Crystallization of YIoQ, a GTPase of unknown function essential for Bacillus subtilis viability

YLoQ is a putative ATP/GTP-binding protein of unknown function identified from the complete sequence of the Bacillus subtilis genome. A gene-knockout programme established that yloQ is one of a set of some 270 indispensable genes for the viability of this organism. Crystals of YloQ have been grown from HEPES-buffered solutions at pH 7.5 containing polyethylene glycol and diffraction data have been collected extending to 2.5 Angstrom spacing

A novel DNA nuclease is stimulated by association with the GINS complex

Chromosomal DNA replication requires the spatial and temporal coordination of the activities of several complexes that constitute the replisome. A previously uncharacterized protein, encoded by TK1252 in the archaeon Thermococcus kodakaraensis, was shown to stably interact with the archaeal GINS complex in vivo, a central component of the archaeal replisome. Here, we document that this protein (TK1252p) is a processive, single-strand DNA-specific exonuclease that degrades DNA in the 5′ → 3′ direction. TK1252p binds specifically to the GINS15 subunit of T. kodakaraensis GINS complex and this interaction stimulates the exonuclease activity in vitro. This novel archaeal nuclease, designated GINS-associated nuclease (GAN), also forms a complex in vivo with the euryarchaeal-specific DNA polymerase D. Roles for GAN in replisome assembly and DNA replication are discussed

The wonders of flap endonucleases: structure, function, mechanism and regulation.

Processing of Okazaki fragments to complete lagging strand DNA synthesis requires coordination among several proteins. RNA primers and DNA synthesised by DNA polymerase α are displaced by DNA polymerase δ to create bifurcated nucleic acid structures known as 5'-flaps. These 5'-flaps are removed by Flap Endonuclease 1 (FEN), a structure-specific nuclease whose divalent metal ion-dependent phosphodiesterase activity cleaves 5'-flaps with exquisite specificity. FENs are paradigms for the 5' nuclease superfamily, whose members perform a wide variety of roles in nucleic acid metabolism using a similar nuclease core domain that displays common biochemical properties and structural features. A detailed review of FEN structure is undertaken to show how DNA substrate recognition occurs and how FEN achieves cleavage at a single phosphate diester. A proposed double nucleotide unpairing trap (DoNUT) is discussed with regards to FEN and has relevance to the wider 5' nuclease superfamily. The homotrimeric proliferating cell nuclear antigen protein (PCNA) coordinates the actions of DNA polymerase, FEN and DNA ligase by facilitating the hand-off intermediates between each protein during Okazaki fragment maturation to maximise through-put and minimise consequences of intermediates being released into the wider cellular environment. FEN has numerous partner proteins that modulate and control its action during DNA replication and is also controlled by several post-translational modification events, all acting in concert to maintain precise and appropriate cleavage of Okazaki fragment intermediates during DNA replication

PCNA dependent cellular activities tolerate dramatic perturbations in PCNA client interactions

Proliferating cell nuclear antigen (PCNA) is an essential cofactor for DNA replication and repair, recruiting multiple proteins to their sites of action. We examined the effects of the PCNA(S228I) mutation that causes PCNA-associated DNA repair disorder (PARD). Cells from individuals affected by PARD are sensitive to the PCNA inhibitors T3 and T2AA, showing that the S228I mutation has consequences for undamaged cells. Analysis of the binding between PCNA and PCNA-interacting proteins (PIPs) shows that the S228I change dramatically impairs the majority of these interactions, including that of Cdt1, DNMT1, PolD3(p66) and PolD4(p12). In contrast p21 largely retains the ability to bind PCNA(S228I). This property is conferred by the p21 PIP box sequence itself, which is both necessary and sufficient for PCNA(S228I) binding. Ubiquitination of PCNA is unaffected by the S228I change, which indirectly alters the structure of the inter-domain connecting loop. Despite the dramatic in vitro effects of the PARD mutation on PIP-degron binding, there are only minor alterations to the stability of p21 and Cdt1 in cells from affected individuals. Overall our data suggests that reduced affinity of PCNA(S228I) for specific clients causes subtle cellular defects in undamaged cells which likely contribute to the etiology of PARD

Two distinct pathways for thymidylate (dTMP) synthesis in (hyper)thermophilic bacteria and archaea

International audienceThe hyperthermophilic anaerobic archaeon Pyrococcus obyssi, which lacks thymidine kinase, incorporates label from extracellular uracil, but not from thymidine, into its DNA. This implies that P. obyssi must synthesize dTMP (thymidylate), an essential precursor for DNA synthesis, de novo. However, iterative similarity searches of the three completed Pyrococcus genomes fail to detect candidate genes for canonical thymidylate synthase ThyA, suggesting the presence of alternative pathways for dTMP synthesis. Indeed, by identifying a novel class of flavin-dependent thymidylate synthases, ThyX, we have recently proven that two distinct pathways for de novo synthesis of dTMP are operational in the microbial world. While both thyX and thyA can be found in hyperthermophilic micro-organisms, the phylogenetic distribution of thyX among hyperthermophiles is wider than that of thyA. In this contribution, we discuss the differences in the distinct mechanisms of dTMP synthesis, with a special emphasis on hyperthermophilic micro-organisms

POS0252 Myofibroblasts maintain TH1 and TC1 polarizations in giant cell arteritis

Background: Giant cell arteritis (GCA) is a large-vessel vasculitis mainly involving the aorta and cranial arteries. It is the most frequent vasculitis in adults over 50 years. When they are stimulated by interferon-gamma (IFN-γ), vascular smooth muscle cells (VSMC) contribute to GCA pathogenesis by producing chemokines triggering the recruitment of pro-inflammatory T cells and monocytes (1).Objectives: Current knowledge about the interaction between resident cells of the vascular wall (VSMC, myofibroblasts [MF]) and immune cells is limited. The aim of our research was to better characterize the interactions between VSMC, MF and T cells in GCA.Methods: Fresh fragments of temporal artery biopsies (TAB) performed at Dijon university hospital (France) were prospectively sent to our research unit. Fresh sections of positive and negative TAB were fixed and embedded in optimal cutting temperature OCT and stored at -80°C. Then, cryostat sections were fixed, permeabilized, blocked and incubated with primary antibodies (anti-alpha smooth muscle actin [α-SMA], anti-myosin heavy chain 11 [MHC11], anti-Desmin, anti CD90, anti-CD45, anti-HLA-DR, anti-phospho STAT1 [pSTAT1] and anti-pSTAT3) and secondary antibodies for confocal microscopy analyses. Fresh sections of healthy TAB were embedded in MATRIGEL and covered by DMEM to obtain vascular cells in culture. Cells were treated with trypsina-EDTA between each passage. Vascular cells were used after 4-7 doubling passages. Cells were analyzed by immunofluorescence, flow cytometry and RT-PCR and their proliferation was evaluated by impedancemetry (iCELLigence system). Peripheral blood mononuclear cells (PBMC) and vascular cells thus obtained were co-cultured for 7 days in different conditions. Vascular cells were cultured in the presence or absence of IFN-γ and tumor necrosis factor alpha (TNF-α) or interleukin-6 (IL-6) and soluble receptor of IL-6 for 72 hours. When cells reached confluence, they were cultured alone or with allogenic PBMC activated with anti-CD3/CD28 microbeads. After 7 days of culture, cells were separated with a treatment with EDTA and studied by flow cytometry.Results: Confocal microscopy analyses of GCA arteries showed that neointima was mainly composed of myofibroblasts (MF) (α-SMA+Desmin+MHC11lowCD90+) in contact with CD45+ cells and that MF expressed HLA-DR, the phosphorylated form of STAT1 (pSTAT1) and in a lesser extent pSTAT3, strongly suggesting the activation of the IFN-γ signaling pathway rather than the IL-6 pathway. The phenotype of cultured vascular cells isolated from fresh TAB was consistent with MF. When MF were exposed to IFN-γ and TNF-α in vitro, their proliferation capacity decreased and their levels of expression of HLA-DR and CD86 increased (median fluorescence intensity [MFI] from 0 to 57 [p=0.03] and from 34 to 103 [p=0.03], respectively). In addition, co-cultures of MF and activated PBMC revealed that MF maintained the polarization of T cells into Th1 and Tc1 cells (p≤0.001) and to a lesser extent into Th17 and Tc17 cells (p=0.03). This effect was even more significant when MF were previously exposed to IFN-γ and TNF-α but not when they were exposed to IL-6.Conclusion: Our results show that myofibroblasts are present in the neointima of GCA patients and that these MF activate signaling pathways indicative of IFN-γ exposure. Moreover, these MF, especially when exposed to IFN-γ, maintain the polarization of T cells into Th1 and Tc1 cells, which contributes to amplify the production of IFN-γ and thus initiate a pro-inflammatory amplification loop that likely participates in vascular inflammation and remodelling.References: [1]Corbera-Bellalta M, Planas-Rigol E, Lozano E, Terrades-Garcia N, Alba MA, Prieto-Gonzalez S, et al. Blocking interferon gamma reduces expression of chemokines CXCL9, CXCL10 and CXCL11 and decreases macrophage infiltration in ex vivo cultured arteries from patients with giant cell arteritis. Ann Rheum Dis 2016;75:1177-86

Contamination de la Seine par les micropolluants organiques : Effet des conditions hydriques et de l'urbanisation

Contamination de la Seine par les micropolluants organiques : Effet des conditions hydriques et de l'urbanisatio