Downregulation of Wip1 phosphatase modulates the cellular threshold of DNA damage signaling in mitosis

Cells are constantly challenged by DNA damage and protect their genome integrity by activation of an evolutionary conserved DNA damage response pathway (DDR). A central core of DDR is composed of a spatiotemporally ordered net of post-translational modifications, among which protein phosphorylation plays a major role. Activation of checkpoint kinases ATM/ATR and Chk1/2 leads to a temporal arrest in cell cycle progression (checkpoint) and allows time for DNA repair. Following DNA repair, cells re-enter the cell cycle by checkpoint recovery. Wip1 phosphatase (also called PPM1D) dephosphorylates multiple proteins involved in DDR and is essential for timely termination of the DDR. Here we have investigated how Wip1 is regulated in the context of the cell cycle. We found that Wip1 activity is downregulated by several mechanisms during mitosis. Wip1 protein abundance increases from G₁ phase to G₂ and declines in mitosis. Decreased abundance of Wip1 during mitosis is caused by proteasomal degradation. In addition, Wip1 is phosphorylated at multiple residues during mitosis, and this leads to inhibition of its enzymatic activity. Importantly, ectopic expression of Wip1 reduced γH2AX staining in mitotic cells and decreased the number of 53BP1 nuclear bodies in G₁ cells. We propose that the combined decrease and inhibition of Wip1 in mitosis decreases the threshold necessary for DDR activation and enables cells to react adequately even to modest levels of DNA damage encountered during unperturbed mitotic progression.     

53BP1 nuclear bodies form around DNA lesions generated by mitotic transmission of chromosomes under replication stress

Completion of genome duplication is challenged by structural and topological barriers that impede progression of replication forks. Although this can seriously undermine genome integrity, the fate of DNA with unresolved replication intermediates is not known. Here, we show that mild replication stress increases the frequency of chromosomal lesions that are transmitted to daughter cells. Throughout G1, these lesions are sequestered in nuclear compartments marked by p53-binding protein 1 (53BP1) and other chromatin-associated genome caretakers. We show that the number of such 53BP1 nuclear bodies increases after genetic ablation of BLM, a DNA helicase associated with dissolution of entangled DNA. Conversely, 53BP1 nuclear bodies are partially suppressed by knocking down SMC2, a condensin subunit required for mechanical stability of mitotic chromosomes. Finally, we provide evidence that 53BP1 nuclear bodies shield chromosomal fragile sites sequestered in these compartments against erosion. Together, these data indicate that restoration of DNA or chromatin integrity at loci prone to replication problems requires mitotic transmission to the next cell generations.     

Resistance to diamide insecticides in diamondback moth,Plutella xylostella (Lepidoptera: Plutellidae) is associated with a mutation in the membrane-spanning domain of the ryanodine receptor

Diamide insecticides such as chlorantraniliprole and flubendiamide are a new class of insecticide that selectively target insect ryanodine receptors (RyR), a distinct class of homo-tetrameric calcium release channels which play a pivotal role in calcium homeostasis in numerous cell types. Resistance to these insecticides has recently been reported in the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), a global lepidopteran pest of cruciferous crops. In the present study a region of the gene encoding the proposed diamide binding site of the RyR from P. xylostella collected from the Philippines and Thailand and found to be over 200-fold resistant to both chlorantraniliprole and flubendiamide compared to susceptible strains, were amplified by RT-PCR and sequenced. Comparison of the sequence with those from several susceptible reference strains revealed non-synonymous mutations in each of the resistant strains that in both cases lead to a glycine to glutamic acid substitution (G4946E) in the protein. The independent evolution of the same amino acid substitution within a highly conserved region of the proposed diamide binding site in two geographically separated resistant strains of P. xylostella strongly suggests a causal association with diamide resistance. Furthermore we designed a pyrosequencing-based diagnostic assay for resistance monitoring purposes that can be used to detect the G4946E mutation in field-collected samples of diamondback moth. The implications of the reported findings for resistance management strategies are discussed.     

Phosphorylation of SDT repeats in the MDC1 N terminus triggers retention of NBS1 at the DNA damage-modified chromatin

DNA double-strand breaks (DSBs) trigger accumulation of the MRE11-RAD50-Nijmegen breakage syndrome 1 (NBS1 [MRN]) complex, whose retention on the DSB-flanking chromatin facilitates survival. Chromatin retention of MRN requires the MDC1 adaptor protein, but the mechanism behind the MRN-MDC1 interaction is unknown. We show that the NBS1 subunit of MRN interacts with the MDC1 N terminus enriched in Ser-Asp-Thr (SDT) repeats. This interaction was constitutive and mediated by binding between the phosphorylated SDT repeats of MDC1 and the phosphate-binding forkhead-associated domain of NBS1. Phosphorylation of the SDT repeats by casein kinase 2 (CK2) was sufficient to trigger MDC1-NBS1 interaction in vitro, and MDC1 associated with CK2 activity in cells. Inhibition of CK2 reduced SDT phosphorylation in vivo, and disruption of the SDT-associated phosphoacceptor sites prevented the retention of NBS1 at DSBs. Together, these data suggest that phosphorylation of the SDT repeats in the MDC1 N terminus functions to recruit NBS1 and, thereby, increases the local concentration of MRN at the sites of chromosomal breakage.     

Robust,universal biomarker assay to detect senescent cells in biological specimens

Cellular senescence contributes to organismal development, aging, and diverse pathologies, yet available assays to detect senescent cells remain unsatisfactory. Here, we designed and synthesized a lipophilic, biotin‐linked Sudan Black B (SBB) analogue suitable for sensitive and specific, antibody‐enhanced detection of lipofuscin‐containing senescent cells in any biological material. This new hybrid histo‐/immunochemical method is easy to perform, reliable, and universally applicable to assess senescence in biomedicine, from cancer research to gerontology.     

Influence of l-isoleucine and pantothenate auxotrophy for l-valine formation in Corynebacterium glutamicum revisited by metabolome analyses

The effect of different amounts of supplemented l-isoleucine and pantothenate has been analysed with the auxotrophic strain Corynebacterium glutamicum ΔilvA ΔpanB, showing that the final biomass concentration of this preliminary l-valine production strain can be controlled by the amount of added l-isoleucine. One gramme cell dry weight is formed from 48 μmol l-isoleucine. Different amounts of available pantothenate affect the intracellular pyruvate concentration. By limiting pantothenate supplementation from 0.8 to 0.1 μM, a 35-fold increase of cytoplasmic pyruvate up to 14.2 mM can be observed, resulting in the increased formation of l-valine, l-alanine and organic acids in the presence of low pantothenate concentrations. These findings can be used to redirect the carbon flux from glycolysis via pyruvate to the TCA cycle towards the desired product l-valine.     

<Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> tailored for high-yield L-valine production

We recently engineered the wild type of Corynebacterium glutamicum for the growth-decoupled production of L: -valine from glucose by inactivation of the pyruvate dehydrogenase complex and additional overexpression of the ilvBNCE genes, encoding the L-valine biosynthetic enzymes acetohydroxyacid synthase, isomeroreductase, and transaminase B. Based on the first generation of pyruvate-dehydrogenase-complex-deficient C. glutamicum strains, a second generation of high-yield L-valine producers was constructed by successive deletion of the genes encoding pyruvate:quinone oxidoreductase, phosphoglucose isomerase, and pyruvate carboxylase and overexpression of ilvBNCE. In fed-batch fermentations at high cell densities, the newly constructed strains produced up to 410 mM (48 g/l) L-valine, showed a maximum yield of 0.75 to 0.86 mol/mol (0.49 to 0.56 g/g) of glucose in the production phase and, in contrast to the first generation strains, excreted neither pyruvate nor any other by-product tested.     

Mammalian G1- and S-phase checkpoints in response to DNA damage

The ability to preserve genomic integrity is a fundamental feature of life. Recent findings regarding the molecular basis of the cell-cycle checkpoint responses of mammalian cells to genotoxic stress have converged into a two-wave concept of the G1 checkpoint, and shed light on the so-far elusive intra-S-phase checkpoint. Rapidly operating cascades that target the Cdc25A phosphatase appear central in both the initiation wave of the G1 checkpoint (preceding the p53-mediated maintenance wave) and the transient intra-S-phase response. Multiple links between defects in the G1/S checkpoints, genomic instability and oncogenesis are emerging, as are new challenges and hopes raised by this knowledge.     

Checking on DNA damage in S phase

The precise replication of the genome and the continuous surveillance of its integrity are essential for survival and the avoidance of various diseases. Cells respond to DNA damage by activating a complex network of the so-called checkpoint pathways to delay their cell-cycle progression and repair the defects. In this review we integrate findings on the emerging mechanisms of activation, the signalling pathways and the spatio-temporal organization of the intra-S-phase DNA-damage checkpoint and its impact on the cell-cycle machinery, and discuss its biological significance.