Silencing of dUTPase in <i>Drosophila</i> larvae and pupae.

<p>Western blots in (A) show that the protein level of dUTPase is under detection limit in silenced animals. Actin served as loading control. (B) Curves show the relative number of silenced and non-silenced animals that have undergone puparium formation at the given time point after egg deposition. Inflection points of the curves represent the mean time of puparium formation characteristic for the given population. dUTPase silencing did not perturb the time interval required for puparium formation. (C) Graph shows the number of counted dead animals relative to number of hatched curly winged control flies. Among these dead animals, three groups with distinct morphological traits characteristic for wandering larvae (w3L), prepupae (preP), and pupal stage P5 (P5) were identified and counted. (D) Genomic uracil content of dUTPase silenced and control tissues from 3^rd larvae.</p

Occurrence of genes encoding dUTPase and UNG in different insects.

<p>The gene for dUTPase is ubiquitous, but the gene of the major uracil–DNA glycosylase, ung is not encoded in the genome of Holometabola species.</p>*<p>In the genome of <i>Aedes aegypti</i> strain Liverpool, an unexpected ung sequence was found, showing very high (87%–94%) similarity to the ung gene of Comamonadaceae, a family of Proteobacteria, arguing for its bacterial origin.</p

<i>D. melanogaster</i> genomic DNA uracil content inversely correlates with dUTPase expression.

<p>(A) Changes of dUTPase mRNA level throughout fruitfly development: embryo (E), 1^st larvae (L1), 2^nd larvae (L2), late 3^rd larvae (L3) and pupae (P). Note that dUTPase is down-regulated in larvae. (B) Comparison of dUTPase RNA level in the larval tissues salivary gland and imaginal tissue. Data are presented as mean of triplicates ± s.e.m. mRNA level was measured by RT-qPCR and dUTPase mRNA level was normalized to Rp49 mRNA level. (C) Uracil content of <i>D. melanogaster</i> genome in different developmental stages: embryo (E), 1^st larvae (L1), 2^nd larvae (L2), late 3^rd larvae (L3) and pupae (P). Embryonic sample was used as reference since it was shown to contain undetectable levels of uracil in DNA (cf. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002738#pgen.1002738.s002" target="_blank">Figure S2</a>). (D) Comparison of genomic uracil content in wild type imaginal disc and salivary gland of 3^rd larvae. Data are presented as mean ± s.e.m.</p

Tolerance and stability of uracil-containing DNA in <i>D. melanogaster</i>.

<p>(A) Dose-response curve upon FdUR treatment followed by Alamar Blue assay. (B) FdUR leads to uracil accumulation in DNA of <i>Drosophila</i> S2 cells. Data indicate that increased level of uracil is well-tolerated in <i>Drosophila</i>, but not in human cells. Data are presented as mean ± s.e.m. (C) <i>Drosophila</i> S2 (top panels) and human HeLa cells (bottom panels) were transfected with normal plasmid (left panels) or uracil-containing plasmid (right panels). Expression of YFP in <i>Drosophila</i> S2 cells or dsRedMonomer in HeLa cells indicates stability of the DNA. (D) Microinjection of uracil-plasmid into <i>Drosophila</i> embryo. Non-injected embryos served as control sample.</p

Stage- and tissue-specific distribution of dUTPase protein levels in <i>D. melanogaster</i>.

<p>Western blotting (A) and immunohistochemistry (B) was performed on selected developmental stages and tissues. Embryo 0–6 h (E1), embryo 0–24 h (E2), 1^st larvae (1L), 2^nd larvae (2L), early 3^rd larvae (3L1), wandering 3^rd larvae (3L2), pupae before head eversion (P1), pupae after head eversion (P2) and pupae 50–60 h after puparium formation (P3). For Western blotting, actin was used as loading control. Note that dUTPase protein levels are down-regulated during larval stages and expression is confined to specific tissues.</p

Morphological consequences of dUTPase silencing.

<p>In larvae (A) and pupae (B). (A) Immunohistochemistry of wing and eye discs, and brain of non-silenced (control) and silenced larvae for dUTPase (red) and DAPI staining for DNA (blue) demonstrate on one hand highly effective silencing; and on the other hand no observable morphological changes within these tissues. (B) Wild type pupae (control) in stage P6 (cf. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002738#pgen.1002738.s005" target="_blank">Figure S5</a>) and dUTPase silenced pupae at corresponding time after puparium formation in dorsal and ventral view are shown, after puparium removal. Wild type traits, Malpighian tubules (white arrows), Yellow Body (white asterix), developing adult eye (white arrowheads) are not observable on silenced animals. Instead, darkened (apoptotic/necrotic or melanized) tissues (red arrowheads) can be visualized on these pupae. Note the basically different inner texture of the everted discs (white boxes) and head sack (white circles).</p

dUTPase silencing results in cell death and DNA strand breaks in larval imaginal discs.

<p>(A) Imaginal discs were isolated from wild type and dUTPase silenced wandering 3^rd larvae and stained for TUNEL assay (shown as red dots). Discs from silenced animals showed highly increased TUNEL staining. (B) TUNEL positive cell counts in imaginal discs from wild type and dUTPase silenced wandering 3^rd larvae. Error bars represent the standard error of mean. (C) Imaginal discs from wild type and dUTPase silenced 3^rd wandering larvae stained against phospho-H2Av foci (white dots, some of these are appointed by white arrowheads). dUTPase depleted discs showed several nuclei with phospho-H2Av foci indicating DNA damage. Scale bar represents 50 µm.</p

Dynamics of re-constitution of the human nuclear proteome after cell division is regulated by NLS-adjacent phosphorylation

<div><p>Phosphorylation by the cyclin-dependent kinase 1 (Cdk1) adjacent to nuclear localization signals (NLSs) is an important mechanism of regulation of nucleocytoplasmic transport. However, no systematic survey has yet been performed in human cells to analyze this regulatory process, and the corresponding cell-cycle dynamics have not yet been investigated. Here, we focused on the human proteome and found that numerous proteins, previously not identified in this context, are associated with Cdk1-dependent phosphorylation sites adjacent to their NLSs. Interestingly, these proteins are involved in key regulatory events of DNA repair, epigenetics, or RNA editing and splicing. This finding indicates that cell-cycle dependent events of genome editing and gene expression profiling may be controlled by nucleocytoplasmic trafficking. For in-depth investigations, we selected a number of these proteins and analyzed how point mutations, expected to modify the phosphorylation ability of the NLS segments, perturb nucleocytoplasmic localization. In each case, we found that mutations mimicking hyper-phosphorylation abolish nuclear import processes. To understand the mechanism underlying these phenomena, we performed a video microscopy-based kinetic analysis to obtain information on cell-cycle dynamics on a model protein, dUTPase. We show that the NLS-adjacent phosphorylation by Cdk1 of human dUTPase, an enzyme essential for genomic integrity, results in dynamic cell cycle-dependent distribution of the protein. Non-phosphorylatable mutants have drastically altered protein re-import characteristics into the nucleus during the G1 phase. Our results suggest a dynamic Cdk1-driven mechanism of regulation of the nuclear proteome composition during the cell cycle.</p></div