Multivalent binding of the tardigrade Dsup protein to chromatin promotes yeast survival and longevity upon exposure to oxidative damage.

Tardigrades are remarkable in their ability to survive extreme environments. The damage suppressor (Dsup) protein is thought responsible for their extreme resistance to reactive oxygen species (ROS) generated by irradiation. Here we show that expression of Ramazzottius varieornatus Dsup in Saccharomyces cerevisiae reduces oxidative DNA damage and extends the lifespan of budding yeast exposed to chronic oxidative genotoxicity. This protection from ROS requires either the Dsup HMGN-like domain or sequences C-terminal to same. Dsup associates with no apparent bias across the yeast genome, using multiple modes of nucleosome binding; the HMGN-like region interacts with both the H2A/H2B acidic patch and H3/H4 histone tails, while the C-terminal region binds DNA. These findings give precedent for engineering an organism by physically shielding its genome to promote survival and longevity in the face of oxidative damage.

A novel single alpha-helix DNA-binding domain in CAF-1 promotes gene silencing and DNA damage survival through tetrasome-length DNA selectivity and spacer function.

The histone chaperone chromatin assembly factor 1 (CAF-1) deposits two nascent histone H3/H4 dimers onto newly replicated DNA forming the central core of the nucleosome known as the tetrasome. How CAF-1 ensures there is sufficient space for the assembly of tetrasomes remains unknown. Structural and biophysical characterization of the lysine/glutamic acid/arginine-rich (KER) region of CAF-1 revealed a 128-Å single alpha-helix (SAH) motif with unprecedented DNA-binding properties. Distinct KER sequence features and length of the SAH drive the selectivity of CAF-1 for tetrasome-length DNA and facilitate function in budding yeast. In vivo, the KER cooperates with the DNA-binding winged helix domain in CAF-1 to overcome DNA damage sensitivity and maintain silencing of gene expression. We propose that the KER SAH links functional domains within CAF-1 with structural precision, acting as a DNA-binding spacer element during chromatin assembly.

A Simple, Improved Method for Scarless Genome Editing of Budding Yeast Using CRISPR-Cas9.

Until recently, the favored method for making directed modifications to the budding yeast genome involved the introduction of a DNA template carrying the desired genetic changes along with a selectable marker, flanked by homology arms. This approach both limited the ability to make changes within genes due to disruption by the introduced selectable marker and prevented the use of that selectable marker for subsequent genomic manipulations. Following the discovery of CRISPR-Cas9-mediated genome editing, protocols were developed for modifying any DNA region of interest in a similar single transformation step without the need for a permanent selectable marker. This approach involves the generation of a DNA double-strand break (DSB) at the desired genomic location by the Cas9 nuclease, expressed on a plasmid which also expresses the guide RNA (gRNA) sequence directing the location of the DSB. The DSB is subsequently repaired via homologous recombination using a PCR-derived DNA repair template. Here, we describe in detail an improved method for incorporation of the gRNA-encoding DNA sequences into the Cas9 expression plasmid. Using Golden Gate cloning, annealed oligonucleotides bearing unique single-strand DNA overhangs are ligated into directional restriction enzyme sites. We describe the use of this CRISPR-Cas9 genome editing protocol to introduce multiple types of directed genetic changes into the yeast genome.

Corrigendum: Commensal bacteria make GPCR ligands that mimic human signalling molecules.

This corrects the article DOI: 10.1038/nature23874.

Commensal bacteria make GPCR ligands that mimic human signalling molecules.

Commensal bacteria are believed to have important roles in human health. The mechanisms by which they affect mammalian physiology remain poorly understood, but bacterial metabolites are likely to be key components of host interactions. Here we use bioinformatics and synthetic biology to mine the human microbiota for N-acyl amides that interact with G-protein-coupled receptors (GPCRs). We found that N-acyl amide synthase genes are enriched in gastrointestinal bacteria and the lipids that they encode interact with GPCRs that regulate gastrointestinal tract physiology. Mouse and cell-based models demonstrate that commensal GPR119 agonists regulate metabolic hormones and glucose homeostasis as efficiently as human ligands, although future studies are needed to define their potential physiological role in humans. Our results suggest that chemical mimicry of eukaryotic signalling molecules may be common among commensal bacteria and that manipulation of microbiota genes encoding metabolites that elicit host cellular responses represents a possible small-molecule therapeutic modality (microbiome-biosynthetic gene therapy).

Thinking Outside the Cell: Replicating Replication In Vitro.

Benchmark studies by Yeeles et al. (2017), Kurat et al. (2017), and Devbhandari et al. (2017) have reconstituted rapid and regulated budding yeast DNA replication on naked and chromatinized templates in vitro, providing key insights into this fundamentally important process.

The aging mouse partially models the aging human spine: lumbar and coccygeal disc height, composition, mechanical properties, and Wnt signaling in young and old mice.

Murine lumbar and coccygeal (tail) regions of spines are commonly used to study cellular signaling of age-related disc diseases, but the tissue-level changes of aging intervertebral discs and vertebrae of each spinal region remain unclear. Furthermore, the impact of aging lumbar and coccygeal discs on Wnt/ß-catenin signaling, which is putatively involved in the catabolism of intervertebral discs, is also unclear. We compared disc/vertebrae morphology and mechanics and biochemical composition of intervertebral discs from lumbar and coccygeal regions between young (4-5 mo) and old (20-22 mo) female C57BL/6 mice. Center intervertebral disc height from both regions was greater in old discs than young discs. Compared with young, old lumbar discs had a lower early viscous coefficient (a measure of stiffness) by 40%, while conversely old coccygeal discs were stiffer by 53%. Biochemically, old mice had double the collagen content in lumbar and coccygeal discs of young discs, greater glycosaminoglycan in lumbar discs by 37%, but less glycosaminoglycan in coccygeal discs by 32%. Next, we compared Wnt activity of lumbar and coccygeal discs of 4- to 5-mo and 12- to 14-mo TOPGAL mice. Despite the disc-specific changes, aging decreased Wnt signaling in the nucleus pulposus from both spinal regions by ≥64%. Compared with young, trabecular bone volume/tissue volume and ultimate force were less in old lumbar vertebrae, but greater in old coccygeal vertebrae. Thus intervertebral discs and vertebrae age in a spinal region-dependent manner, but these differential age-related changes may be uncoupled from Wnt signaling. Overall, lumbar and coccygeal regions are not interchangeable in modeling human aging.