Molecular dissection of the checkpoint kinase Hsl1p.
Ontology highlight
ABSTRACT:
SUBMITTER: Crutchley J
PROVIDER: S-EPMC2663927 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The essential yeast kinases Mec1 and Rad53, or human ATR and Chk1, are crucial for checkpoint responses to exogenous genotoxic agents, but why they are also required for DNA replication in unperturbed cells remains poorly understood. Here we report that even in the absence of DNA-damaging agents, the rad53-4AQ mutant, lacking the N-terminal Mec1 phosphorylation site cluster, is synthetic lethal with a deletion of the RAD9 DNA damage checkpoint adaptor. This phenotype is caused by an inability of rad53-4AQ to activate the downstream kinase Dun1, which then leads to reduced basal deoxynucleoside triphosphate (dNTP) levels, spontaneous replication fork stalling, and constitutive activation of and dependence on S phase DNA damage checkpoints. Surprisingly, the kinase-deficient rad53-K227A mutant does not share these phenotypes but is rendered inviable by additional phosphosite mutations that prevent its binding to Dun1. The results demonstrate that ultralow Rad53 catalytic activity is sufficient for normal replication of undamaged chromosomes as long as it is targeted toward activation of the effector kinase Dun1. Our findings indicate that the essential S phase function of Rad53 is comprised by the combination of its role in regulating basal dNTP levels and its compensatory kinase function if dNTP levels are perturbed.
| S-EPMC3753913 | biostudies-literature
Project description:Budding yeast asymmetric cell division relies upon the precise coordination of spindle orientation and cell cycle progression. The spindle position checkpoint (SPOC) is a surveillance mechanism that prevents cells with misoriented spindles from exiting mitosis. The cortical kinase Kin4 acts near the top of this network. How Kin4 kinase activity is regulated and maintained in respect to spindle positional cues remains to be established. Here, we show that the bud neck-associated kinase Elm1 participates in Kin4 activation and SPOC signaling by phosphorylating a conserved residue within the activation loop of Kin4. Blocking Elm1 function abolishes Kin4 kinase activity in vivo and eliminates the SPOC response to spindle misalignment. These findings establish a novel function for Elm1 in the coordination of spindle positioning with cell cycle progression via its control of Kin4.
| S-EPMC3101594 | biostudies-literature
Project description:The discovery of bacterial conductive structures, termed nanowires, has intrigued scientists for almost a decade. Nanowires enable bacteria to transfer electrons over micrometer distances to extracellular electron acceptors such as insoluble metal oxides or electrodes. Nanowires are pilus based and in Geobacter sulfurreducens are composed of the type IV pilin subunit PilA. Multiheme c-type cytochromes have been shown to attach to nanowire pili. Two hypotheses have been proposed for electron conduction in nanowires. The first (termed the metal-like conductivity or MLC hypothesis) claims that the pilus itself has the electron-conductive properties and the attached cytochromes mediate transfer to the final electron acceptor, whereas the second hypothesis (termed the superexchange conductivity or SEC hypothesis) suggests that electrons are "hopping" between heme groups in cytochromes closely aligned with the pilus as a scaffold. In their recent article in mBio, Vargas et al. [M. Vargas, N. S. Malvankar, P.-L. Tremblay, C. Leang, J. A. Smith, P. Patel, O. Snoeyenbos-West, K. P. Nevin, and D. R. Lovley, mBio 4(2):e00210-13, 2013] address this ambiguity through an analysis of strain Aro-5, a G. sulfurreducens PilA mutant lacking aromatic residues in the nonconserved portion of PilA. These residues were suspected of involvement in electron transport according to the MLC hypothesis. The G. sulfurreducens mutant had reduced conductive properties, lending important support to the MLC hypothesis. The data also highlight the need for further and more conclusive evidence for one or the other hypothesis.
| S-EPMC3663193 | biostudies-literature
Project description:The spindle checkpoint is a cell cycle surveillance system that ensures the fidelity of chromosome segregation. In mitosis, it elicits the "wait anaphase" signal to inhibit the anaphase-promoting complex or cyclosome until all chromosomes achieve bipolar microtubule attachment and align at the metaphase plate. Because a single kinetochore unattached to microtubules activates the checkpoint, the wait anaphase signal is thought to be generated by this kinetochore and is then amplified and distributed throughout the cell to inhibit the anaphase-promoting complex/cyclosome. Several spindle checkpoint kinases participate in the generation and amplification of this signal. Recent studies have begun to reveal the activation mechanisms of these checkpoint kinases. Increasing evidence also indicates that the checkpoint kinases not only help to generate the wait anaphase signal but also actively correct kinetochore-microtubule attachment defects.
| S-EPMC2708832 | biostudies-literature
Project description:Knowledge about an organismâs cell and tissue-specific transcriptional repertoire is essential for understanding the gene regulatory circuits that control key developmental events. The shoot apical meristem (SAM) is responsible for development of all the above ground parts of plants. Our understanding of SAM at the molecular level is far from complete. The present work investigates the global gene expression repertoire of SAMs in the garden pea (Pisum sativum). To this end, 10,346 EST sequences representing 7611 unique genes were generated from pea SAM cDNA libraries. These sequences, together with previously reported ESTs, were used to construct a 12K oligonucleotide array used to identify genes exhibiting differential SAM expression, as compared to the axillary meristem, root apical meristem, and non-meristematic tissues. We identified a number of genes that are predominantly expressed in specific cell layers or domains of the SAM, and thus are likely components of the gene networks involved in stem cell maintenance and initiation of lateral organ primordial cells. In situ hybridization confirmed the spatial localisation of some of these key genes within the SAM. Our data also indicate the diversification of some gene expression patterns and functions in legume crop plants. RNA samples were hybridized to two-colour Combimatrix arrays manufactured with a custom library of 11958 probes. The limma software package for R (Smyth, 2005) was used in subsequent statistical analysis of the data generated. On examination of the data during quality control assessment, it became evident that the Cy3 channel showed little dynamic range and background correction and normalisation could not correct for this. Therefore only the Cy5 channel was used in the subsequent analysis. This resulted in somewhat unbalanced sample sizes with six replicates of SAM and one each of RAM, AM and NM. Nevertheless, linear modelling and empirical Bayes moderated t-statistics (Smyth, 2004) used in the statistical analysis allows variance information to be borrowed across both samples and genes, resulting in reliable statistical inference even for the groups with only one sample.
2010-06-20 | E-GEOD-13451 | biostudies-arrayexpress
Project description:RNA expression data was generated as part of a colon cancer study. Samples were obtained from patients, including primary colon cancer, polyps, metastases, and matched normal mucosa (obtained from the margins of the resection). The RNA was extracted from tissue samples obtained from resections and hybridized to Affymetrix HG-U133 arrays. RNA expression data was also obtained for a few cell lines. notte-00422 Assay Type: Gene Expression Provider: Affymetrix Array Designs: HG-U133A Organism: Homo sapiens (ncbitax) Material Types: cell, total_RNA, synthetic_RNA, organism_part, whole_organism Disease States: colon cancer, colon polyp, Colon Carcinoma, colon adenocarcinoma, breast cancer, Normal, Prostate Adenocarcinoma
2015-05-02 | E-GEOD-68468 | biostudies-arrayexpress
Project description:The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation during mitosis by delaying the activation of the anaphase-promoting complex/cyclosome (APC/C) in response to unattached kinetochores. The Mad2 protein is essential for a functional checkpoint because it binds directly to Cdc20, the mitotic co-activator of the APC/C, thereby inhibiting progression into anaphase. Mad2 exists in at least 2 different conformations, open-Mad2 (O-Mad2) and closed-Mad2 (C-Mad2), with the latter representing the active form that is able to bind Cdc20. Our ability to dissect Mad2 biology in vivo is limited by the absence of monoclonal antibodies (mAbs) useful for recognizing the different conformations of Mad2. Here, we describe and extensively characterize mAbs specific for either O-Mad2 or C-Mad2, as well as a pan-Mad2 antibody, and use these to investigate the different Mad2 complexes present in mitotic cells. Our antibodies validate current Mad2 models but also suggest that O-Mad2 can associate with checkpoint complexes, most likely through dimerization with C-Mad2. Furthermore, we investigate the makeup of checkpoint complexes bound to the APC/C, which indicate the presence of both Cdc20-BubR1-Bub3 and Mad2-Cdc20-BubR1-Bub3 complexes, with Cdc20 being ubiquitinated in both. Thus, our defined mAbs provide insight into checkpoint signaling and provide useful tools for future research on Mad2 function and regulation.
| S-EPMC4966835 | biostudies-literature
Project description:The 2.1-kb cryptic plasmid pCIBAO89 from Bifidobacterium asteroides harbors a 1.4-kb segment which is sufficient for its autonomous replication. The segment is divided into two parts, the presumed replication origin, ori89, and the rep gene encoding the putative 41-kDa Rep89 replication initiation protein. This minimal replication region of pCIBAO89 was functionally dissected by transcriptional analyses as well as by DNA-binding studies, and the information obtained was exploited to create a number of Escherichia coli-Bifidobacterium shuttle vectors capable of transforming various bifidobacteria with an efficiency of up to 10(6) transformants/mug DNA.
| S-EPMC2168138 | biostudies-literature
Project description:Immunotherapeutics represent highly promising agents with the potential to improve patient outcomes in a variety of cancer types. Unfortunately, single-agent immunotherapy has achieved limited clinical benefit to date in patients suffering from pancreatic ductal adenocarcinoma (PDAC). This may be due to the presence of a uniquely immunosuppressive tumor microenvironment (TME) present in PDACs, which creates a barrier to effective immune surveillance. Critical obstacles to immunotherapy in PDAC tumors include the dense desmoplastic stroma that acts as a barrier to T-cell infiltration and the high numbers of tumor-associated immunosuppressive cells. We have identified hyperactivated focal adhesion kinase (FAK) activity in neoplastic PDAC cells as a significant regulator of the fibrotic and immunosuppressive TME. We found that FAK activity was elevated in human PDAC tissues and correlates with high levels of fibrosis and poor CD8+ cytotoxic T-cell infiltration. Single-agent FAK inhibition (VS-4718) dramatically limited tumor progression, resulting in a doubling of survival in the p48-Cre/LSL-KrasG12D/p53Flox/+ (KPC) mouse model of human PDAC. This alteration in tumor progression was associated with dramatically reduced tumor fibrosis, decreased numbers of tumor-infiltrating immature myeloid cells and immunosuppressive macrophages. We postulated that these desirable effects of FAK inhibition on the TME might render PDAC tumors more sensitive to immunotherapy. Accordingly, we found that VS-4718 rendered the previously unresponsive KPC mouse model responsive to anti-PD1 and anti-CTLA4 antagonists leading to a nearly tripling of survival times. These data suggest that FAK inhibition increases immune surveillance by overcoming the fibrotic and immunosuppressive PDAC TME thus rendering tumors more responsive to immunotherapy. We treated KP orthotopic tumor-bearing mice with vehicle and FAK inhibitor (FAKi) for 14 days, then extracted total RNA from tumor tissues.
2015-11-21 | E-GEOD-75233 | biostudies-arrayexpress
Project description:Immunoglobulin (Ig) diversification by somatic hypermutation in germinal center B cells is instrumental for maturation of the humoral immune response, but also bears the risk of excessive or aberrant genetic changes. Thus, introduction of DNA damage by activation-induced cytidine deaminase as well as DNA repair by multiple pathways need to be tightly regulated during the germinal center response to prevent lymphomagenesis. In the present study, we show that DNA damage checkpoint signaling via checkpoint kinase 1 (Chk1) negatively regulates somatic hypermutation. Chk1 inhibition in human B cell lymphoma lines as well as inactivation of Chk1 alleles by gene targeting in DT40 B cells leads to increased somatic hypermutation. This is apparently due to changes in DNA repair pathways regulated by Chk1, such as a decreased homologous recombination efficiency that also leads to decreased Ig gene conversion in DT40. Our data show that Chk1 signaling plays a crucial role in regulation of Ig diversification and sheds unexpected light on potential origins of aberrant somatic hypermutation in B cell lymphomagenesis.
| S-EPMC3973322 | biostudies-literature