal of the broken chromosome arm and to break-induced replication of the intact arm from the centromere, a considerable distance from the initial lesion. This process also promotes genome-wide copy number variation. A genetic screen revealed Rhp51, Rhp55, Rhp57 and the MRN complex to suppress both isochromosome formation and chromosome loss, in accordance with these events resulting from extensive end processing associated with failed homologous recombination repair.

Saving your rookie managers from themselves.

Most organizations promote employees into managerial positions based on their technical competence. But very often, that kind of competence does not translate into good managerial performance. Many rookie managers fail to grasp how their roles have changed: that their jobs are no longer about personal achievement but about enabling others to achieve, that sometimes driving the bus means taking a backseat, and that building a team is often more important than cutting a deal. Even the best employees have trouble adjusting to these new realities, and that trouble can be exacerbated by the normal insecurities that may make rookie managers hesitant to ask for help. The dynamic unfolds something like this: As rookie managers internalize their stress, their focus, too, becomes increasingly internal. They become insecure and self-focused and cannot properly support their teams. Invariably, trust breaks down, staff members become alienated, and productivity suffers. In this article, coach and management consultant Carol Walker, who works primarily with rookie managers and their supervisors, addresses the five problem areas that rookie managers typically face: delegating, getting support from senior staffers, projecting confidence, thinking strategically, and giving feedback. You may think these elements sound like Management 101, and you'd be right, Walker writes. But these basic elements are also what trip up most managers in the early stages of their careers (and even, she admits, throughout their careers). The bosses of rookie managers have a responsibility to anticipate and address these problems; not doing so will hurt the rookie, the boss, and the company overall.

Stress-activated protein kinase pathway functions to support protein synthesis and translational adaptation in response to environmental stress in fission yeast.

The stress-activated protein kinase (SAPK) pathway plays a central role in coordinating gene expression in response to diverse environmental stress stimuli. We examined the role of this pathway in the translational response to stress in Schizosaccharomyces pombe. Exposing wild-type cells to osmotic stress (KCl) resulted in a rapid but transient reduction in protein synthesis. Protein synthesis was further reduced in mutants disrupting the SAPK pathway, including the mitogen-activated protein kinase Wis1 or the mitogen-activated protein kinase Spc1/Sty1, suggesting a role for these stress response factors in this translational control. Further polysome analyses revealed a role for Spc1 in supporting translation initiation during osmotic stress, and additionally in facilitating translational adaptation. Exposure to oxidative stress (H2O2) resulted in a striking reduction in translation initiation in wild-type cells, which was further reduced in spc1- cells. Reduced translation initiation correlated with phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) in wild-type cells. Disruption of Wis1 or Spc1 kinase or the downstream bZip transcription factors Atf1 and Pap1 resulted in a marked increase in eIF2alpha phosphorylation which was dependent on the eIF2alpha kinases Hri2 and Gcn2. These findings suggest a role for the SAPK pathway in supporting translation initiation and facilitating adaptation to environmental stress in part through reducing eIF2alpha phosphorylation in fission yeast.

Saving your rookie managers from themselves.

Rookie managers have a real knack for allowing immediate tasks to overshadow overarching initiatives. You've promoted your star performer into management. Now help him avoid the classic errors that beginners so often make.

STAR Day: one hospital's solution to educational challenges.

To answer the challenge of 24-hour staffing coupled with required contact hour requirements, an all-day educational program is offered to all 1,300 employees of this suburban Level II trauma center. Providing flexible session attendance options while retaining the advantages of interpersonal adult learning make this a useful option for the continuing education of hospital staff.