An acetylated form of histone H2A.Z regulates chromosome architecture in Schizosaccharomyces pombe

Histone variant H2A.Z has a conserved role in genome stability, although it remains unclear how this is mediated. Here we demonstrate that the fission yeast Swr1 ATPase inserts H2A.Z (Pht1) into chromatin and Kat5 acetyltransferase (Mst1) acetylates it. Deletion or an unacetylatable mutation of Pht1 leads to genome instability, primarily caused by chromosome entanglement and breakage at anaphase. This leads to the loss of telomere-proximal markers, though telomere protection and repeat length are unaffected by the absence of Pht1. Strikingly, the chromosome entanglement in pht1Delta anaphase cells can be rescued by forcing chromosome condensation before anaphase onset. We show that the condensin complex, required for the maintenance of anaphase chromosome condensation, prematurely dissociates from chromatin in the absence of Pht1. This and other findings suggest an important role for H2A.Z in the architecture of anaphase chromosomes

CMM-Based Appraisal for Internal Process Improvement (CBA IPI) Version 1.2 Method Description

This document is a high-level overview of the Capability Maturity Model (CMM)-Based Appraisal for Internal Process Improvement (CBA IPI) V1.2 assessment method and is an update to the CBA IPI V1.1 Method Description. It provides a brief history of SEI appraisal methods, and establishes appraisals in the context of the IDEAL approach to software process improvement. CBA IPI is a diagnostic tool that supports, enables, and encourages an organization's commitment to process improvement. The method helps an organization to gain insight into its software development capability by identifying strengths and weaknesses of its current processes related to the Capability Maturity Model for Software V1.1. The method focuses on identifying software improvements that are most beneficial, given an organization's business goals and current maturity level. Brief descriptions of the method activities, roles, and responsibilities are provided. In addition, guidelines are provided for establishing resource requirements for conducting a CBA IPI. The SEI Appraiser Program is discussed, detailing the requirements for persons qualified to lead CBA IPIs

The Unique Evolutionary Distribution of Eukaryotic Elongation Factor 3

Translation, the mechanism by which proteins are synthesized based on the information encoded in mRNA, is an essential process in all living organisms. Consisting of initiation, elongation and termination phases, many aspects of this process are conserved across bacteria and eukaryotes. The elongation phase, in particular, has several well-conserved steps and universally requires two protein elongation (EF) factors. However, fungal translation elongation was determined to be unique in its absolute requirement for a third factor, the ATPase eEF3. While the exact function of eEF3 is unclear, eEF3 binds close to the E-site of the ribosome and has been proposed to facilitate the removal of deacylated tRNA from the E-site. Originally described as a “fungal-specific factor,” recent bioinformatic analysis of eEF3 distribution challenges this designation as eEF3-like proteins are found in other lower order eukaryotes. In agreement with its role as an ATPase, all the putative eEF3 homologs identified have two ABC domains. Critical residues of the two ABC domains involved in nucleotide binding and hydrolysis were highly conserved in all the putative eEF3 homologs identified, supporting the functional role of the homologs as ATPases. The HEAT and chromodomain regions, both of which have been implicated in ribosomal interactions, are less conserved than the ABC domains. Further analysis of these putative eEF3s may facilitate the elucidation of the critical functions of eEF3 in translation elongation and shed light on how the protein synthesis machinery evolved from bacteria to fungi to higher eukaryotes

The Schizosaccharomyces pombe Checkpoint Kinases Chk1 and Cds1 Are Important for Cell Survival in Response to Cisplatin

Background: DNA damage checkpoints insure that the integrity of genomic DNA is faithfully maintained throughout the eukaryotic cell cycle. In the presence of damaged DNA, checkpoints are triggered to delay cell cycle progression to allow for DNA repair. In fission yeast, the kinases Chk1 and Cds1 are major components of these DNA damage checkpoint pathways. Both Chk1 and Cds1 are important for viability in the presence of several DNA damaging agents. In this study we hypothesized that Chk1 and Cds1 play a vital role in fission yeast cells ability to survive exposure to the DNA damaging agent cisplatin. Cisplatin is a potent chemotherapeutic drug that interacts with DNA and causes both inter- and intra-strand DNA cross-links. Methodology/Principal Findings: Here, we demonstrated that treatment with cisplatin in fission yeast causes a Chk1dependent DNA damage signal. chk1 2 cells were sensitive to cisplatin and Chk1 was phosphorylated in response to cisplatin treatment. We also showed that a Chk1-dependent DNA damage checkpoint pathway is activated in a dosedependent fashion in cells challenged with cisplatin. Furthermore the Cds1 checkpoint kinase was also important for viability in cisplatin challenged cells. In cds1 2 cells, cisplatin treatment reduced cell viability and this phenotype was exacerbated in a chk1 2 /cds1 2 background. Conclusions/Significance: Thus, we conclude that the concerted effort of both major checkpoint kinases in fission yeast

The S. cerevisiae Rrm3p DNA helicase moves with the replication forkand affects replication of allyeast chromosomes

The Saccharomyces cerevisiae DNA helicase Rrm3p is needed for normal fork progression through >1000 discrete sites scattered throughout the genome. Here we show that replication of all yeast chromosomes was markedly delayed in rrm3 cells. Delayed replication was seen even in a region that lacks any predicted Rrm3p-dependent sites. Based on the pattern of replication intermediates in two-dimensional gels, the rate of fork movement in rrm3 cells appeared similar to wild-type except at known Rrm3p-dependent sites. These data suggest that although Rrm3p has a global role in DNA replication, its activity is needed only or primarily at specific, difficult-to-replicate sites. By the criterion of chromatin immunoprecipitation, Rrm3p was associated with both Rrm3p-dependent and -independent sites, and moved with the replication fork through both. In addition, Rrm3p interacted with Pol2p, the catalytic subunit of DNA polymerase ε, in vivo. Thus, rather than being recruited to its sites of action when replication forks stall at these sites, Rrm3p is likely a component of the replication fork apparatus

Demonstration of Translation Elongation Factor 3 Activity From a Non-Fungal Species, Phytophthora infestans

In most eukaryotic organisms, translation elongation requires two highly conserved elongation factors eEF1A and eEF2. Fungal systems are unique in requiring a third factor, the eukaryotic Elongation Factor 3 (eEF3). For decades, eEF3, a ribosome-dependent ATPase, was considered “fungal-specific”, however, recent bioinformatics analysis indicates it may be more widely distributed among other unicellular eukaryotes. In order to determine whether divergent eEF3-like proteins from other eukaryotic organisms can provide the essential functions of eEF3 in budding yeast, the eEF3-like proteins from Schizosaccharomyes pombe and an oomycete, Phytophthora infestans, were cloned and expressed in Saccharomyces cerevisiae. Plasmid shuffling experiments showed that both S. pombe and P. infestans eEF3 can support the growth of S. cerevisiae in the absence of endogenous budding yeast eEF3. Consistent with its ability to provide the essential functions of eEF3, P. infestans eEF3 possessed ribosome-dependent ATPase activity. Yeast cells expressing P. infestans eEF3 displayed reduced protein synthesis due to defects in translation elongation/termination. Identification of eEF3 in divergent species will advance understanding of its function and the ribosome specific determinants that lead to its requirement as well as contribute to the identification of functional domains of eEF3 for potential drug discovery

Cisplatin causes Chk1 to become phosporylated and activates a Chk1-dependent DNA damage checkpoint.

<p>A and B. Cultures from <i>chk1Δ</i>, <i>chk1-HA</i>, <i>rad3Δchk1-HA</i>, and <i>rad1-1chk1-HA</i> were grown to mid-log phase in YEA media at 30°C and treated with 200 µM cisplatin for one hour then harvested and analysed by western blot analysis with an antibody directed against the HA epitope tag at the C- terminus of Chk1. C. The chk1cdc25-22 and chk1Δcdc25-22 strains were grown to mid-log phase at 25°C and shifted to 36.5°C for 3 hours to synchronize cells in G2 phase of the cell cycle. This was followed by treatment with either 100 µM or 200 µM cisplatin for one hour at 36.5°C and then the strains were released to 25°C to permit cycling. The percentage of binulceate cells was scored over a 4 hour time period as an indication of cells progressing into and thru mitosis.</p

Deletion of <i>chk1⁺</i> results in cisplatin sensitivity.

<p>A. The wildtype, <i>chk1Δ</i>, and <i>rad3Δ</i> cells were grown to mid-log phase in YEA media, treated with 200 µM cisplatin for 1 hour, resuspended to 1×10⁸cells/ml and serially diluted and spotted on YEA plates. Plates were photographed after three days of growth at 30°C. B. The wildtype and <i>chk1Δ</i> cells transformed with an either empty pSP1 or pSP1-<i>chk1⁺</i> were grown to mid-log phase in SC-leu media, treated with 200 µM cisplatin for 1 hour, resuspended to 1×10⁷cells/ml and serially diluted and spotted on SC-leu plates. Plates were photographed after four days of growth at 30°C. C. The wildtype, <i>chk1Δ</i>, and <i>rad3Δ</i> cells were grown to mid-log phase in YEA media, treated with either 100 µM or 200 µM cisplatin for 1 hour and cells plated in triplicate on YEA and incubated for 3 days. Mean survival was calculated as a percentage of colonies appearing on untreated plates. Data represented here is typical of one of 3 independent experiments. Error bars represent standard deviation of the mean.</p

Both Chk1 and Cds1 are required to maintain cell viability in the presence of cisplatin induced damage.

<p>A. The wildtype, <i>chk1Δ</i>, <i>cds1Δ</i>, <i>chk1Δcds1Δ</i> and <i>rad3Δ</i> cells were grown to mid-log phase in YEA media, treated with 200 µM cisplatin for 1 hour, resuspended to 1×10⁷cells/ml and serially diluted and spotted on YEA plates. Plates were photographed after three days of growth at 30°C. B. The wildtype, <i>chk1Δ</i>, <i>cds1Δ</i>, <i>chk1ΔcdsΔ</i>, and <i>rad3Δ</i> cells were grown to mid-log phase in YEA media, treated with or 200 µM cisplatin for 1 hour and cells plated in triplicate on YEA and incubated for 3 days. Mean survival was calculated as a percentage of colonies appearing on untreated plates. Data represented here is typical of one of 2 independent experiments. Error bars represent standard deviation of the mean.</p