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  • In the present work we ask whether rNMP

    2020-05-22

    In the present work, we ask whether rNMP incorporation is an evolutionarily conserved property of DNA synthesis in Archaea. For this purpose, we used the best characterized hyperthermophilic anaerobe, Pyrococcus abyssi GE5 [47]. This strain duplicates its genome as fast as Bacteria, supported by eukaryotic-like replication proteins [48]. Roles have been proposed whereby RNA priming is performed by the p41/p46 complex [31], followed by leading and lagging strand DNA synthesis by PolB and PolD, respectively [35], [49], [50],. Maturation of Okazaki fragments likely involves PolD but precludes any contribution of PolB in the absence of RNase HII [34]. Beyond these crucial roles in DNA replication, all three DNA polymerizing enzymes seem to work in DNA transactions such as repair, damage signaling or tolerance [31], [42], [43], [51]. We further analyzed whether rNMP incorporation by PolB, PolD and the p41/p46 complex is conserved during DNA synthesis. At the physiological dNTP and rNTP levels, we demonstrate that PolD is the main DNA Pol able to insert rNMPs in an error-free manner. In addition, we report that single embedded rNMPs are bypassed by all three DNA Pols with variable nucleotide incorporation/misincorporation proficiency regardless of the nature of the o6 and sequence context. Unexpectedly, we also discovered that PolD can incorporate a single rNMP opposite template ribonucleotides. These results suggest that rNMP incorporation into DNA represents a conserved feature of archaeal DNA Pols, which may have various consequences for their genome integrity.
    Results
    Discussion
    Materials and Methods
    Acknowledgments Mélanie Lemor thanks the French Institute for the Exploitation of the Sea (Ifremer) and the Regional Council of Brittany for funding. The dNTP/rNTP measurements were performed in the laboratory of Dr. Chabes, Umeå University. This work was supported by the French National Research Agency (ANR-10-JCJC-1501-01 to G.H.). Funding for the open access charge was provided by the French National Research Agency (ANR). Declaration of Interest: None.
    Introduction Phosphatidylinositol-3-phosphate (PI3P) is o6 a lipid that accumulates in the early endosomal membrane. PI3P is recognized by several PI3P-binding domains, such as FYVE and PX, and recruits PI3P-binding proteins to early endosomes. The functions of proteins that contain an FYVE domain are diverse and include endocytosis, autophagy, and signal transduction [[1], [2], [3], [4], [5], [6]]. In addition, a role for PI3P itself in actin dynamics has also been reported. An in vitro actin polymerization assay revealed that polymerization is stimulated in PI3P/phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-containing liposomes by recruiting Cdc42, N-WASP, and the Arp2/3 complex to liposome membranes [7,8]. Furthermore, actin nucleation and actin puncta formation also occur in autophagosomes; both processes are also dependent on PI3P [9,10]. So, the depletion of PI3P or the perturbation of its function can affect a broad range of cellular events. The turnover of PI3P is regulated by phosphatidylinositol kinases and phosphatases, and may become perturbed under various conditions. For example, hydrogen peroxide depletes intracellular PI3P and causes the dissociation of EEA1 from endosomes. The dissociation is induced by the inhibition of the recruitment of Vps34, a class III PI3 kinase that creates PI3P from phosphatidylinositol (PI), to early endosomes in a p38 MAPK–dependent manner [11,12]. We also showed a decrease in the PI3P level in early endosomal membranes in diabetic model cells [13]. We previously established diabetic model cells (referred to as Db model cells) by a cell-resealing technique using a cholesterol-dependent cytolysin, streptolysin O (SLO) [13,14]. SLO forms pores, 30 nm in diameter, in the plasma membrane that enable the introduction of various molecules into cells. Such permeabilized cells maintain the integrity of the cytoskeleton and organelles, and are called “semi-intact cells”. We introduced cytosol, prepared from the livers of wild-type (WT) or diabetic (db/db) model mice, into semi-intact cells, to change cytosolic conditions to healthy or diabetic liver ones, respectively. After introducing the cytosol, the plasma membrane injury was repaired by the addition of CaCl2. The resealing process was dependent on exocytosis/endocytosis/bleb formation, which removed SLO-mediated pores from the cell membrane [[15], [16], [17]]. These cells were used as WT or Db model cells in our study, the phenotypes of which were compared to reveal Db-specific aberrant phenotypes, i.e. disease-specific markers.