Proteomics of a fuzzy organelle: interphase chromatin

Chromatin proteins mediate replication, regulate expression and ensure integrity of the genome. So far, a comprehensive inventory of interphase chromatin has not been determined. This is largely due to its heterogeneous and dynamic composition, which makes conclusive biochemical purification difficult, if not impossible. As a fuzzy organelle it defies classical organellar proteomics and cannot be described by a single and ultimate list of protein components. Instead we propose a new approach that provides a quantitative assessment of a protein’s probability to function in chromatin. We integrate chromatin composition over a range of different biochemical and biological conditions. This resulted in interphase chromatin probabilities for 7635 human proteins, including 1840 previously uncharacterized proteins. We demonstrate the power of our large-scale data-driven annotation during the analysis of CDK regulation in chromatin. Quantitative protein ontologies may provide a general alternative to list-based investigations of organelles and complement Gene Ontology

PrimPol-deficient cells exhibit a pronounced G2 checkpoint response following UV damage

PrimPol is a recently identified member of the archaeo-eukaryote primase (AEP) family of primase-polymerases. It has been shown that this mitochondrial and nuclear localised enzyme plays roles in the maintenance of both unperturbed replication fork progression and in the bypass of lesions after DNA damage. Here, we utilised an avian (DT40) knockout cell line to further study the consequences of loss of PrimPol (PrimPol-/-) on the downstream maintenance of cells after UV damage. We report that PrimPol-/- cells are more sensitive to UV-C irradiation in colony survival assays than Pol η-deficient cells. Although this increased UV sensitivity is not evident in cell viability assays, we show that this discrepancy is due to an enhanced checkpoint arrest after UV-C damage in the absence of PrimPol. PrimPol-/- arrested cells become stalled in G2, where they are protected from UV-induced cell death. Despite lacking an enzyme required for the bypass and maintenance of replication fork progression in the presence of UV damage, we show that PrimPol-/- cells actually have an advantage in the presence of a Chk1 inhibitor due to their slow progression through S-phase

Aurora A and Aurora B jointly coordinate chromosome segregation and anaphase microtubule dynamics

Aurora A and Aurora B have nonredundant functions during mitosis in chromosome segregation and anaphase microtubule dynamics

Analytical biochemistry of DNA–protein assemblies from crude cell extracts

Purification of specific DNA–protein complexes is a challenging task, as the involved interactions can be both electrostatic/H-bond and hydrophobic. The chromatographic stringency needed to obtain reasonable purifications uses salts and detergents. However, these components elicit the removal of proteins unspecifically bound to the chromatographic support itself, thus contaminating the purification products. In this work, a photocleavable linker connected the target oligonucleotidic sequence to the chromatographic beads so as to allow the irradiation-based release of the purified DNA–protein complexes off the beads. Our bioanalytical conditions were validated by purifying the tetracycline repressor protein onto a specific oligonucleotide. The purification factor was unprecedented, with a single contaminant. The robustness of our method was challenged by applying it to the purification of multiprotein assemblies forming onto DNA damage-mimicking oligonucleotides. The purified components were identified as well-known DNA repair proteins, and were shown to retain their enzymatic activities, as seen by monitoring DNA ligation products. Remarkably, kinase activities, also monitored, were found to be distinct on the beads and on the purified DNA–protein complexes, showing the benefits to uncouple the DNA–protein assemblies from the beads for a proper understanding of biochemical regulatory mechanisms involved in the DNA–protein assemblies

PP2A/B55 and Fcp1 regulate Greatwall and Ensa desphorylation during mitotic exit

Entry into mitosis is triggered by activation of Cdk1 and inactivation of its counteracting phosphatase PP2A/B55. Greatwall kinase inactivates PP2A/B55 via its substrates Ensa and ARPP19. Both Greatwall and Ensa/ARPP19 are regulated by phosphorylation, but the dynamic regulation of Greatwall activity and the phosphatases that control Greatwall kinase and its substrates are poorly understood. To address these questions we applied a combination of mathematical modelling and experiments using phospho-specific antibodies to monitor Greatwall, Ensa/ARPP19 and Cdk substrate phosphorylation during mitotic entry and exit. We demonstrate that PP2A/B55 is required for Gwl dephosphorylation at the essential Cdk site Thr194. Ensa/ARPP19 dephosphorylation is mediated by the RNA Polymerase II carboxy terminal domain phosphatase Fcp1. Surprisingly, neither Fcp1 nor PP2A appear to essential to dephosphorylate the bulk of mitotic Cdk1 substrates following Cdk1 inhibition. Taken together our results suggest a hierarchy of phosphatases coordinating Greatwall, Ensa/ARPP19 and Cdk substrate dephosphorylation during mitotic exit

The human DNA ends proteome uncovers an unexpected entanglement of functional pathways

International audienceDNA ends get exposed in cells upon either normal or dysfunctional cellular processes or molecular events. Telomeres need to be protected by the shel-terin complex to avoid junctions occurring between chromosomes while failing topoisomerases or clustered DNA damage processing may produce double-strand breaks, thus requiring swift repair to avoid cell death. The rigorous study of the great many proteins involved in the maintenance of DNA integrity is a challenging task because of the innumerous unspe-cific electrostatic and/or hydrophobic DNA––protein interactions that arise due to the chemical nature of DNA. We devised a technique that discriminates the proteins recruited specifically at DNA ends from those that bind to DNA because of a generic affinity for the double helix. Our study shows that the DNA ends proteome comprises proteins of an unexpectedly wide functional spectrum, ranging from DNA repair to ribosome biogenesis and cytoskeleton, including novel proteins of undocumented function. A global mapping of the identified proteome on published DNA repair protein networks demonstrated the excellent specificity and functional coverage of our purification technique. Finally, the native nucleopro-teic complexes that assembled specifically onto DNA ends were shown to be endowed with a highly efficient DNA repair activity

Characterizing Gwl, Ensa/ARPP19 and SP dephosphorylation during mitotic exit.

<p>(<b>A</b>) HeLa cells were synchronized in mitosis by Eg5 inhibition using 5 µM STLC and pretreated for one hour with 1 µM OA, 10 µM TC or both. Samples were taken for extraction and immunoblot analysis at indicated timepoints following treatment with 10 µM RO3306. (<b>B</b>) Quantification of relative Cdk substrate (phospho-SP) dephosphorylation in OA and TC treated cells. Error bars indicate standard deviation calculated from three independent experiments. (<b>C</b>) Immunoblot analysis of STLC arrested cells following one hour treatment with 100 nM Calyculin A (CalA). Extracts were taken at indicated times after Cdk1 inhibition by 10 µM RO3306. (<b>D</b>) MBP Kinase assays with immuno-precipitated Flag-Gwl that was transiently expressed in HeLa cells. Flag-Gwl was purified from STLC arrested cells before and 30 minutes after RO3306 treatment. Cells were pretreated for one hour with the indicated phosphatase inhibitors. (<b>E</b>) Quantification of MBP kinase activity following Cdk1 inhibition (+RO) relative to mitotic cells before Cdk1 inhibition (-RO). Error bars indicate the standard deviation calculated from three independent experiments.</p

The regulatory network and the dynamics of the mitotic switch.

<p>(<b>A</b>) Model of the Cdk1 activation switch. Cdk1 activity is regulated by inhibitory Tyr15 phosphorylation modulated through the activities of Wee1 and Cdc25. The activity of these modifying enzymes are regulated both directly and indirectly (through Gwl, Ensa/ARPP19 and PP2A/B55) by Cdk1. (<b>B–J</b>) Simulation of mitotic entry and exit using a mathematical model of the regulatory network with the assumption included that Gwl is dephosphorylated by an OA-insensitive phosphatase (left panels), by an OA-sensitive phosphatase (middle panels), or specifically by PP2A/B55 (right panels). The simulation of mitotic entry is shown from the initial condition obtained using Cdk1 inhibition and either removal of Cdk1 inhibition (B–D) or PP2A inhibition by OA (E–G) promotes mitotic entry. The mitotic exit (H–J) is shown from the initial condition corresponding to metaphase state and Cdk1 inhibition promotes mitotic exit.</p

Identifying the phosphatases required for Gwl and Ensa/ARPP19.

<p>(A) Dephosphorylation of Gwl and SP sites in B55 depleted cells. HeLa cells were transfected with combinations of B55α and δ siRNAs and synchronized in mitosis with STLC as above (see Materials & Methods). Cell extracts were sampled before and 30 minutes after RO3306 treatment and analyzed by immuno-blotting with indicated antibodies. (B) Dephosphorylation of Gwl, Ensa/Arpp19 and SP sites in Fcp1 depleted cells. HeLa cells were transfected with Fcp1 siRNA and synchronized in mitosis with STLC (see Materials & Methods). Cell extracts were sampled before and 30 minutes after RO3306 treatment and analyzed by immune-blotting with indicated antibodies. (C) Gwl phosphatase assay with immuno-precipitated Flag-B55α. Purified his-Gwl was phosphorylated by Cdk2/cycA and γ³²P ATP. Cdk2/cycA was removed from the reaction by further purification with Ni-Agarose beads and radiolabeled phospho-Gwl was incubated with immunoprecipitated B55α. The presence of the PP2A/B55α complex in the immune-precipitate was confirmed by immuno-blotting. Phosphatase activity was measured by scintillation counting of released <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004004#pgen.1004004-Visconti1" target="_blank">[³²]</a>P phosphate. (D) Similar phosphatase assay as in (C) with recombinant <i>in vitro</i> phosphorylated Ensa and immuno-precipitated GFP-Fcp1.</p