Impaired tRNA Nuclear Export Links DNA Damage and Cell-Cycle Checkpoint

SummaryIn response to genotoxic stress, cells evoke a plethora of physiological responses collectively aimed at enhancing viability and maintaining the integrity of the genome. Here, we report that unspliced tRNA rapidly accumulates in the nuclei of yeast Saccharomyces cerevisiae after DNA damage. This response requires an intact MEC1- and RAD53-dependent signaling pathway that impedes the nuclear export of intron-containing tRNA via differential relocalization of the karyopherin Los1 to the cytoplasm. The accumulation of unspliced tRNA in the nucleus signals the activation of Gcn4 transcription factor, which, in turn, contributes to cell-cycle arrest in G1 in part by delaying accumulation of the cyclin Cln2. The regulated nucleocytoplasmic tRNA trafficking thus constitutes an integral physiological adaptation to DNA damage. These data further illustrate how signal-mediated crosstalk between distinct functional modules, namely, tRNA nucleocytoplasmic trafficking, protein synthesis, and checkpoint execution, allows for functional coupling of tRNA biogenesis and cell-cycle progression

Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p

<p>Abstract</p> <p>Background</p> <p>Histone modifications have been implicated in the regulation of transcription and, more recently, in DNA replication and repair. In yeast, a major conserved histone acetyltransferase, Hat1p, preferentially acetylates lysine residues 5 and 12 on histone H4.</p> <p>Results</p> <p>Here, we report that a nuclear sub-complex consisting of Hat1p and its partner Hat2p interacts physically and functionally with the origin recognition complex (ORC). While mutational inactivation of the histone acetyltransferase (HAT) gene <it>HAT1 </it>alone does not compromise origin firing or initiation of DNA replication, a deletion in <it>HAT1 </it>(or <it>HAT2</it>) exacerbates the growth defects of conditional <it>orc-ts </it>mutants. Thus, the ORC-associated Hat1p-dependent histone acetyltransferase activity suggests a novel linkage between histone modification and DNA replication. Additional genetic and biochemical evidence points to the existence of partly overlapping histone H3 acetyltransferase activities in addition to Hat1p/Hat2p for proper DNA replication efficiency. Furthermore, we demonstrated a dynamic association of Hat1p with chromatin during S-phase that suggests a role of this enzyme at the replication fork.</p> <p>Conclusion</p> <p>We have found an intriguing new association of the Hat1p-dependent histone acetyltransferase in addition to its previously known role in nuclear chromatin assembly (Hat1p/Hat2p-Hif1p). The participation of a distinct Hat1p/Hat2p sub-complex suggests a linkage of histone H4 modification with ORC-dependent DNA replication.</p

Global Functional Atlas of \u3cem\u3eEscherichia coli\u3c/em\u3e Encompassing Previously Uncharacterized Proteins

One-third of the 4,225 protein-coding genes of Escherichia coli K-12 remain functionally unannotated (orphans). Many map to distant clades such as Archaea, suggesting involvement in basic prokaryotic traits, whereas others appear restricted to E. coli, including pathogenic strains. To elucidate the orphans’ biological roles, we performed an extensive proteomic survey using affinity-tagged E. coli strains and generated comprehensive genomic context inferences to derive a high-confidence compendium for virtually the entire proteome consisting of 5,993 putative physical interactions and 74,776 putative functional associations, most of which are novel. Clustering of the respective probabilistic networks revealed putative orphan membership in discrete multiprotein complexes and functional modules together with annotated gene products, whereas a machine-learning strategy based on network integration implicated the orphans in specific biological processes. We provide additional experimental evidence supporting orphan participation in protein synthesis, amino acid metabolism, biofilm formation, motility, and assembly of the bacterial cell envelope. This resource provides a “systems-wide” functional blueprint of a model microbe, with insights into the biological and evolutionary significance of previously uncharacterized proteins

Genetic Interaction Maps in Escherichia coli Reveal Functional Crosstalk among Cell Envelope Biogenesis Pathways

As the interface between a microbe and its environment, the bacterial cell envelope has broad biological and clinical significance. While numerous biosynthesis genes and pathways have been identified and studied in isolation, how these intersect functionally to ensure envelope integrity during adaptive responses to environmental challenge remains unclear. To this end, we performed high-density synthetic genetic screens to generate quantitative functional association maps encompassing virtually the entire cell envelope biosynthetic machinery of Escherichia coli under both auxotrophic (rich medium) and prototrophic (minimal medium) culture conditions. The differential patterns of genetic interactions detected among >235,000 digenic mutant combinations tested reveal unexpected condition-specific functional crosstalk and genetic backup mechanisms that ensure stress-resistant envelope assembly and maintenance. These networks also provide insights into the global systems connectivity and dynamic functional reorganization of a universal bacterial structure that is both broadly conserved among eubacteria (including pathogens) and an important target

An Unbiased Chemical Proteomics Method Identifies FabI as the Primary Target of 6‑OH-BDE-47

Determination of the physical interactions of environmental chemicals with cellular proteins is important for characterizing biological and toxic mechanism of action. Yet despite the discovery of numerous bioactive natural brominated compounds, such as hydroxylated polybrominated diphenyl ethers (OH-PBDEs), their corresponding protein targets remain largely unclear. Here, we reported a systematic and unbiased chemical proteomics assay (Target Identification by Ligand Stabilization, TILS) for target identification of bioactive molecules based on monitoring ligand-induced thermal stabilization. We first validated the broad applicability of this approach by identifying both known and unexpected proteins bound by diverse compounds (anticancer drugs, antibiotics). We then applied TILS to identify the bacterial target of 6-OH-BDE-47 as enoyl-acyl carrier protein reductase (FabI), an essential and widely conserved enzyme. Using affinity pull-down and in vitro enzymatic assays, we confirmed the potent antibacterial activity of 6-OH-BDE-47 occurs via direct binding and inhibition of FabI. Conversely, overexpression of FabI rescued the growth inhibition of <i>Escherichia coli</i> by 6-OH-BDE-47, validating it as the primary in vivo target. This study documents a chemical proteomics strategy for identifying the physical and functional targets of small molecules, and its potential high-throughput application to investigate the modes-of-action of environmental compounds

An Unbiased Chemical Proteomics Method Identifies FabI as the Primary Target of 6‑OH-BDE-47

Determination of the physical interactions of environmental chemicals with cellular proteins is important for characterizing biological and toxic mechanism of action. Yet despite the discovery of numerous bioactive natural brominated compounds, such as hydroxylated polybrominated diphenyl ethers (OH-PBDEs), their corresponding protein targets remain largely unclear. Here, we reported a systematic and unbiased chemical proteomics assay (Target Identification by Ligand Stabilization, TILS) for target identification of bioactive molecules based on monitoring ligand-induced thermal stabilization. We first validated the broad applicability of this approach by identifying both known and unexpected proteins bound by diverse compounds (anticancer drugs, antibiotics). We then applied TILS to identify the bacterial target of 6-OH-BDE-47 as enoyl-acyl carrier protein reductase (FabI), an essential and widely conserved enzyme. Using affinity pull-down and in vitro enzymatic assays, we confirmed the potent antibacterial activity of 6-OH-BDE-47 occurs via direct binding and inhibition of FabI. Conversely, overexpression of FabI rescued the growth inhibition of <i>Escherichia coli</i> by 6-OH-BDE-47, validating it as the primary in vivo target. This study documents a chemical proteomics strategy for identifying the physical and functional targets of small molecules, and its potential high-throughput application to investigate the modes-of-action of environmental compounds

Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p-8

<p><b>Copyright information:</b></p><p>Taken from "Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p"</p><p>http://www.biomedcentral.com/1741-7007/5/38</p><p>BMC Biology 2007;5():38-38.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2140264.</p><p></p>d from strains carrying either a TAP-tagged version of Orc1p, Orc2p, Orc4p, Orc5p, or Orc6p. For comparison, lane 2 shows marker proteins (MWs are 45.0, 66.2, and 97.4 kDa). The positions of the respective subunits of the ORC and Hat1p complex are indicated. Hat1-CBP: Hat1p with calmodulin binding protein (CBP) after TEV digestion of the TAP-tag. (B) Summary of proteins identified in purifications of TAP-tagged baits. Components of ORC and Hat1p complexes that were detected at least once with either MALDI-TOF or liquid chromatography-mass spectrometry (LC-MS) with high confidence (>90%) are indicated (M/L). (C) Architecture of the Hat1p complexes by differential tagging and subunit deletions. Strains were (BSY675), (BSY681), (BSY679), (BSY720), (BSY682). Lane 1 shows marker proteins (45.0, 66.2, and 97.4 kDa). (D) Western blot from two series of TAP purifications (Figure 1C for left panel, Additional file for right panel), probed with a-Hat1p, a-Orc2p, a-Orc3p, and a-Orc5p antibodies. (E) histone acetyltransferase activities of Hat1p complexes. Concentrated eluate (10-fold) from indicated TAP-tag purifications from Figure 1C was used for HAT-assays with C acetyl-CoA and chicken erythrocyte histones. The upper panel shows C incorporation into histones shown in the lower panel by Coomassie strain. (F) association of acetylated histone H4 with Hat1p sub-complexes from Figure 1C). Eluates from TAP-tag purifications were analyzed for Lys12 acetylated histone H4 by Western blot (a-Acetyl H4 Lys12)

Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p-3

<p><b>Copyright information:</b></p><p>Taken from "Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p"</p><p>http://www.biomedcentral.com/1741-7007/5/38</p><p>BMC Biology 2007;5():38-38.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2140264.</p><p></p>) and wild-type strain (JRY2334) were shifted to the nonpermissive temperature for 0–5 h. Viability was measured as the fraction of microcolonies that formed after the incubation at 36°C within 1–2 days at permissive temperature. (B) Percentage of , , and wild-type cells that form viable microcolonies when synchronized cultures were shifted to the nonpermissive temperature. Cells were arrested in G1 (α-factor) or in S-phase (hydroxyurea) at 23°C and then maintained at restrictive temperature (36°C) for 0 to 3 h in G1 phase, in S-phase or from G1 to S-phase arrest. Averages of two independent experiments are shown. (C) FACS analysis of wild-type, (BSY539) (BSY568) and (BSY569) cells at semi-permissive temperature (26°C). Cells were arrested in α-factor (5 mg/ml) and release was performed at 26°C for 0, 10, 20, 30, 40, 50, 60, 90, 120, 150, and 190 min. (D) Cell-cycle progression of and wild-type strain at restrictive temperature for . G1-arrested cells were held at 36°C (restrictive temperature) for 1 h and then released into fresh (36°C) medium. Samples for FACS were taken at times indicated. For a detailed list of strains, see Additional file

Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p-1

<p><b>Copyright information:</b></p><p>Taken from "Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p"</p><p>http://www.biomedcentral.com/1741-7007/5/38</p><p>BMC Biology 2007;5():38-38.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2140264.</p><p></p>, Hat1–13myc (BSY676), and Orc5–13myc (BSY677). IPs were performed with monoclonal a-Orc3p and a-GFP antibodies. One twentieth of the extracts were loaded as whole cell extract (WCE). Immunoprecipitated Hat1–13myc was detected with mouse α-myc as a primary antibody (9E10). Note that equal amounts and concentrations of extracts were used for all IPs. (B) ORC binds Hat1p/Hat2p but not Hif1p. Strains were: untagged control, Hat1–13myc, Hat2–13myc (BSY691), and Hif1–13myc (BSY692). One twentieth of the extracts were loaded as WCE control. IPs were performed with monoclonal α-Orc3p and α-GFP control antibodies. (C) IP of Hat1p-13myc with α-Orc3p antibody in synchronized cells. Log-phase cells were either arrested in 3 μg/ml α-factor, 200 mM hydroxyurea, or 15 μg/ml nocodazole. Cells were also released from α-factor arrest and samples were taken at 20, 30, and 40 min after release (lanes 4–6). Lanes 7–12 show WCE controls. (D) FACS control of samples in panel C. Time after release is indicated (Rel)

Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p-4

<p><b>Copyright information:</b></p><p>Taken from "Association with the origin recognition complex suggests a novel role for histone acetyltransferase Hat1p/Hat2p"</p><p>http://www.biomedcentral.com/1741-7007/5/38</p><p>BMC Biology 2007;5():38-38.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2140264.</p><p></p>s either labeled with P (panels A and C) or visualized by ethidium bromide (panels B and D). (A) Binding of Hat1-TAP and Cdc45-TAP to and to control sequence at the time of origin firing and later in S-phase. Strains were (BSY679) and (BSY680). One representative experiment with input and immunoprecipitate and the percentage of precipitated DNA is shown. (B) Recruitment of Hat1p to is coincident with Cdc45p and dependent on functional ORC. Strains used for chromatin immunoprecipitation are , , and (BSY699). Strains were held in a-factor at 36°C to inactivate and then released at 23°C. (C) Recruitment of Hat1p (Hat1-TAP) to late replication origin and comparison with sequence. (D) Recruitment of Hat1p to is affected by the allele. Strains used for chromatin immunoprecipitation are , , and (BSY734)