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Replicative helicases are ring-shaped hexamers that encircle DNA for duplex unwinding. The currently accepted view of hexameric helicase function is by steric exclusion, where the helicase encircles one DNA strand and excludes the other, acting as a wedge with an external DNA unwinding point during translocation. Accordingly, strand-specific blocks only affect these helicases when placed on the tracking strand, not the excluded strand. We examined the effect of blocks on the eukaryotic CMG and, contrary to expectations, blocks on either strand inhibit CMG unwinding. A recent cryoEM structure of yeast CMG shows that duplex DNA enters the helicase and unwinding occurs in the central channel. The results of this report inform important aspects of the structure, and we propose that CMG functions by a modified steric exclusion process in which both strands enter the helicase and the duplex unwinding point is internal, followed by exclusion of the non-tracking strand.

Neuroscience is entering a collaborative era in which powerful new technologies, generated by large scientific projects in many countries, will have a dramatic impact on science, medicine, and society. Coordinating these international initiatives and ensuring broad distribution of novel technologies and open accessibility of the generated data will multiply their value, while tapping creativity and expertise from every source.

The vertebrate-conserved RNA-binding protein DND1 is required for the survival of primordial germ cells (PGCs), as well as the suppression of germ cell tumours in mice(1-5). Here we show that in mice DND1 binds a UU(A/U) trinucleotide motif predominantly in the 3' untranslated regions of mRNA, and destabilizes target mRNAs through direct recruitment of the CCR4-NOT deadenylase complex. Transcriptomic analysis reveals that the extent of suppression is dependent on the number of DND1-binding sites. This DND1-dependent mRNA destabilization is required for the survival of mouse PGCs and spermatogonial stem cells by suppressing apoptosis. The spectrum of target RNAs includes positive regulators of apoptosis and inflammation, and modulators of signalling pathways that regulate stem-cell pluripotency, including the TGFa superfamily, all of which are aberrantly elevated in DND1-deficient PGCs. We propose that the induction of the post-transcriptional suppressor DND1 synergizes with concurrent transcriptional changes to ensure precise developmental transitions during cellular differentiation and maintenance of the germ line.

Cutaneous lymphocyte-associated antigen (CLA(+)) T cells are specialized for skin homing and represent the main T-cell population in atopic dermatitis (AD) lesions. CLA(+) is expressed on the surface of circulating CD45RO(+) memory T cells and most skin-infiltrating T cells. Mechanistic studies and thus treatment advancements are limited by the need of large number of skin biopsies. Circulating CLA(+) T cells may be a reliable surrogate marker of the inflammatory events occurring in the skin, and thus, the evaluation of CLA(+) T cells in the blood may eliminate the need for skin biopsies. Preliminary work in AD has established that disease-associated T-cell abnormalities can be approached by either a study of skin lesions or activated CLA(+) T-cell subsets in peripheral blood. Future studies in adults and children, across different skin disorders, correlating blood and skin phenotypes and determining skin-homing T-cell functional properties are needed to establish whether CLA(+) memory subsets can be used as biomarkers and a substitute for skin biopsies. This review summarizes the latest advancements reached on circulating CLA(+) in AD and the great potential they harbor in understanding AD mechanisms.

Innate lymphoid cells (ILCs) represent innate versions of T helper and cytotoxic T cells that differentiate from committed ILC precursors (ILCPs). How ILCPs give rise to mature tissue-resident ILCs remains unclear. Here, we identify circulating and tissue ILCPs in humans that fail to express the transcription factors and cytokine outputs of mature ILCs but have these signature loci in an epigenetically poised configuration. Human ILCPs robustly generate all ILC subsets in vitro and in vivo. While human ILCPs express low levels of retinoic acid receptor (RAR)-related orphan receptor C (RORC) transcripts, these cells are found in RORC-deficient patients and retain potential forEOMES(+) natural killer (NK) cells, interferon gamma-positive (IFN-gamma(+)) ILC1s, interleukin (IL)-13(+) ILC2s, and for IL-22(+), but not for IL-17A(+) ILC3s. Our results support a model of tissue ILC differentiation ("ILC-poiesis''), whereby diverse ILC subsets are generated in situ from systemically distributed ILCPs in response to local environmental signals.

With a single exception, all isolates of hepatitis C virus (HCV) require adaptive mutations to replicate efficiently in cell culture. Here, we show that a major class of adaptive mutations regulates the activity of a cellular lipid kinase, phosphatidylinositol 4-kinase IIIa (PI4KA). HCV needs to stimulate PI4KA to create a permissive phosphatidylinositol 4-phosphate-enriched membrane microenvironment in the liver and in primary human hepatocytes (PHHs). In contrast, in Huh7 hepatoma cells, the virus must acquire loss-of-function mutations that prevent PI4KA overactivation. This adaptive mechanism is necessitated by increased PI4KA levels in Huh7 cells compared with PHHs, and is conserved across HCV genotypes. PI4KA-specific inhibitors promote replication of unadapted viral isolates and allow efficient replication of patient-derived virus in cell culture. In summary, this study has uncovered a long-sought mechanism of HCV cell-culture adaptation and demonstrates how a virus can adapt to changes in a cellular environment associated with tumorigenesis.

The shortening of human telomeres has two opposing effects during cancer development. On the one hand, telomere shortening can exert a tumour-suppressive effect through the proliferation arrest induced by activating the kinases ATM and ATR at unprotected chromosome ends. On the other hand, loss of telomere protection can lead to telomere crisis, which is a state of extensive genome instability that can promote cancer progression. Recent data, reviewed here, provide new evidence for the telomere tumour suppressor pathway and has revealed that telomere crisis can induce numerous cancer-relevant changes, including chromothripsis, kataegis and tetraploidization.

ADP-ribosylation is a post-translational modification that is known to be involved in cellular homeostasis and stress but has been challenging to analyze biochemically. To facilitate the detection of ADP-ribosylated proteins, we show that an alkyne-adenosine analog, N-6-propargyl adenosine (N(6)pA), is metabolically incorporated in mammalian cells and enables fluorescence detection and proteomic analysis of ADP-ribosylated proteins. Notably, our analysis of N(6)pA-labeled proteins that are upregulated by oxidative stress revealed differential ADP-ribosylation of small GTPases. We discovered that oxidative stress induced ADP-ribosylation of Hras on Cys181 and Cys184 in the C-terminal hypervariable region, which are normally S-fatty-acylated. Downstream Hras signaling is impaired by ADP-ribosylation during oxidative stress, but is rescued by ADP-ribosyltransferase inhibitors. Our study demonstrates that ADP-ribosylation of small GTPases not only is mediated by bacterial toxins but is endogenously regulated in mammalian cells. N(6)pA provides a useful tool to characterize ADP-ribosylated proteins and their regulatory mechanisms in cells.

The membrane ring that equatorially circumscribes the nuclear pore complex (NPC) in the perinuclear lumen of the nuclear envelope is composed largely of Pom152 in yeast and its ortholog Nup210 (or Gp210) in vertebrates. Here, we have used a combination of negative-stain electron microscopy, nuclear magnetic resonance, and small-angle X-ray scattering methods to determine an integrative structure of the similar to 120 kDa luminal domain of Pom152. Our structural analysis reveals that the luminal domain is formed by a flexible string-ofpearls arrangement of nine repetitive cadherin-like Ig-like domains, indicating an evolutionary connection between NPCs and the cell adhesion machinery. The 16 copies of Pom152 known to be present in the yeast NPC are long enough to form the observed membrane ring, suggesting how interactions between Pom152 molecules help establish and maintain the NPC architecture.

The bacterial sigma factors confer promoter specificity to the RNA polymerase (RNAP). One alternative sigma factor, sigma(N), is unique in its structure and functional mechanism, forming transcriptionally inactive promoter complexes that require activation by specialized AAA(+) ATPases. We report a 3.4- angstrom resolution X-ray crystal structure of a sigma(N) fragment in complex with its cognate promoter DNA, revealing the molecular details of promoter recognition by sigma(N). The structure allowed us to build and refine an improved sigma(N)-holoenzyme model based on previously published 3.8-angstrom resolution X-ray data. The improved sigma(N)-holoenzyme model reveals a conserved interdomain interface within sigma(N) that, when disrupted by mutations, leads to transcription activity without activator intervention (so-called bypass mutants). Thus, the structure and stability of this interdomain interface are crucial for the role of sigma(N) in blocking transcription activity and in maintaining the activator sensitivity of sigma(N).