March 13th, 2010 / No Comments » / by Mitchell DR
Polyglutamylation: The GLU that Makes Microtubules Sticky.
Curr Biol. 2010 Mar 9;20(5):R234-R236
Authors: Mitchell DR
The beating of cilia and flagella depends on microtubule sliding generated by dynein motors, but the interaction of these motors with their tracks is still under investigation. New evidence suggests that some dynein motors will not function properly unless their track has been modified by a specific post-translational modification.
PMID: 20219172 [PubMed - as supplied by publisher]
Posted in: Curr Biol
March 13th, 2010 / No Comments » / by Chan AC, Borts RH, Hoffmann E
Temperature-dependent modulation of chromosome segregation in msh4 mutants of budding yeast.
PLoS One. 2009;4(10):e7284
Authors: Chan AC, Borts RH, Hoffmann E
BACKGROUND: In many organisms, homologous chromosomes rely upon recombination-mediated linkages, termed crossovers, to promote their accurate segregation at meiosis I. In budding yeast, the evolutionarily conserved mismatch-repair paralogues, Msh4 and Msh5, promote crossover formation in conjunction with several other proteins, collectively termed the Synapsis Initiation Complex (SIC) proteins or 'ZMM's (Zip1-Zip2-Zip3-Zip4-Spo16, Msh4-Msh5, Mer3). zmm mutants show decreased levels of crossovers and increased chromosome missegregation, which is thought to cause decreased spore viability. PRINCIPAL FINDINGS: In contrast to other ZMM mutants, msh4 and msh5 mutants show improved spore viability and chromosome segregation in response to elevated temperature (23 degrees C versus 33 degrees C). Crossover frequencies in the population of viable spores in msh4 and msh5 mutants are similar at both temperatures, suggesting that temperature-mediated chromosome segregation does not occur by increasing crossover frequencies. Furthermore, meiotic progression defects at elevated temperature do not select for a subpopulation of cells with improved segregation. Instead, another ZMM protein, Zip1, is important for the temperature-dependent improvement in spore viability. CONCLUSIONS: Our data demonstrate interactions between genetic (zmm status) and environmental factors in determining chromosome segregation.
PMID: 19816584 [PubMed - indexed for MEDLINE]
Posted in: PLoS One
March 13th, 2010 / No Comments » / by Pagliuca C, Draviam VM, Marco E, Sorger PK, De Wulf P
Roles for the conserved spc105p/kre28p complex in kinetochore-microtubule binding and the spindle assembly checkpoint.
PLoS One. 2009;4(10):e7640
Authors: Pagliuca C, Draviam VM, Marco E, Sorger PK, De Wulf P
BACKGROUND: Kinetochores attach sister chromatids to microtubules of the mitotic spindle and orchestrate chromosome disjunction at anaphase. Although S. cerevisiae has the simplest known kinetochores, they nonetheless contain approximately 70 subunits that assemble on centromeric DNA in a hierarchical manner. Developing an accurate picture of the DNA-binding, linker and microtubule-binding layers of kinetochores, including the functions of individual proteins in these layers, is a key challenge in the field of yeast chromosome segregation. Moreover, comparison of orthologous proteins in yeast and humans promises to extend insight obtained from the study of simple fungal kinetochores to complex animal cell kinetochores. PRINCIPAL FINDINGS: We show that S. cerevisiae Spc105p forms a heterotrimeric complex with Kre28p, the likely orthologue of the metazoan kinetochore protein Zwint-1. Through systematic analysis of interdependencies among kinetochore complexes, focused on Spc105p/Kre28p, we develop a comprehensive picture of the assembly hierarchy of budding yeast kinetochores. We find Spc105p/Kre28p to comprise the third linker complex that, along with the Ndc80 and MIND linker complexes, is responsible for bridging between centromeric heterochromatin and kinetochore MAPs and motors. Like the Ndc80 complex, Spc105p/Kre28p is also essential for kinetochore binding by components of the spindle assembly checkpoint. Moreover, these functions are conserved in human cells. CONCLUSIONS/SIGNIFICANCE: Spc105p/Kre28p is the last of the core linker complexes to be analyzed in yeast and we show it to be required for kinetochore binding by a discrete subset of kMAPs (Bim1p, Bik1p, Slk19p) and motors (Cin8p, Kar3p), all of which are nonessential. Strikingly, dissociation of these proteins from kinetochores prevents bipolar attachment, even though the Ndc80 and DASH complexes, the two best-studied kMAPs, are still present. The failure of Spc105 deficient kinetochores to bind correctly to spindle microtubules and to recruit checkpoint proteins in yeast and human cells explains the observed severity of missegregation phenotypes.
PMID: 19893618 [PubMed - indexed for MEDLINE]
Posted in: PLoS One
March 13th, 2010 / No Comments » / by Nelissen H, De Groeve S, Fleury D, Neyt P, Bruno L, Bitonti MB, Vandenbussche F, Van der Straeten D, Yamaguchi T, Tsukaya H, Witters E, De Jaeger G, Houben A, Van Lijsebettens M
Plant Elongator regulates auxin-related genes during RNA polymerase II transcription elongation.
Proc Natl Acad Sci U S A. 2010 Jan 26;107(4):1678-83
Authors: Nelissen H, De Groeve S, Fleury D, Neyt P, Bruno L, Bitonti MB, Vandenbussche F, Van der Straeten D, Yamaguchi T, Tsukaya H, Witters E, De Jaeger G, Houben A, Van Lijsebettens M
In eukaryotes, transcription of protein-encoding genes is strongly regulated by posttranslational modifications of histones that affect the accessibility of the DNA by RNA polymerase II (RNAPII). The Elongator complex was originally identified in yeast as a histone acetyltransferase (HAT) complex that activates RNAPII-mediated transcription. In Arabidopsis thaliana, the Elongator mutants elo1, elo2, and elo3 with decreased leaf and primary root growth due to reduced cell proliferation identified homologs of components of the yeast Elongator complex, Elp4, Elp1, and Elp3, respectively. Here we show that the Elongator complex was purified from plant cell cultures as a six-component complex. The role of plant Elongator in transcription elongation was supported by colocalization of the HAT enzyme, ELO3, with euchromatin and the phosphorylated form of RNAPII, and reduced histone H3 lysine 14 acetylation at the coding region of the SHORT HYPOCOTYL 2 auxin repressor and the LAX2 auxin influx carrier gene with reduced expression levels in the elo3 mutant. Additional auxin-related genes were down-regulated in the transcriptome of elo mutants but not targeted by the Elongator HAT activity showing specificity in target gene selection. Biological relevance was apparent by auxin-related phenotypes and marker gene analysis. Ethylene and jasmonic acid signaling and abiotic stress responses were up-regulated in the elo transcriptome and might contribute to the pleiotropic elo phenotype. Thus, although the structure of Elongator and its substrate are conserved, target gene selection has diverged, showing that auxin signaling and influx are under chromatin control.
PMID: 20080602 [PubMed - indexed for MEDLINE]
Posted in: Proc Natl Acad Sci U S A
March 13th, 2010 / No Comments » / by Koepp DM
The replication stress response and the ubiquitin system: a new link in maintaining genomic integrity.
Cell Div. 2010 Mar 10;5(1):8
Authors: Koepp DM
ABSTRACT: Maintenance of genomic integrity is important for cellular viability and proliferation. During DNA replication, cells respond to replication stress by activating checkpoint pathways that stabilize replication forks and prevent cell cycle progression. The Saccharomyces cerevisiae F-box protein Dia2 is a ubiquitin ligase component required for genomic stability and may help replication complexes negotiate damaged DNA or natural fragile sites. We recently implicated Dia2 in the replication stress response. We demonstrated that Dia2 is targeted for ubiquitin-mediated proteolysis and that activation of the S-phase checkpoint inhibits Dia2 protein turnover. S-phase checkpoint mutants fail to stabilize the Dia2 protein and checkpoint mutants that lack Dia2 exhibit increased sensitivity to replication stress. We also showed that Dia2 protein turnover is not the result of an autocatalytic mechanism. Instead, an N-terminal 20 amino acid motif that is also required for nuclear localization is necessary for Dia2 proteolysis. Dia2 mutants lacking this motif but modified with an exogenous strong nuclear localization signal are both nuclear and stable and disrupt cell cycle dynamics. In summary, our studies suggest that inhibition of Dia2 proteolysis is a novel target of the S-phase checkpoint. We think that this work will help to identify the mechanisms that function downstream of checkpoint activation and that intersect with cell cycle control pathways.
PMID: 20219119 [PubMed - as supplied by publisher]
Posted in: Cell Div
March 13th, 2010 / No Comments » / by Koren A, Soifer I, Barkai N
MRC1-dependent scaling of the budding yeast DNA replication timing program.
Genome Res. 2010 Mar 10;
Authors: Koren A, Soifer I, Barkai N
We describe the DNA replication timing programs of fourteen yeast mutants with an extended S phase identified by a novel genome-wide screen. These mutants are associated with the DNA replication machinery, cell-cycle control and dNTP synthesis and affect different parts of S phase. In thirteen of the mutants, origin activation time scales with the duration of S phase. A limited number of origins becomes inactive in these strains, with inactive origins characterized by small replicons and are distributed throughout S phase. In sharp contrast, cells deleted of MRC1, a gene implicated in replication fork stabilization and in the replication checkpoint pathway, maintained wild-type firing times despite over two-fold lengthening of S phase. Numerous dormant origins were activated in this mutant. Our data suggests that most perturbations that lengthen S phase affect the entire program of replication timing, rather than a specific subset of origins, maintaining the relative order of origin firing time and delaying firing with relative proportions. Mrc1 emerges as a regulator of this robustness of the replication program.
PMID: 20219942 [PubMed - as supplied by publisher]
Posted in: Genome Res
March 13th, 2010 / No Comments » / by Kim KY, Truman AW, Caesar S, Schlenstedt G, Levin DE
Yeast Mpk1 Cell Wall Integrity MAPK Regulates Nucleocytoplasmic Shuttling of the Swi6 Transcriptional Regulator.
Mol Biol Cell. 2010 Mar 10;
Authors: Kim KY, Truman AW, Caesar S, Schlenstedt G, Levin DE
Monitoring Editor: Karsten Weis The yeast SBF transcription factor is a heterodimer comprised of Swi4 and Swi6 that has a well-defined role in cell cycle-specific transcription. SBF serves a second function in the transcriptional response to cell wall stress in which activated Mpk1 MAPK of the cell wall integrity signaling pathway forms a complex with Swi4, the DNA binding subunit of SBF, conferring upon Swi4 the ability to bind DNA and activate transcription of FKS2. Although Mpk1/Swi4 complex formation and transcriptional activation of FKS2 does not require Mpk1 catalytic activity, Swi6 is phosphorylated by Mpk1 and must be present in the Mpk1/Swi4 complex for transcriptional activation of FKS2. Here we find that Mpk1 regulates Swi6 nucleocytoplasmic shuttling in a biphasic manner. First, formation of the Mpk1/Swi4 complex recruits Swi6 to the nucleus for transcriptional activation. Second, Mpk1 negatively regulates Swi6 by phosphorylation on Ser238, which inhibits nuclear entry. Ser238 neighbors a nuclear localization signal (NLS) whose function is blocked by phosphorylation at Ser238 in a manner similar to the regulation by Cdc28 of another Swi6 NLS, revealing a mechanism for the integration of multiple signals to a single endpoint. Finally, the Kap120 beta-importin binds the Mpk1-regulated Swi6 NLS, but not the Cdc28-regulated NLS.
PMID: 20219973 [PubMed - as supplied by publisher]
Posted in: Mol Biol Cell
March 13th, 2010 / No Comments » / by Fonville NC, Bates D, Hastings PJ, Hanawalt PC, Rosenberg SM
Role of RecA and the SOS Response in Thymineless Death in Escherichia coli.
PLoS Genet. 2010;6(3):e1000865
Authors: Fonville NC, Bates D, Hastings PJ, Hanawalt PC, Rosenberg SM
Thymineless death (TLD) is a classic and enigmatic phenomenon, documented in bacterial, yeast, and human cells, whereby cells lose viability rapidly when deprived of thymine. Despite its being the essential mode of action of important chemotherapeutic agents, and despite having been studied extensively for decades, the basic mechanisms of TLD have remained elusive. In Escherichia coli, several proteins involved in homologous recombination (HR) are required for TLD, however, surprisingly, RecA, the central HR protein and activator of the SOS DNA-damage response was reported not to be. We demonstrate that RecA and the SOS response are required for a substantial fraction of TLD. We show that some of the Rec proteins implicated previously promote TLD via facilitating activation of the SOS response and that, of the roughly 40 proteins upregulated by SOS, SulA, an SOS-inducible inhibitor of cell division, accounts for most or all of how SOS causes TLD. The data imply that much of TLD results from an irreversible cell-cycle checkpoint due to blocked cell division. FISH analyses of the DNA in cells undergoing TLD reveal blocked replication and apparent DNA loss with the region near the replication origin underrepresented initially and the region near the terminus lost later. Models implicating formation of single-strand DNA at blocked replication forks, a SulA-blocked cell cycle, and RecQ/RecJ-catalyzed DNA degradation and HR are discussed. The data predict the importance of DNA damage-response and HR networks to TLD and chemotherapy resistance in humans.
PMID: 20221259 [PubMed - in process]
Posted in: PLoS Genet
March 13th, 2010 / No Comments » / by Kang MS, Yu SL, Lim HS, Choi B, Park CS, Kang JH, Lee SK
Mitotic catastrophe induced by overexpression of budding yeast Rad2p.
Yeast. 2010 Mar 10;
Authors: Kang MS, Yu SL, Lim HS, Choi B, Park CS, Kang JH, Lee SK
Mitotic catastrophe provokes endopolyploidy, giant cell formation and, eventually, delayed cell death. Mitotic catastrophe is induced by defective cell cycle checkpoints and by some anticancer drugs, ionizing radiation and microtubule-destabilizing agents. RAD2 is a yeast homologue of XPG, which is a human endonuclease involved in nucleotide excision repair. Here we show that Rad2p overexpression alone, in the absence of extrinsic DNA damage, causes cell growth arrest and mitotic catastrophe. Interestingly, Rad2p-induced cell growth arrest is not caused by the catalytic activity of Rad2p but rather by its C-terminal region. Cells growth-arrested by Rad2p induction do not show apoptotic phenotypes and deletion of YCA1, a yeast caspase homologue, does not affect cell growth arrest by Rad2p induction. However, Rad2p-induced cell growth arrest is released by rad9 deletion but is not affected by downstream DNA damage checkpoint genes. These observations suggest that RAD2 has a function in coordinating cell cycle regulation and damaged DNA repair. Copyright (c) 2010 John Wiley & Sons, Ltd.
PMID: 20222011 [PubMed - as supplied by publisher]
Posted in: Yeast
March 13th, 2010 / No Comments » / by Steinboeck F, Hubmann M, Bogusch A, Dorninger P, Lengheimer T, Heidenreich E
The relevance of oxidative stress and cytotoxic DNA lesions for spontaneous mutagenesis in non-replicating yeast cells.
Mutat Res. 2010 Mar 8;
Authors: Steinboeck F, Hubmann M, Bogusch A, Dorninger P, Lengheimer T, Heidenreich E
Mutations arising during times of cell cycle arrest may considerably contribute to aging and cancerogenesis. Endogenous oxidative stress could be one of the major triggers for these mutations. We used Saccharomyces cerevisiae cells, arrested by starvation for the essential amino acid lysine, to study the occurrence of reactive oxygen species (ROS), abasic (AP) sites and double strand breaks (DSBs). Furthermore, we analyzed the mutation frequencies in resting wild type cells and in cells deficient for Apn1 (with an impaired base excision repair) or Dnl4 (with an inactivated non-homologous end joining (NHEJ) DSB repair pathway) by monitoring reversions of an auxotrophy-causing frameshift in the LYS2 gene. By fluorescence methods, we observed a distinct increase of ROS-affected cells in the course of starvation-induced cell cycle-arrest. In addition, we could reveal that AP sites and DSBs accumulated under these conditions. The frequency of spontaneous frameshift mutations in wild type cells was decreased to 50% upon addition of 6mM N-acetyl cysteine. However, this radical scavenger had no effect in Dnl4-deficient cells. Our results support the hypothesis that (via an active NHEJ DSB repair pathway) the incidence of spontaneous frameshift mutations in a cell cycle-arrested state is considerably governed by oxidative stress.
PMID: 20223252 [PubMed - as supplied by publisher]
Posted in: Mutat Res
